Abstracts of the papers presented in the international conference of Indian Virological Society, VIROCON 2022 on “Emerging and re-emerging viral infections impacting humans, animals, plants, fish and environment” held during 05–06 November, 2022 at Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Srinagar (J&K), India

Abstract

Kameshwar Sahai Bhargava Oration Award Perspective on research approaches for characterization of plant viruses in India V. K. Baranwal Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi-110012 India The first plant virus causing mosaic disease in tobacco was characterized based on the infectious nature of sap that passed through bacteria proof filter obtained from diseased plants in the late 19th Century. It was followed by local lesion assay, crystallization of virus particles, shape- and size-based virion characterization through electron microscopy, transmission assays. Serological reaction-based assay and production of antibodies using recombinant coat protein gene and synthetic peptides have paved the way for inhouse production of serological diagnostic assay. More sensitive nucleic acid-based assays particularly recombinase polymerase amplification has simplified the detection of viruses even in resource constrained laboratory. The first-generation sequencing technologies unveiled the first complete genome sequence of cauliflower mosaic virus in 1980. Since then, sequencing technologies became ultimate tool to characterize viruses. The advent of next generation sequencing (NGS) technologies has revolutionized the field of plant virology as more and more new viruses are being discovered in different hosts, in different geographical locations and this has facilitated better understanding of viruses. Our recent analysis of public transcriptome data sets as well as sRNA or mRNA sequencing have shown the association of several known/unknown/novel viruses with various plant species. Application of deep sequencing approaches alleviates the problem of false negatives due to high genetic variability or low titer. It has great potential for virus indexing, prevention of introduction of viruses through planting material in newer areas and production of certified planting material in clonally propagated crops. Medical Virology (Oral Presentations) Influenza: Experiences from Kashmir Dr. Parvaiz A Koul Director and Ex-Officio Secretary to Govt. of J&K, SKIMS, Soura, Srinagar Seasonal flu is caused by influenza viruses which circulate in all parts of the world. There were no published data on the existence or circulation of influenza in Kashmir (northern part of India) until 2010. Influenza lab in Sheri Kashmir Institute of Medical Sciences was setup in 2010 by Dr Parvaiz Koul. The laboratory was one of the eight facilities in India and was the outcome of joint cooperative agreement between Centre for Disease Control and Prevention (CDC, USA) Atlanta and Indian Council of Medical Research (ICMR). Initially, the facility was supposed to conduct a “dozen odd tests” a week. However, it soon emerged as an an emergency centre to manage crises as and when it emerged especially during H1N1 epidemic following the 2009–2010 pandemic, the resurgence in 2012 and a new clade associated resurgence in the year 2015. Also outbreak studies were conducted as and when required like an Influenza B outbreak in the nomadic community in Pulwama. Apart from the epidemic studies, we have conducted various surveillance studies in high-risk groups including pregnant females and have also demonstrated the contribution of Influenza in Community acquired pneumonia and acute exacerbation of COPD. We have also explored the Knowledge, Attitude and Practices associated with the Influenza vaccination among Health-care workers as well as corporate employees as well as high risk groups like those with pregnancy, COPD, diabetes, chronic heart failure, etc. The laboratory undertook influenza surveillance during 2011–2012 in 2 cities in northern India, Srinagar and New Delhi, and found evidence for discrete seasonality related to the latitudes of these cities, a finding that has implications for influenza vaccination policy and timing and led WHO to change the vaccination strategy for India. A six-year study from 2010 to 2016 from SKIMS regarding the circulation of Influenza B in Kashmir concluded that Influenza B exhibits a northern hemispherical seasonality in temperate northern India with co-circulation of the 2 lineages of influenza B. These findings exhibited relevance for vaccine effectiveness and argued for vaccination with a Quadrivalent influenza vaccine. Recently a study by our group revealed poor vaccine effectiveness due to a genetic mismatch between the circulating strain and the vaccine strain and called for efforts to continue genomic surveillance for influenza for appropriate matching of the vaccine. SKIMS is the only center in the country to have conducted vaccine effectiveness studies in collaboration with the GIHSN network. After ten years, the influenza lab at SKIMS, in association with the GIHSN, France carries out regular surveillance of influenza and other respiratory viruses in addition to other research projects on vaccination uptake and vaccine effectiveness. Currently the flu lab is involved with various influenza and other respiratory pathogen related projects in Kashmir. All of these studies have been published in high impact journals. SKIMS also has the distinction of developing guidelines for influenza vaccination by various physician bodies like the Indian Chest Society, Geriatric Society fo India, Association of Physicians of India, Indian Association of Occupational Health, etc. PI from the lab, Dr Koul has participated in the only influenza effectiveness studies from the country and recently have not only hinted at sublineage related vaccine effectiveness but also at redefining the influenza equator globally. The published works from the group have earned Dr Koul the Vice Chairmanship of the Middle East Eurasia North Africa Influenza Stake Holders Network (ME’NA ISN)., a global influenza study society. Development of a colorimetric isothermal (LAMP) assay for rapid detection of Monkeypox virus Dr. Shyam Sundar Nandi, Ms. Sonali Ankush Sawant, Dr. Upendra Lambe, Dr. Pragya D. Yadav, Dr. Anita Shete, Dr. Jagadish Deshpande ICMR- National Institute of Virology, (Mumbai Unit), Dept. of Health Research, Ministry of Health and Family Welfare, Govt. of India Monkeypox virus (MPXV) causes a smallpox-like disease in non-human primates and humans. The technology discloses a loop-mediated isothermal amplification (LAMP) for rapid and low cost detection of Monkeypox virus. According to the WHO requirement, the diagnosis of the Monkeypox requires detection of a Orthopoxvirus genus specific gene and confirmation based on Monkeypox virus specific genes. The oligonucleotide primers have been designed targeting the genus specific H2R gene belonging to putative viral trans-membrane protein. While the B6R (Envelope protein), F3L (double-strand RNA-binding protein), F8L (DNA Polymerase) and Rpo18 (DNA dependent RNA polymerase subunit 18) species specific genes have been targeted for development of this assay. The principle of this assay is by using pH sensitive dyes to exploit the change in the pH resulting from proton accumulation while incorporation of dNTPs. Change in the color of reaction can be detected by naked eyes. No sophisticated instruments are required for the performance and results interpretation. By application of the LAMP for detection as described in this invention, the demerits of real-time RT-PCR such as long detection period, complex operation, and sophisticated instrument requirement have been overcome. This assay is suitable to be performed as a point of care diagnostic assay at the health care centers, surveillance laboratories, airports, frontier ports and other areas and has important significance for preventing and controlling the spread of Monkeypox virus. Outbreaks of different viral etiologies amidst COVID-19 pandemic *Pragya D. Yadav ICMR-National Institute of Virology, Pune Maharashtra, India, Pin-411021 The COVID-19 pandemic has severely affected public health system and surveillance of other communicable diseases across the globe. The lockdown, travel constraints and COVID phobia turned down the number of people with illness visiting to the clinics or hospitals. Besides this, the heavy workload of SARS-CoV-2 diagnosis has led to the reduction in differential diagnosis of other diseases. Consequently, it added to the underlying burden of many diseases which remained under-diagnosed. Amidst the pandemic, the rise of emerging and re-emerging infectious diseases was observed worldwide and reported to the World Health Organization i.e., Crimean Congo Hemorrhagic Fever (2022, Iraq; 2021 India), Nipah virus (2021, India), Zika virus (2021, India), and H5N1 influenza (2021, India), Monkeypox (2022, multi-country outbreak), Ebola virus disease (2022, DRC, Uganda; 2021, DRC, Guinea; 2020, DRC), Marburg (2022, Ghana; 2021, Guinea), Yellow fever (2022, Uganda, Kenya, West and Central Africa; 2021, Ghana, Venezuela, Nigeria; 2020, Senegal, Guinea, Nigeria, Gabon; 2020, Ethiopia, Sudan, Uganda), Dengue (2022, Nepal, Pakistan, Sao Tome, Temor-Leste; 2021, Pakistan), Middle east respiratory syndrome coronavirus (2022, Oman, Qatar; 2021, Saudi Arabia, UAE; 2020, Saudi Arabia, UAE), Rift valley fever (2021, Kenya; 2020, Mauritania), wild poliovirus type 1 (2022, Mozambique), Lassa fever (2022, Guinea, Togo, Nigeria; 2020, Nigeria), Avian Influenza (H3N8) (2022, China), Avian Influenza (H5N1) (2022, USA), H10N3 influenza (2021, China), Hepatitis E virus (2022, Sudan), Measles (2022, Malawi, Afghanistan; 2020, Burundi, Mexico), Mayaro virus disease (2020, French Guiana), Oropouche virus disease (2020, French Guiana). All these diseases were associated with high morbidity and burdened the public health system during the COVID-19 pandemic. During this critical public health menace, majority of the laboratory workforce was mobilized to the SARS-CoV-2 diagnosis. This has limited the surveillance efforts that likely led to under diagnosis and under-detection of many infectious pathogens. Lockdowns and travel limitations also put a hold on human and animal surveillance studies to assess the prevalence of these zoonotic viruses. In addition, lack of supplies and laboratory personnel and an overburdened workforce negatively impacted differential diagnosis of the diseases. This is especially critical given the common symptoms between COVID-19 and other pathogens causing respiratory illnesses. Additionally, the vaccination programs against various vaccine preventable diseases were also hampered which might have added to the disease burden. Despite these challenges, the world is better prepared to detect and respond to emerging/re-emerging pathogens. India now has more than 3000 COVID-19 diagnostic laboratories and an enhanced hospital infrastructure. In addition, mobile BSL-3 facilities are being validated for onsite sampling and testing in remote areas during outbreak situations and surveillance activities. This will undoubtedly be valuable as the COVID-19 pandemic evolves as well as during future outbreaks and epidemics. In conclusion, an increase in the emergence and re-emergence of viruses demonstrates that other infectious diseases have been neglected during the COVID-19 pandemic. Lessons learned from the infrastructure strengthening, collaborations with multiple stakeholders, increased laboratory and manufacturing capacity, large-scale COVID-19 surveillance, extensive network for laboratory diagnosis, and intervention strategies can be implemented to provide quick, concerted responses against the future threats associated with other zoonotic pathogens. Circulation of genetically diverse non-polio enteroviruses in respiratory samples during COVID-19 pandemic period (2021–22) Tatte Vaishali, Wagh Saptesh, Potdar Varsha, Lavania Mallika and Mullick Jayati* Poliovirus Group, ICMR-NIV, Pune Enteroviruses, beyond poliovirus, are important pathogens. Several non-polio enteroviruses (NPEVs) are causing epidemics all around the world. Limited data is available on the prevalence and diversity of these viruses from India. Objectives: Detection and characterization of NPEVs in respiratory samples during the COVID-19 pandemic period. Materials and Methods: COVID-19 negative samples from acute respiratory infections (ARI) [n = 105] and severe-acute respiratory infections (SARI) [n = 148] during the period 2021–22 were screened for NPEVs. Detection was carried out using the one step RT-PCR method targeting the 5’UTR region followed by molecular analysis. Results and Conclusions: Total positivity of NPEVs was noted in 35.23% and 31.08% of the ARI and SARI cases, respectively. Comparison within the two groups studied, showed significant difference in the age-wise distribution for cases > 18 years of age. Year round seasonality for ARI cases while autumn seasonality for SARI cases was observed. Sequencing of representative samples of ARI cases showed prevalence of Rhinovirus A (RVA), Rhinovirus B (RVB), Rhinovirus C (RVC) and Echovirus, while predominance of RVC followed by RVA was observed for the SARI cases. Phylogenetic analysis of all the strains showed clustering of RVC strains in different clusters. Divergence was also noted in RVA and RVB strains studied. Circulation of a rare Echovirus-29 strain was noted in the ARI cases. The study highlighted significant divergence in the Rhinovirus strains studied. It warrants the need for surveillance of NPEVs, whole-genome sequencing of the circulating strains for better understanding of biodiversity among the NPEVs and the potential health burden. Molecular Characterization of dengue viruses circulating in Pune district, Maharashtrafrom 2009–2022 JA Patil, MB Kakade, M Bote, YK Gurav, D Parashar and K Alagarasu Dengue and Chikungunya Group, ICMR-National Institute of Virology, Pune-411001, Maharashtra, India Email: [e-mail] Dengue, a vector borne viral disease caused by four serotypes of dengue virus (DENV), is endemic in Pune district of Maharashtra, Western India. The magnitude of incidence of dengue is influenced by changes in the circulating serotypes and genotypes within each serotype of DENV. ICMR-National institute of Virology (NIV) is an apex referral laboratory for molecular surveillance of DENV in Maharashtra. ICMR-NIV has tested 6394 samples from 2009 to 2021 using conventional semi nested RT-PCR and real-time RT-PCR for detection of DENV serotypes. Among these samples, 1811 samples were positive at least one serotype of DENV. Further, the envelope gene of representative samples positive for each serotype were sequenced to find out the genotypes of each serotype. The result revealed that DENV-2 was the dominant serotype during the period of 2009 to 2015 and re-merged in 2019 and 2021 as the most prevalent serotype. DENV-3 was codominant with DENV-2 from 2009–2013 and re-emerged as dominant serotype in 2018. DENV-4 was co-dominant with DENV-2 in 2014 and 2015. DENV-1 was codominant with DENV-3 in 2017 and 2018 and with DENV-2 in 2019. Phylogenetic analysis of envelope gene sequences revealed that GIV as the circulating genotype for DENV-1 and introduction of G1 of DENV-1 was observed in 2018. The cosmopolitan genotype, GIII and GI were the circulating genotypes for DENV-2, DENV-3 and DENV-4. Within the cosmopolitan genotype of DENV-2, multiple lineages were found to be circulating. The study underscores the importance of molecular surveillance of DENV and might be useful in predicting outbreaks when changes in the dominant serotypes/genotypes were detected early during dengue season. Evolutionary analysis of all eleven genes of species C rotaviruses circulating in humans and domestic animals Madhuri S. Joshi 1, Atul M. Walimbe 2, Shalu A. Arya 1, Varanasi Gopalkrishna 1 1Enteric Viruses Group, ICMR- National Institute of Virology, Pune, India. 2Bioinformatics Group, ICMR- National Institute of Virology, Pune, India Email: [e-mail] Background: Species C rotaviruses (RVC) are the second most common rotavirus species known to cause gastroenteritis in humans and pigs, and have occurred in bovine, canine, ferret, and sloth bears. Despite the host-specific nature of RVC genotypes, cross-species transmission, reassortment, and recombination events are documented. Objectives: The main aim of the present study was to elucidate the evolutionary relationships and time scale stasis existing in RVC strains circulating in different hosts globally. Material and Methods: RVC nucleotide sequence data obtained from multiple countries, hosts and periods (1970–2017) along with three strains previously reported from ICMR-NIV (India) were shortlisted for the phylo-geographical analysis with country and host as traits. For each gene, the Maximum Clade Credibility (MCC) tree was constructed by using the Bayesian Markov Chain Monte Carlo (MCMC) algorithm as implemented in BEAST 1.8.4. Results and Conclusions: The human strains were majorly monophyletic and further grouped into two lineages. The porcine strains were monophyletic only for the NSP5 and VP1 genes and the bovine strains for all 11 genes. The root mean age for all the genes indicated the circulation of RVC for over 800 years. Overall, the most recent common ancestor of human strains dated back to the beginning of the nineteenth century. Most of the genes of human strains showed their origin in Japan or China, with the exception of the VP3-M2 genotype and the NSP1 gene in India. The most probable ancestral host for human strains was porcine for the majority of the genes. Isolation and genomic characterization of Cell fusing agent virus from Aedes aegypti mosquitoes from Assam, India Abhranil Gangopadhayya, Onkar Ghuge, Ashwini Ramdasi, Asmita Kamble, Surendra Kumar, Sreelakshmi PR, Sudeep Balan, SS Cherian, KS Lole Indian Council of Medical Research-National Institute of Virology, Pune Background: India has a diverse population of haematophagous arthropods, the virus populations inhabiting which are hitherto poorly characterized, including that of Aedes aegypti, a nearly ubiquitous vector for major human pathogens. During our study on metagenomic analysis of Aedes mosquito viromes from different parts of India, we identified a wide range of insect-specific viruses (ISVs). Of these, some ISVshave been reported to influence replication of human pathogenic viruses in mosquitoes. Cell Fusing Agent Virus (CFAV), belonging to family Flaviviridae is one of such ISVs. It was detected in mosquitoes from Assam and West Bengal. Objectives: To isolate Cell fusing agent virus (CFAV) from Ae. aegypti mosquitoes, perform genetic characterization of the virus and study growth kinetics of the virus in insect cells and mosquitoes. Materials and Methods: ISV isolation, cytopathic effect (CPE) production, and growth kinetics studies were done using the C6/36 Aedes albopictus cell line. WGS was done using a multiplexPCR tiling approach. Phylogenetic analyses were done using MAFFT and MEGA 5.2 software. Results and Conclusion: Mosquito cell line, C6/36was inoculatedwithmosquito lysate containing CFAV and passaged blindly. CPE and characteristic cell fusion pattern was seen in infected cells after three passages. Analysis of CFAV WGSshowed highest homology (~ 96.66%) with 2014Australian isolate while lowest homology (~ 94.28%) was seen with 2015 Cambodian isolate. It would be insightful to know whether CFAV has any effect on the replication and transmission of clinically important arboviruses such as dengue, chikungunya and zika. Integration of HBV Receptor NTCP into Hepatoma Cell Using Grnome Editing Pooja Bhatia, Naga Suresh Veerapu Department of Life Sciences, Shiv Nadar Institution of Eminence, NH91, Gautam Buddha Nagar, UP 201314 Background: Hepatitis B virus (HBV) is an enveloped DNA virus that causes acute and chronic hepatitis. Recently, sodium taurocholate co-transporting polypeptide (NTCP) has been identified as the cellular receptor for the attachment of HBV. It is liver specific bile acid transporter that plays role in the enterohepatic circulation of bile acids. CRISPR-Cas9 is a powerful, highly specific, and efficient genetic tool for a genome editing. CRISPR-Cas9 plasmids with the homology arms target AAVS1 loci on the chromosome 19 of the human genome. The homology arms in plasmid are recognized by the HDR mechanism, and the entire cassette containing the gene of interest between the left and right homology arms gets integrated. Objective: We aimed to generate a stable NTCP-expressing Huh7 cell line to make the cell susceptible and permissive to HBV infection using genome editing approach. Methodology: Huh7 cells were co-electroporated with the donor plasmid that has NTCP gene template and sgRNA carrying plasmid for the integration of NTCP. NTCP induced using tetracycline and its expression verified by RT-PCR, immunofluorescence and western blotting. Results: Stable NTCP expression has been observed after puromycin selection and tetracycline induction of Huh7 cells. Huh7-NTCP cells expressed NTCP in a dose dependent manner. A 1 Kb long specific NTCP gene detected using RT-PCR. NTCP was also detected in immunofluorescence assay and a ~ 40KDa NTCP was detected through western blotting. Conclusion: To understand the HBV infection at the entry-level and complete life cycle, an efficient cell culture model development is necessary that will show high levels of infection. It is anticipated that new technology of using the CRISPR-Cas9 tool to integrate the NTCP gene in the hepatoma cells may allow for better cell culture systems by making cells more permissive to HBV entry. Through the cells expressing NTCP, the mechanism behind early steps in the HBV life cycle can be studied and therapeutic studies can be applied in-vitro to inhibit HBV from causing infection. Structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase Ravi Kumar, Akshay Pareek, Pravindra Kumar, Shailly Tomar Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India – 247667 Email: [e-mail] Background: Alphavirus nsP4 replication protein possesses RNA dependent RNA polymerase (RdRp) activity and is a potential antiviral target. Alphaviruses are continuously re-emerging and are a global threat to human health. Until date, no antiviral drug is commercially available against alphaviruses. Objectives: Here, in this study for the first time small molecule inhibitors targeting nsP4 replication protein were identified and characterized as an effective antiviral against alphaviruses. Materials and Methods: RdRp Δ97 protein was purified and biophysical techniques were employed for analyzing interaction of various ligands with target nsP4 protein. Further, Binding to purified protein were analysed using surface plasmon resonance (SPR). For identification of potential inhibitors, molecular docking was done using 3D model of chimeric RdRp domain of chikungunya nsP4 generated using Phyre2. Further, evaluation of antiviral activity of these molecules was done against alphaviruses (Sindbis and Chikungunya virus) using in vitro cell-based antiviral assay. Results and Conclusions: Four potential inhibitors, piperine (PIP), 2-thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA) targeting the nucleotide-binding site in RdRp were identified. SPR experiments validated the binding of all four molecules to RdRp and affinity of PIP, 2TU, PZA, and CGA, were 0.08, 0.13, 0.66, and 9.87 µM, respectively. These were effectively inhibiting the Sindbis and Chikungunya virus and the EC50 values of PIP, 2TU, PZA, and CGA, were 6.68, 27.88, 35.56, and 53.62 µM, respectively. In conclusion, these non-nucleoside small molecules have the potential to be used for the development of an effective antiviral therapy for treatment of alphaviral infections. Lytic reactivation of Epstein-Barr Virus occurs via LMP1-DNMT1- ID3 lineage and modulates CpG methylation patterns in host leading to carcinogenesis Nabanita Roy Chattopadhyay* and Tathagata Choudhuri Department of Biotechnology, Visva-Bharati, Santiniketan, West Bengal, PIN: 731235 *Corrspondence: [e-mail], Mobile: [phone] Background: EBV-associated cancers, like nasopharyngeal carcinoma, involve hundreds of host genes to become modified, either by direct interactions between host and viral proteins, or by modulation of CpG methylation patterns of the host/viral genes leading to silencing of some beneficial genes for preventing cancers. Objective: We have tried to determine the mechanism for EBV-mediated aberration of CpG methylation patterns in the host. Materials and Methods: 1. Raji (EBV positive) and Ramos (EBV negative) cell lines were established. 2. RNA extraction, DNA extraction, and total protein extraction were done as per the published protocols. 3. PCR amplifications of LMP1, ID3, HDAC1, and DNMT1 genes were done for both cell lines. 4. Presence of various proteins (LMP1, ID3, HDAC1, and DNMT1) were checked in both cell lines by standard western blotting techniques. Results: 1. EBV infection does not alter the presence/amount of both DNMT1 and HDAC1 in host cells. 2. In both RT-PCR results and western blot results, it is seen that presence of LMP1 alters the expression of ID3 of host. In EBV-positive cells, LMP1 is active and it lowers/silences ID3 of the host. But in EBV-negative cells, where LMP1 is not expected to be present, the presence and amount of ID3 is clearly seen. Conclusion: LMP1 of EBV might have a negative regulation for host-ID3 gene which plays the key role for EBV-reactivation and the latent-lytic switch. Our findings clearly indicate that EBV-mediated carcinogenesis (like the nasopharyngeal carcinoma as our model) occurs via the LMP1-DNMT-ID3 lineage. This will help prognosis of such cancers by modulating the host-ID3 suppression via EBV infection. Hepatitis B Virus Genome Targeting Using CRISPR/Cas9based Gene Editing Tool Baibaswata Nayak, Geetanjali Lal, Afnan Quadri, Rutumbara Das, Shalimarand Anoop Saraya Department of Gastroenterology & Human Nutrition, All India Institute of Medical Sciences, New Delhi, India Background: HepatitisB virus (HBV) belongs to genus Orthohepadnavirus of the family Hepadnaviridae. The HBV genome is partially dsDNA circular genome of 3.2 kb long and four partially overlapping genes encode core, polymerase, surface and HBx proteins. Chronic HBV infection responsible for liver fibrosis, cirrhosis and hepatocellular carcinoma. There is no curative therapy for HBV. The potent nucleos(t) ide analogs therapy effective only in reducing viral load but not episomalcccDNA archived in the cell nucleus. TheCRISPR/Cas9 based gene editing is now promising tool to target cccDNAof HBV and subsequent cleavage. This gene editing tool consists of the single guide RNA (sgRNA) that guide Cas9 endonuclease to cut specific DNA sequence by recognizing the protospacer-adjacent motif (PAM) at target DNA sequence. Methods: The guide sequences[5’-N (20) NGG-3’]were predicted with high specificity to surfaceand core region of HBV and less off targets on human genome hg 37 was determined using online software tool. The plasmid based system (pGuide-it CRISPR/Cas9) was used for mammalian expression of target specific sgRNA, Cas9 and reporter green fluorescent protein (ZsGreen1) for monitoring transfection efficiency. The custom oligos containing 20 nt long target sequence was synthesized with flanking overhangsfor cloning. Full length HBV 1.3 mer replicon and specific sgRNA CRISPR plasmids were co-transfected in 293 T and Huh7 cell lines. Levels of reduction in HBsAg and HBeAg were detected by Vidas based ELFA system. Results: Co-transfection HBV1.3mer and CRISPR/Cas9 plasmids was carried out and transfection efficacy confirmed by zsGreenfluorescence protein. Levels of HBsAg and HBeAg were determined in the supernatant of co-transfected cells by ELFA. Almost 85–90% reduction in HBsAg and HBeAg levels was observed in cells transfected with CRISPR constructs, at transfection ratio of 1:1. Conclusion: Novel CRISPR-Cas9 based strategies to target the viral genome in both its replicative and episomal formmay emerge as a curative alternative by disrupting the viral genome leading to loss of function. Genome characterization of monkeypox cases detected in India: Identification of three sub clusters among A.2 lineage Anita M. Shete1, Pragya D. Yadav1, Abhinendra Kumar1, Savita Patil1, Deepak Y. Patil1, Yash Joshi1, Triparna Majumdar1, Vineet Relhan2, Rima R. Sahay1, Meenakshy V3, Pranita Gawande1, Ajay Verma1, AnukumarBala Krishnan4, Shubin Chenayil5, Suresh Kumar2, Priya Abraham1 1Indian Council of Medical Research-National Institute of Virology, Pune, Maharashtra.2Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India, Pin-110002. 3Public Health Department of Kerala, Directorate of Health Services, Thiruvananthapuram, 4Indian Council of Medical Research-National Institute of Virology, Alappuzha, Kerala, India Background: Monkeypox virus (MPXV) have a genetic core of about 120 kbwhich is highly conserved; while the termini region is plastic. Low mutation rates serve as a reminder that even poxviruses, which are DNA viruses that have a tendency to evolve. This necessitates complete genome sequencing of monkeypox cases to understand the evolutionary pattern of the circulating MPXV strain. Here, we report the findings of genomic characterization and phylogenetic analysis of ten monkeypox cases detected in India during July–August 2022 from Kerala (n = 5 UAE travel history) and Delhi (n = 5 without travel history) and identification of three sub clusters among A.2 lineage. Objectives: Genomic characterization of monkeypox cases detected in India. Methods: We performed complete genome analysis of ten monkeypox positive cases from Kerala (n = 5 travel from UAE) and Delhi (n = 5 no travel history) using next-generation sequencing. Results: The phylogenetic analysis placed ten genome sequences from India under lineage A.2 of clade IIb. Further, they diverge into three sub clusters of A.2 lineage. In this sub cluster, five sequences from Kerala were designated as A.2.1 based on the lineage defining mutations in the position C 25072 T, A 140492 C, C 179537 T. Two sequences from Delhi are lacking these three mutations hence still defined into A.2 lineage. These mutations were also lacking in the 3 sequences of Delhi from sub cluster II which aligned with sequences of lineage A.2 reported from USA 2022 (USA_2022_VA001). Delhi MPXV sequences in sub cluster I and II are showing divergence which needs to be further explored. Conclusions: The study emphasizes need of enhancing MPXV genomic surveillance to understand themutationto understand evolution of the MPXV genome. Keywords: Monkeypox cases; Genomic characterization; Phylogeny, India Genetic characterization of human Bocavirus strains isolated from patients with acute lower respiratory tract infections in tertiary care hospital of north India Meenakshi Rana1, Subhabrata Sarkar1, Mannat Kang1, Vikrant Sharma1, Shankar Prasad2, Rishi Chetanya1, Ishani Bora1, Suresh Kumar2, Muralidharan Jayashree2, R K Ratho1* Department of Virology1 and Advanced Paediatrics Centre2 *Corresponding author Background: Human bocavirus (HBoV) was first described in 2005 and found worldwide in respiratory samples, mainly from children of 6–24 months of age with acute respiratory infections. In India, the scientific information about the circulating hBoV genotype is scanty. Objectives: To detect circulating human bocavirus DNA in children with lower respiratory tract infections and determine circulating genotype followed by phylogenetic analysis. Materials and methods: The study focused on the detection of human Bocaviral DNA in respiratory samples collected from patients with acute lower respiratory tract infections targeting the NP1 gene and determination of circulating genotypes of Bocavirus through amplification of VP1-VP2 gene followed by Sanger sequencing. The phylogenetic trees generated with several molecular algorithms available with MEGA7 are evaluated for the log-likelihood value using https://evomics.org/resources/likelihood-ratio-test/ and the highest log likelihood was chosen for the display. The study described the clinical course and outcomes of Bocavirus-infected patients. Results: One hundred and sixty-seven children (between 2 months to 12 years of age) with clinically suspected viral pneumonia, bronchiolitis, and ARDS from July 2021 to July 2022 were enrolled in the study with positivity of 6.5% (11/167). Out of the 11 bocavirus-infected patients, 81.81% (9/11) patients needed PICU management with a mean duration of 5.6 days (ranging from 3 to 11 days), and 77.77% (7/9) patients needed mechanical ventilation support and 1 patient expired. Pneumonia was the most common presentation (7 cases), followed by disseminated viral illness with encephalopathy (3 cases) and Acute gastroenteritis with severe acute malnutrition (1 case). hBOV strains (n = 11) could be amplified for the VP1–VP2 gene and phylogenetic analysis revealed all the strains belong to hBoV1 genogroup St1. Percent identity matrix of hBOV1 St1 reference strain (DQ000495) ranged from 98.51% to 99.5% and with hBOV1 St2 reference strain (DQ000496) ranged from 98.1% to 99.5%. Conclusion: Information about the seasonality trend, synonymous-nonsynonymous mutations, and circulating genotypes may be useful in the futurefor vaccine development to prevent emerging viral infection. Immunogenetic studies on SARS-CoV-2: Our experience at SKIMS Malik Gawharul Haq, Rabiyah Maqbool, Zafar A Shah, S Mudasir Qadri, Fouzia Rashid Presenter: Dr. Zafar A Shah, MVSc., PhD. Designation: Professor and Head (Immunology & Molecular Medicine, SKIMS, Srinagar) Email: [e-mail] Background: The pathophysiology of viral-infections is highly complex and involves host immunocompetence, host genetics, and gene-environment interactions. We hypothesized that polymorphic variants in host genes, blood group and previous vaccination status against H1N1 may affect the clinical course of covid-19 infection. Methods: A total of 202 subjects who were RT-PCR negative after Covid-19 infection were recruited. We investigated association between Covid-19 infection (Severity and recovery period) and multiple factors including ABO and Rh blood groups, H1N1 vaccination, polymorphism in Viral susceptibility genes (ACE2 G8790A), and polymorphism in host response genes (ACE I/D rs4646994, IL6-174G/C, GSTT1/GSTM1 I/D and GSTP1 Ile 105 Val). Results: B-ve and O-ve ABO and Rh blood groups had significantly higher Covid-19 recovery period applied on one-vs.-all in a non-parametric t-test (p < 0.05). Subjects who had vaccinated themselves against H1N1 presented with a lower recovery-period (p < 0.05). Both variables (blood group and H1N1 vaccination) were not however associated with Covid-19 severity. Out of the studied polymorphisms, ACE2 G8790A and GSTT1/GSTM1 were significantly associated with covid-19 infection. Our results indicated that G/G genotype of ACE2 G8790A (OR 3.52, P 0.007) and GSTT1/GSTM1 null (M1 − / − OR = 3.98, P = 0.0004; T1 − / − OR 3.84, P = 0.004) and double null (M1 − / − /T1 − / − OR = 9.66, P = 0.001) are likely to be associated with an increased risk for severe–critical outcomes in individuals with COVID-19. Other polymorphisms analyzed in this study were found to have no significant association with Covid-19 outcome. Conclusion: This study suggests that outcome of Covid-19 infection is affected by both clinical and genetic factors. Thus it seems plausible to utilize these factors as prediction and susceptibility markers in the prognosis of COVID-19, which may help to personalize the treatment. Detection of SARS-CoV-2 RNA in stool and urine specimens of COVID-19 positive patients by Droplet Digital RT-PCR (dd RT- PCR) Mallika Lavania1, Manohar Shinde1, Jatin Rawal1, Aditi Kaledhonkar2, Pooja Shinde1, Nutan Chavan1 and Varsha Potdar2 1Enteric Viruses Group, ICMR-National Institute of Virology, Pune. 2National Influenza Centre, ICMR-National Institute of Virology, Pune Email address of presenting author: [e-mail] SARS-CoV-2 infected cases diagnosis is based on the count of real-time reverse transcription-polymerase chain reaction (RT-PCR). The widely used reverse transcription-polymerase chain reaction (RT-PCR) method has some limitations for clinical diagnosis and treatment. However, there are only few reports on the detection of the viral load in the stool and urine samples. While information about other modes of transmission is relatively less, some published literature supporting the possibility of a faecal-oral mode of transmission has been accumulating. Objective: The current study's objective was to assess the performance of real-time RT-qPCR assay and a droplet digital RT-PCR (dd RT-PCR) for detecting SARS-CoV-2 in stool and urine specimens. Methodology: One hundred and seven paired samples from 107 COVID-19-confirmed patients were analysed by dd RT-PCR and RT-PCR based target gene (N1 and N2). Stool and urine were collected from COVID Care Centers of Pune Region. RNA was isolated using MagMax magnetic beads base procedure for further analysis. Real Time RT-PCR and DD PCR was performed from all the patients. Results: In 107 patients, all the stool samples showed 100% positive concordance by both methods, the average of 28.88 cycle threshold (Ct) of RT-PCR was highly correlated with the average copy number of 327.10 copies/µl analyzed in ddPCR. Whereas 27.1% urine samples were tested positive in ddPCR & 1.86% were positive with the average of 36.41 cycle threshold (Ct) in RT-PCR. Using Pangolin COVID-19 Lineage Assigner variants were analyzed and found to be delta prevalent. Conclusion: In the context of the COVID-19 pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. The findings of this study not only show that SARS-CoV-2 is present in urine and faeces, but they also raise the possibility that low concentrations of the viral target may make it easier to identify positive samples and help resolve situations of inconclusive diagnosis. Structure-Based Identification and Analysis of Viral Protease Inhibitors Targeting SARS CoV-2 Virus Replication Ankur Singh1, Sanketkumar Nehul1, Ruchi Rani1, Manidipa Banerjee2, Pravindra Kumar1, Gaurav Sharma3, Shailly Tomar*1 1Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India. 2KUSUMA Schoolof Biological Sciences, Indian Institute of Technology Delhi, New Delhi, 110016, India. 3Centre for Animal Disease Research and Diagnosis, ICAR-Indian Veterinary Research Institute. Izatnagar, Bareilly, Uttar Pradesh, 243122, India E-mail: [e-mail] The re-emergence of SARS-CoV, known as SARS-CoV-2, has proven extremely infectious that has infected a huge population worldwide. SARS-CoV-2 genome is translated into polyproteins that is processed by virus-specific protease enzymes. 3CLprotease is named as the main protease (Mpro) enzyme that cleaves nsp4 to nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. Till date, there is no effective commercially available drug against COVID-19 and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. We have used a high-throughput virtual screening approach to examine FDA approved library, natural compound library, and LOPAC 1280 (Library of Pharmacologically Active Compounds, Sigma-Aldrich, St. Louis, MO) library against Mpro. Primary screening identified potential drug molecules for the target, among which ten molecules were studied further using biophysical and biochemical techniques. SPR was used to validate the binding of inhibitors to purified Mpro and using FRET-based biochemical protease assay these inhibitors were confirmed to have Mpro inhibitory activity. Based on the kinetic studies, the antiviral efficacy of these compounds was further analysed by cell-culture based antiviral assays. Four out of ten molecules inhibited SARS-CoV-2 replication in Vero cells at a concentration range of 12.5 to 50 µM. The antiviral activity was evaluated by RT-PCR assay and TCID50 experiments. The co-crystallization of Mpro in complex with inhibitor for determining their structures is being carried out. Collectively, this study will provide valuable mechanistic and structural insights for development of effective antiviral therapeutics against SARS-CoV-2. Antiviral Drugs Used for COVID-19 Pharmacotherapy: A Narrative Review Dr Ghulam Mohmad Bhat, Prof. (Dr.) Samina Farhat, Dr Zubair Ashai Department of Pharmacology, Government Medical College, Srinagar Background: A number of research articles has been published evaluating safety and efficacy of drugs against COVID-19. Objectives: This study was undertaken to collate and review the information regarding common proposed antiviral drugs for easy reference. Methods: The literature search was done using terms like severe acute respiratory syndrome or SARS-CoV-2 or 2019-nCoV or SARS-CoV or COVID-19 in combination with drugs or treatment or pharmacotherapy using PubMed and google scholar to identify relevant articles. Results: Despite showing good early results, hydroxychloroquine and lopinavir-ritonavir has not shown clinical benefit in randomized controlled trials. However lopinavir in combination with other drugs specially interferon is being investigated. Remdesivir has shown positive effect in terms of clinical improvement and continued to being investigated alone or in combination with other drugs. Favipiravir has shown mixed results and more data from adequately powered study is needed to prove its efficacy. Conclusions: Many drugs which showed positive effect in initial studies could not replicate the same benefit in large randomized controlled trials. There is need to evaluate efficacy and safety of drugs based on high quality evidence before allowing it to be used in general population. Keywords: COVID-19; favipiravir, hydroxychloroquine, lopinavir-ritonavir, pharmacotherapy, remdesivir Adverse Events Following Oxford-Astrazeneca’s COVID-19 Vaccine among Health Care Workers in a Tertiary Care Teaching Hospital: A Prospective Observational Study Dr Rizwan ul Rashid, Prof. (Dr.) Samina Farhat Department of Pharmacology, Government Medical College, Srinagar Background: The ubiquitous elixir for mortality and morbidity inflicted by severe acute respiratory syndrome virus (SARS-CoV-2) has been a vaccine. These vaccines were approved for emergency use authorization by health authorities based on limited data from clinical trials. Hence, there was a need for active surveillance of vaccinees to monitor for safety. Objectives: This study reports adverse events following immunization with Oxford-AstraZeneca’s COVID-19 vaccine (COVISHIELD). Materials and Methods: The present study is an observational follow-up study to assess any adverse event occurrence following immunization (AEFI) within 7 days of vaccination among all eligible participants who were vaccinated. A structured safety surveillance questionnaire was administered consecutively to 714 participants. Vaccinees were observed for thirty minutes and followed telephonically for adverse events. Results: The overall incidence of any AEFI within 7 days was found to be 136/1000 vaccinations for the first dose. Out of total, 97 recipients reported with adverse events, 76.3% had AEFI within 24 h with fever as the most common symptom reported. The incidence of AEFI’s was found to be associated with gender (P < 0.02), age group (P < 0.05) and occupation (P < 0.05). No cases of hospitalization, disability or death were reported. Conclusion: Most of the adverse events were short-lived and observed in the first 24 h of vaccination. Incidence decreased in subsequent days and as no significant life-threatening adverse event was observed, this study might help reduce hesitancy for vaccination among the population and thus help reduce transmission of this highly contagious disease. Keywords: COVID-19, Adverse events following immunization, covishield vaccine, healthcare workers Utilization of prophylactic drugs for covid-19 infection among health care workers at government medical college Srinagar-a retrospective observational study 1Arjumand Maqbool, 2Sameena Farhat, 3Rehana Tabassum, 4Irfan Yousuf 1Post Graduate Scholar, Department of Pharmacology, GMC Srinagar. 2HOD, Department of Pharmacology, GMC Srinagar. 3Professor, Department of Pharmacology, GMC Srinagar. 4Assistant Professor, Department of Medicine, GMC Srinagar Introduction: Corona virus disease (COVID-19) was declared as a Pandemic by WHO on March 11, 2020. Since health care workers play an important role in providing care to infected patients, they are exposed to unprecedented levels of risk. At the initial phase of this pandemic, no definitive treatment was available, the only way to combat this disease was prevention. A number of prophylactic drugs were being studied during that time for use by health care workers. On 23rd March 2020, Government of India issued recommendation through National Task Force for Covid-19, for using Hydroxychloroquine as prophylaxis for SARS COV-2. Preclinical studies of Azithromycin have shown immunomodulation and in vitro activity against SARS-COV-2, that has led to its widespread usage during COVID-19. Ivermectin, an antiparasitic drug was reported to have an in vitro activity against SARS-COV-2. This orally administered drug was included in India’s revised National COVID-19 treatment protocol for people with mild infection.. Vitamin C, a water soluble vitamin has been considered for potential beneficial effects in COVID-19 disease. Many animal studies have indicated that a daily intake of vitamin C may prevent infections. Aim: To evaluate the pattern of drugs (HCQ, AZITHROMYCIN, IVERMECTIN,and VITAMIN C) used for COVID-19 prophylaxis among health care workers at GMC, Srinagar. MATERIALS AND METHODS: This study is being conducted by using a survey questionnaire. A survey questionnaire in English has been developed after literature review. The responses will be analyzed using descriptive statistics of frequency and percentage. Keywords: Covid-19, prophylactic drugs, health care workers Impact of COVID-19 pandemic on burden of dog bite cases at a Tertiary Care Hospital in Kashmir Presenting Author: Dr Shifana Ayoub; Co-author: Prof. (Dr.) S.M. Salim Khan Background: Dog bite is a public health problem in Kashmir incurring huge cost of treatment. Objective: To see the impact of COVID-19 pandemic on the burden of dog bite cases and the profile of patients, comparing with that of the years before the three peak waves of COVID-19 and after that at Shri Maharaja Hari Singh Hospital. Methodology: This is a retrospective record review of the dog bite cases in the year 2018 to 2022 in Anti Rabies Clinic, SMHS Hospital Srinagar. The target population of the study were people living in Srinagar city. Proportion of dog bite according to demographic and clinical variables were calculated. Univariate and multivariate analysis were done to look for risk factors responsible for dog bite in COVID infection waves compared to pre and post pandemic times. Results and Conclusion: The dog bite cases in the prepandemic time period and during the three waves of COVID 19 infection were recorded at 3.3% and 2.4% respectively. There was male predominance and highest incidence of the dog bite was in age group of 20–59 years. The commonest site of bite were lower limbs and stray dog bites were highest risk exposure. Less dog bite cases reported in 2020, 2021 which may be due to the impact of pandemic. Majority of the cases were stray dog bites and high incidence of dog bites can be a major concern for health, social and economic wellbeing of the nation which needs urgent intervention. Evaluation of Knowledge, Attitude, Practice and Hospital Experience Regarding COVID-19 among Pregnant and Post-partum Mothers at a Tertiary Care Hospital in Kashmir: A Cross-sectional Study Dr Sahila Nabi (GMC, Srinagar) Background: Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. The virus can spread from an infected person when they cough, sneeze, speak, or breathe through mouth or nose. Majority of the people who get infected with the virus will experience mild to moderate illness. However, some will become severely ill and require medical attention. Pregnancy is associated with increased risk for severe COVID-19. Few studies have examined knowledge, attitudes, and practices (KAP) during the pandemic especially the high-risk groups like pregnancy and post-partum period. Aim: To analyze the knowledge, attitude, practice and hospital experience regarding COVID-19 among pregnant and post-partum mothers at a tertiary care hospital in Kashmir. Methods: A cross-sectional study was conducted among 403 research participants from a tertiary care hospital of Kashmir in year 2020–2021 using a semi-structured questionnaire. The confidentiality and anonymity of respondents was maintained. The data was entered in Microsoft Excel and analysed using Statistical Package for Social Sciences version 25. The findings were presented as percentages (95% confidence intervals; CI), median, means and Standard deviation. Results: A total of 403 post-partum women participated in the study. Almost all the participants had heard about COVID-19 (96.6%). A majority of them were aware about how COVID-19 gets transmitted and its preventive measures. Most of the participants (62%) knew that COVID-19 has effects on pregnancy. Almost all of participants (97%) wore mask during hospital stay. All of the women washed their hands with soap water or alcohol based sanitizer. A fewer of the mothers (20%) wore mask while breastfeeding their baby. The results of binary logistic regression analysis found associations between knowledge and marital status (OR = 4.983, 95% CI 1.894–13.107). Correlation analysis found a weak positive correlation between knowledge and practice scores (r = 0.210, p-value = 0.01). Conclusion: As the COVID-19 cases are still increasing globally, participants overall had high KAP scores. This study can guide public health strategies regarding pregnant women and COVID-19. We recommend that interventions to improve and attitude and practice scores. Knowledge, Attitude, and Practices Related to COVID 19 Pandemic among Social Media Users in J&K, India Iqra Nisar Chowdri Department of Community Medicine, Government Medical College, Srinagar Background: A series of measures have been suggested to reduce Covid-19 infection, including knowledge training for prevention and control, isolation, disinfection, classified protections at different degrees in infection areas, and protection of confirmed cases. Objectives: We conducted this study with an aim to assess the knowledge, attitude and practice among the general population regarding COVID-19. Methods: This was a cross-sectional study carried out by the Department of Community Medicine, Government Medical College, Srinagar in the month of April 2020. The questionnaire had four segments to collect data regarding social-demographic details, knowledge regarding Covid-19, attitude and practice based questions. The questionnaire was shared via social media applications like face book and Whatsapp to reach the target population. Continuous variables were summarized as frequency and percentage. All the analysis was done using Microsoft Excel 2016. Results: Among participants who responded, 1252 (82%) were in the age group of 18–40 years and 912 (60%) from urban areas. A total of 934 (61%) respondents had heard details on COVID 19 from the social media, 1358 (89%) knew all ways of coronavirus transmission, 602 (40%) felt that COVID 19 is a serious disease, 1184 (78%) responded that they totally agree with the lockdown decision, and 1296 (85%) responded that lockdown is helping in reducing the number of cases. The majority, i.e. 1318 (87%), followed advisories and reported washing hands with soap and water regularly, 1108 (73%) reported regularly wearing masks, 1344 (89%) reported following lockdown guidelines, and 1306 (87%) reported maintaining social distancing. The respondents exhibited good knowledge, positive attitude, and sensible practices regarding COVID 19. Conclusion: Our study showed that the respondents have exhibited good knowledge, positive attitude and sensible practices regarding covid-19 during the pandemic. Keywords: Attitude, COVID 19, Knowledge, Practice, UT of J and K. Attitude towards covid-19 vaccines and vaccine hesitancy in antenal and breast-feeding women in tribal and urban communities of Kashmir-A cross-sectional study Mehvish Khan Background: The World Health Organization indicated vaccine hesitancy as one of the top 10 threats to global health. The success of a vaccine depends not only on its efficacy but also on its acceptance. Pregnant women are at high risk of severe illness, intensive care unit admission (3 times more likely), and invasive ventilation (1.5 times more likely) when compared with non-pregnant women of the same age. Objectives: To study attitude towards covid 19 vaccines in antenatal and breastfeeding women Proportion of vaccine hesitancy among antenatal and breast-feeding women in tribal and urban communities of Kashmir. Material and Methods: Study design: Cross-sectional study Study period: August 2021 to December 2021 Study population: Antenatal and breast-feeding women in tribal and urban communities of block Hazratbal, Kashmir. Sample size: A total of 414 participants were recruited for the study. Sampling technique: snowball sampling. Study tool: VAX (vaccine attitude scale) scale was used to asses attitude of study participants towards vaccine. Information about socio demographic variables and reasons for vaccine hesitancy was also obtained. Results: A) Among the reasons assessed for uncertainty or unwillingness to vaccinate (n = 350) 65% of woman were worried about possible side effects both for themselves and for fetus/infant. B) In Response to VAX (Vaccine attitude scale) scale n = 414 % of women had high level of negative attitude towards protection after getting vaccinated and 79.9% of women had high level of negative attitude towards safety of covid-19 vaccination. Conclusion: The dissemination of professional and reliable information regarding the safety and efficacy of COVID-19 vaccine uptake by qualified health care personnel can significantly increase the level of trust and public awareness regarding the safety and efficacy of COVID-19 vaccine uptake in pregnancy and while breastfeeding. COVID-19 vaccine acceptance in pregnant women of block Hazratbal, Kashmir Dr Mudasir SPM, GMC Srinagar Background: Coronavirus disease 2019 (COVID-19) is a disease caused by a novel coronavirus (2019-nCoV) that was first reported in Wuhan, Hubei Province, China in December. Since then, there have been over 62300396 cases of COVID-19 infections worldwide, with 6550033 deaths. Therefore, it is important to avoid infection. In the absence of an effective treatment for coronavirus disease 2019 (COVID-19) non-pharmaceutical interventions are the only available methods of disease control. Social distancing, face masks, and personal hygiene are the most effective precautions, but maintaining these actions is not practicable in the long term. As a result, herd immunity by vaccination becomes the most effective eradication method, as in other viral epidemic diseases in the past. Research into development of a vaccine for SARS-CoV-2 was undertaken immediately after the disease was identified. The success of a vaccine depends not only on its efficacy, but also its acceptance. However, vaccine hesitancy has become an important threat to global health, which was pointed out by WHO in 2019.4 Several key factors behind vaccine hesitancy include fear or mistrust of the vaccine, underestimation of the value of the vaccine, and lack of access to the vaccine. Objective: To determine vaccine acceptance and hesitancy attitudes toward coronavirus disease 2019 (COVID-19) vaccines in pregnant women. Methods: 250 pregnant women were surveyed face to face with 40 questions. Socio demographic characteristics, vaccination history, perception of risk for the COVID-19 pandemic, the impact of the COVID-19 pandemic, and acceptance of and attitude toward future COVID-19 vaccination were prospectively evaluated. Among all participants, 93 (37%) stated their intent to receive the vaccine if it were recommended for pregnant women. Most common refusal reasons were lack of data about COVID-19 vaccine safety in pregnant populations and possibility of harm to the fetus. Conclusion: The present study reported low acceptance of COVID-19 vaccination in a sample of pregnant women. Concern about vaccine safety was the major reason for hesitancy. Identifying attitudes among priority groups will be useful for creating vaccination strategies that increase uptake during the current pandemic. Study of Anxiety, stress and Depression Associated with Breastfeeding in COVID-positive Mothers of block Hazratbal, Kashmir. Dr Nazia SPM, GMC Srinagar Background: Coronavirus disease-2019 (COVID-19) has rapidly disseminated worldwide, with a wide variety of clinical manifestations ranging from mild respiratory symptoms to severe pneumonia. Since then, there have been over 62300396 cases of COVID-19 infections worldwide, with 6550033 deaths. Coronavirus disease has presented the world to uncertainty and clinical dilemma with developing and constantly changing management guidelines and protocols. In the backdrop of this pandemic, it thus becomes crucial to study the effects of the infection on pregnancy, childbirth, and the postpartum period. In this study, we analyzed experiences of breastfeeding mothers during the COVID-19 pandemic, specifically concerning how COVID-positive status affected their infant’s feeding decisions. Objective: To study anxiety, fear and depression associated with breastfeeding in coronavirus disease (COVID)-positive mothers. Methods: The following DASS scale was used to measure depression anxiety, and stress of coronavirus disease-2019 (COVID-19) among postpartum women along with a self-made breastfeeding questionnaire to assess the association with breastfeeding. Results: Among the total of 77 respondents, 13% showed symptoms of depression, 16% anxiety and 9% stress. The breastfeeding questionnaire suggested that most women are afraid of transmitting the infection to their newborns and they lack the knowledge about the importance of breast milk in warding off other infections. Also, women found it difficult to take care of their newborns on their own. Conclusion: With this study, we could determine the effects of this pandemic on anxiety depression, and stress levels of COVID infection in postpartum women. It clearly showed that being COVID positive created, affected, and exacerbated mental health issues for mothers. So, there is an urgent need to provide emotional and psychosocial support to this group of the population during the crisis. Otherwise, the adverse outcome is possible involving both mother and newborn. Impact of thymosin alpha on biochemical markers and mortality in covid 19 patients: a retrospective study Shahid Majid, Adnanza, Hena Mustafa, Khurshid Ahmed Dar Background: Immune-mediated lung injury and complex changes of the immune system, such as lymphopenia and cytokine storm, that have been associated with adverse outcomes underlining a fundamental role of host response in severe acute respiratory syndrome coronavirus 2 infection and the pathogenesis of the disease. Thymosin alpha 1 (Tα1) is one of the molecules used in the management of COVID-19, because it is known to restore the homeostasis of the immune system during infections and cancer. Aim: To study the impact of thymosin alpha on the biochemical markers and mortality in covid 19 patients. Methodology: A retrospective, single-centred study including 127 patients with laboratory detected moderate to severe SARS-CoV-2 infection admitted to designated COVID-19 centre in a tertiary care hospital from September 2021 to March 2022 was done. 52 patients received thymosin alpha 1 and their results were compared with 75 patients who received standard care without thymosin alpha. Clinical records, laboratory data, and radiological findings were analysed of patients treated with thymosin alpha 1 to evaluate the role of treatment outcome. Results: hospital mortality was 7.6% (n = 4) in the thymosin group as compared to 9.3% (n = 7) in the non-thymosin group. 40 patients in the thymosin group had increased CRP levels on day 1 as compared to 61 in the non-thymosin group. On day 5, 11 patients in thymosin group had increased levels as compared to 47 patients in the non-thymosin group with a significant p-value of < 0.001. Statistically significant results were obtained on day 10, only 7 patients in the thymosin group had increased levels as compared to 30 in the non-thymosin group. On day 1, 46 patients in the thymosin group had increased level of IL-6 as compared to 53 in the non-thymosin group. Serial monitoring on day 5 showed that in thymosin group, 18 patients had increased levels as compared to 44 patients in the non-thymosin group (with a significant of < 0.05). Again, on day 1difference was statistically significant when in thymosin group only 5 patients had elevated levels as compared to 23 in non-thymosin group. Conclusion: Significant difference was seen in terms of biochemical parameters but that could not be translated in clinical improvement in terms of mortality rates. Knowledge Attitude and Practice Related to COVID Appropriate Behaviour and COVID-19 Vaccine Acceptance Among Pregnant Women attending a District Hospital in Kashmir India: A Mixed Method Study Introduction: WHO recommends that pregnant women should receive a vaccine against COVID-19 as it has been observed that the clinical course of COVID-19 infection in pregnant women is worse than in non-pregnant women. Vaccines are effective intervention to reduce the burden of the disease, however, public hesitancy is a problem for public health authorities. Aim: To determine Knowledge Attitude and Practice Related to COVID Appropriate Behaviour and COVID-19 Vaccine acceptance among pregnant women attending the Antenatal care Clinic at District Hospital Shopian. Material and Methods: A Hospital-based mixed-method approach comprising both quantitative part (Cross-Sectional method) and qualitative part (In depth interviews). Study participants were all pregnant women attending antenatal care clinic at District Hospital Shopian during the study period (1st January-31st March 2022). Results: All of the 262 study participants (100%) reported that they had heard about the corona virus pandemic. Attitude and practice related to COVID-19 preventive behaviour was average. The COVID-19 vaccine acceptance was found to be 77.4%. The highest number (40.3%) of COVID-19 vaccine hesitance found was because the respondents believed that the COVID-19 vaccine would harm their fetus. Conclusion: Health care providers should pay extensive attention to the dissemination of accurate vaccination information and address misinformation to boost vaccine acceptance among pregnant women. An Audit of Clinico-demographic Profile of Covid-19 Deaths in a Tertiary Care Hospital Asma Rafi1, Muzaffar Maqbool1, Tabindah Shah1, Rakesh Kumar Koul1, Masood Tanvir1 Postgraduate Department of Medicine, Government Medical College, Srinagar Background: Coronavirus disease (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus with more than 6.5 million deaths worldwide. Objectives: The aim of this study was to analyze the clinical and demographic profile of covid-19 deaths in Government SMHS Hospital, an associated hospital of Government Medical College, Srinagar. Materials and Methods: A retrospective audit of hospital record of Covid-19 patients who expired over a period of 30 months (April 2020 to October 2022) was done to study their clinical and demographic profile. Detailed history, investigations and co-morbidities were studied to assess their impact on overall mortality. Results: Out of a total of 5300 admissions, 3339 (63%) were males and 1961 (37%) were females. Total Covid-19 deaths were 821 (15.5%), of which 15.03% (502/3339) were males and 16.26% (319/1961) were females. Mortality rate in severe Covid not-critically-ill was 12.3% while it was 42.4% in critically-ill Covid-19 patients. Majority patients belonged to 60–74 years age-group. Hypertension followed by Diabetes Mellitus were the most common co-morbidities in expired patients. Out of 119 patients managed on Non-Invasive ventilation, 21 (17.6%) died while out of 102 patients managed on Invasive Ventilation, 74 (72.54%) died. Only 24 (5.9%) out of expired 403 patients during 2nd wave of Covid were fully vaccinated. Conclusions: The overall in-hospital mortality of Covid-19 patients was 15.5%. Advanced age was a significant predictor of mortality. Vaccinated patients had a significantly lower mortality. Materno-Fetal Outcome Among SARS-CoV-2 positive pregnant women: Experience from Covid 19 dedicated Maternity Hospital, MCH, Srinagar Azmat Jahan COVID-19 has affected the population worldwide drastically with a tremendous impact on obstetric population which has led to serious concerns regarding maternal and fetal outcomes. Although there are recommended guidelines regarding delivery and management of complications, due to changes in characteristics of COVID-19 infection, they are constantly changing and evolving. Methods: Prospective cohort study done during the covid pandemic from 1st April 2020 to 15th Feb 2022 in the department of Obstetrics & Gynecology, SKIMS MCH Srinagar J&K. The parameters measured were severity of covid disease, maternal age, gestational age, parity, blood investigations, mode of delivery, APGAR score, neonatal infection status and post-delivery complications. Results: A total of 311 pregnant covid 19 positive patients were included in the study who were actively managed.239 (76.85%) were delivered by casearean section and 72 (23.15%) by NVD. 92% patients had mild symptoms only, 8% had severe symptoms with 1.6% rate of ICU admission and 1.2% mortality rate. 83% delivered at term, 17% had preterm deliveries.8% patients had pneumonitis with positive findings on CT scan.24% patients had anemia, 12% had GDM, 10% had PIH, 10% had IHCOP, 5% had PPH, 1.6% had APH. All the neonates were negative for covid 19. 80% babies had an APGAR score of ≥ 8/10 at 1 min of birth with a mean birth weight of 2400 g ± 500. No postdelivery complication was noted. Conclusion: Our study concludes that SARS‐CoV‐2 infection can lead to unfavorable maternal and perinatal outcomes. Keywords: Covid 19, pregnancy, matero-fetal effect Global Status of Covid-19 Vaccination Dr Tanzeela Bashir Qazi Department of Community Medicine, Government Medical College Srinagar Background: Covid 19 vaccination has substantially altered the course of the pandemic, saving tens of millions of lives globally. However, inadequate access to vaccines in low income countries has limited the impact in these settings, reinforcing the need for global vaccine equity and coverage. The present study was aimed to assess the global data of covid 19 vaccination from a secondary data source. Material and Methods: It is a secondary data analysis of worldwide covid 19 vaccination data obtained from World Health Organization Website. Data updated upto October 2022 on WHO website was collected. Results: Variables included name of countries, total vaccinations, total vaccinations per 100, person vaccinated one plus dose, booster dose, booster dose per 100, type of vaccine etc. Afghnaistan reported a total vaccinations of 11951964 till 11-10-2022 whereas India reported vaccinations of 2190969572 till 22 October 2022. Conclusion: Total vaccination per 100 in Afghnaistan is 30.702 and for India it is 158.7. SARS-CoV-2 antibody avidity responses in natural infection and vaccination Gururaj Rao Deshpande, Atharva Athavle, Gajanan N Sapkal*, Shankar M Vidhate, B. N Tilekar, Nilesh Pawar, Pradnya Shinde, Nitali Tadkalkar Diagnostic Virology Group, ICMR- National Institute of Virology, Maharashtra, India Background: In ongoing SARS CoV-2 pandemic, understanding antibody responses have played a key role in measuring extent of exposure, protection from reinfection, vaccine efficacy and serodiagnosis. Antibody avidity is total binding strength of immunoglobulin G (IgG) toward its target epitope. High antibody avidity has been correlated with effective neutralization of the SARSCoV-2 virus. However, the data on avidity responses against COVID-19 infection and vaccination are limited. Objectives: To understand the avidity responses among sera of naturally infected, recovered COVID-19 patients; naive Covaxin, Covishield vaccinees and breakthrough infections. Materials and Methods: In this study, we utilized an in-house developed SARS-CoV-2 anti-spike receptor binding domain (SRBD) IgG ELISA to optimize the avidity assay. A panel of anti-SARS-CoV-2 SRBD IgG positive serum samples were treated with known concentration of a chaotropic agent (urea) for disruption of the non-covalent interactions of the antigen–antibody complex. This disruption causes low avidity antibodies to dissociate which gives the percentage of high avidity antibodies present in a serum sample. Additionally, the optimized assay was used to understand the avidity responses among sera belonging to individuals naturally infected and recovered after COVID-19, naive Covaxin and Covishield vaccinees; followed by breakthrough infections. Results and conclusion: The anti-SRBD avidity progressively elevated over a period of twelve months. Moreover, overall antibody avidity responses were similar in the case of natural infection and naive two doses of Covaxin and Covishield vaccinated individuals. However, avidity responses were high among individuals with a breakthrough infection as compared to naive vaccinees. Keywords: SARS-CoV-2, COVID-19, antibody avidity, SRBD, Covishield, Covaxin, urea, affinity maturation Respiratory Syncytial Virus (RSV) infections in children: recent concepts in immunopathogenesis and diagnostic dilemma Dr R K Ratho Prof and Head, Dept of Virology and Sub Dean (Research), PGIMER, Chandigarh Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013–2017 were subjected for IL-17A, IFN-γ, TNF-α, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-α) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p < 0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p < 0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p < 0.001). Thus, the study highlights the role of TNF –α, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020; n = 166) and during COVID-19 Pandemic (July 2021 to July 2022; n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p < 0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p < 0.001); and significantly lower positivity for hRV (p < 0.05), hMPV (p < 0.05), and hBoV (p ≤ 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. Cell Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory-developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCR-based technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and –B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing. Nipah virus pathogenicity in Syrian hamster model Sreelekshmy Mohandas*, Anita Shete, Prasad Sarkale, Abhinendra Kumar, Chandrasekhar Mote, Pragya Yadav ICMR-National Institute of Virology, Pune Background: Nipah virus is a zoonotic high risk group viral agent known to cause fatal infection. The Malaysian and Bangladesh strains of virus differ in terms of incubation period, fatality rate, transmission pattern and symptoms. Bangladesh virus strains are less studied. The virus was isolated for the first time in India from the 2018 outbreak in Kerala. The phylogenetic analysis the isolate showed it sub clustering within the Bangladesh Nipah virus strains and proposing it as a separate genotype as ‘I’. Objectives: To study the pathogenicity of Nipah virus (Indian isolate) in hamster model. Materials and Methods: Nipah virus (isolated from 2018 Kerala outbreak in Kerala) propagated and titrated in the Vero CCL-81 cells was used for the study. The isolate characterization was performed by growth kinetics in cell lines, titration and by next generation sequencing. Lethal dose 50 was determined in adult, Golden Syrian hamsters. A time point study was performed to understand, virus shedding, viral organ load and host immune response by real time RT-PCR and tissue tropism by histopathology by intraperitoneal and intranasal infection in hamster model. Results: Nipah virus isolate was found pathogenic in hamster model by both intraperitoneal and intranasal infection. Acute respiratory disease as well as a late onset encephalitis disease were observed in hamsters. Viral RNA could be detected in the swab samples, brain, lungs, kidney, liver, heart, trachea, spleen and intestine. Multisystemic infection was observed with predominant vascular changes in lungs, brain and kidney by intraperitoneal infection. Gliosis, perivascular cuffing were observed in the brain and lungs. Intranasal infection resulted in pneumonia. IL-4, IL-6, IL-12 and IFN-Gamma were found elevated in infected animals. Conclusions: Nipah virus, Indian isolate infection in the hamster model produced respiratory disease and encephalitis resembling that of human Nipah virus infection similar to that reported for the Malaysian and Bangladesh isolates. Spectrum of adverse maternal, fetal and neonatal outcomes following Dengue infection during pregnancy in a tertiary care hospital of north India: Evidence suggestive of Perinatal transmission Mannat Kang1, Amanjot Kaur2, Vikrant Sharma1, Tanvi Katoch2, Saurabh Dutta3, Keerti Chauhan1, Bhartendu Singh1, Amanjit Bal4, Manish Rohilla5, Ishani Bora1, Neelam Aggarwal2, R K Ratho1* Departments of Virology1, Obstetrics and Gynaecology2, Pediatrics3, Histopathology4 and Cytology and Gynaepathology5 *Corresponding author Background: Dengue infection in pregnancy may lead to adverse maternal and fetal outcomes. Objectives: To study the incidence of tropical infections [Dengue (DENV), Chikungunya, Scrub Typhus, Leptospira, Zikavirus] in pregnant women presenting with acute febrile illness (AFI) during DENV transmission season and their neonates and determine their outcomes. To describe the serotypes and genotypes of DENV strains isolated. Among infected mothers, to detect DENV RNA in the placenta, cord blood (CB) and high vaginal swabs (HVS) and describe pathological changes in the placenta. Methods: 83 mothers presenting with AFI in the 3rd trimester were screened by Trioplex RT-PCR (CDC) for DENV, Chikungunya and Zikavirus; and by ELISA tests for DENV NS1 Ag, DENV IgM, Chkv IgM, Scrub typhus IgM and Leptospira IgM. Representative DENV RT-PCR positive samples underwent serotype analysis followed by DNA sequencing. 14 neonates born to DENV positive mothers were enrolled. Placenta, CB and HVS were collected from representative DENV infected mothers to investigate for perinatal transmission. Results: 50.6% (42/83) pregnant women with AFI were positive for DENV by serology and RT-PCR. Dengue mono-infection was present in 36/42 (85.7%) patients. Other infectious causes of AFI were observed in 16.8% (14/83) mothers. 52.3% (22/42) DENV positive mothers developed thrombocytopenia, of whom 59% (13/22) had platelet count < 50,000. Four of the five mothers that died in the study tested DENV positive. DENV RNA was detected in 30% (3/10) of Placenta, 25% (1/4) HVS and 42.8% (3/7) CB samples. Histopathological examination of 10 placentas showed immature chorionic villi, multifocal intervillous fibrin, chorioangiosis etc. 50% (21/42) of DENV positive mothers delivered within the viremic period resulting in 3 intrauterine fetal deaths and 61.9% (13/21) low birth weight (< 2500 g) neonates. Of 14 neonates born to DENV positive mothers, 6 tested positive for DENV. Only two mother-baby pairs were available for sequencing. One of each pair was infected with DENV type 2 and type 4 respectively. Conclusion: Perinatal transmission of dengue infection in pregnancy could lead to adverse neonatal outcomes. Burden and probable risk factors of Hepatitis C virus infection in a tertiary care hospital Bashir Fomda1, Amrit Pal2, Junaid Ahmad2, Sofia Zaffar*1 1Department of Microbiology; Sher-i-Kashmir Institute of Medical Sciences, Soura-190011. 2Government medical college, Baramulla, J&K, India 1* Email: [e-mail] Background: Hepatitis C is a major public health problem and a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma with an estimated global prevalence of 3%. Limited studies have been conducted regarding epidemiology of HCV infection in our state and the associated risk factors. The present study was conducted to determine the burden and risk factors associated with HCV infection. Objectives: To determine the burden and to investigate probable risk factors associated with HCV infection Material and methods: Our study included 3000 patients, admitted, or attending OPD, who required Hepatitis C testing, with relevant information. Detection of Anti-HCV antibody in serum was done by third-generation ELISA and all seropositive samples were further processed by Real-time RT-PCR to detect viral load. Results: Seroprevalence of 2.3% was observed in our study and 75.71% of these patients had detectable viremia. Higher seroprevalence was noted in the age group 20–40 years (2.9%), in males (1.53%) and in patients with Chronic Kidney Disease (16.32%) followed by haematological malignancies (7.17%) and solid organ tumors (5.66%). Conclusion: Better prevention, screening and treatment methods for Hepatitis C infection need to be identified and described. There is a need to educate the general population about the virus and various associated risk factors. Dog bites the poor: Exposure of rabies in poverty prone areas in Kashmir an ecological study Dr. Salib Hamid, Dr. Kouser Sideeq Department of Pediatrics Govt. Medical College Srinagar Introduction: India annually has around 20,000 rabies deaths (more than 1/3rd of global statistics). In Kashmir dog bite is the main exposure for rabies, annually around more than 6000 dog bites are reported to the only established anti Rabies clinic in Kashmir (SMHS hospital GMC Srinagar), but rabies not being a notifiable disease in India, official mortality statistics are not available but the recent numbers estimated is around 3 deaths since November 2020 due to rabies in Kashmir. Globally rabies outbreak is rampant among impoverished and vulnerable population. Our study wanted to test whether poverty is associated with the higher number of dog bite cases in Kashmir. Methodology: An ecological study was designed. The data for the number cases of dog bite coming from 9 districts of Kashmir was accessed from the anti rabies clinic at Government Medical College Srinagar for the period of 8 years from 2013 to 2021. District Srinagar was not involved in the study, as Srinagar reports around 4 times the number of dog bite cases as compared to any other district to the anti rabies clinic as the clinic is in the same district which may lead to bias in the results. The statistics for people living below poverty line in each of these districts was obtained from the Economic survey J&K 2013–2014 report. These data sets where analyzed to find out the association between poverty and dog bite cases from each of these 9 districts. The social science statistical software was used to analyze the results with p value of < 0.1 taken as statistically significant. Result: A total of 14,583 cases where reported in 8 years from the 9 districts of Kashmir with maximum number of cases from district Budgam followed by district Bandipora which are among the districts having highest percentage of the people living below poverty line; Bandipora being on the second number after Kupwara. The Pearsons Coefficient was calculated for each year for poverty and number of dog bite cases. The relationship was significant for each year except for two years. From 2013 to 2021 the r values for each year were 0.6122, 0.5663, 0.6106, 0.5921, 0.5876, 0.6546, 0.5867, and 0.4919 respectively. The two years with non significant results were those when the situation was not in normal conditions in Kashmir one being 2014 when Kashmir was in floods and the other being 2020–21 (lockdown period). Conclusion: Our study found significant association of poverty and number of dog bite cases in Kashmir, which points towards the need to address the preventive as social aspects of Rabies and animal bite cases in the impoverished areas of Kashmir. Seroprevalence of Hepatitis B and hepatitis C virus among individuals attending a tertiary care hospital in northern India Background: Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are major causes of acute and chronic liver disease (cirrhosis and hepatocellular carcinoma) globally, and cause an estimated 1.4 million deaths annually. At present, 296 million people are living with chronic HBV infection and around 80 million have active HCV infection. The burden of these infections is disproportionately high in Asia and Africa. Thus, early diagnosis and accurate treatment save millions of lives annually. Objectives: To find out the hospital based prevalence of HBV and HCV virus infections and associated clinical and demographic variables among individuals attending a tertiary care hospital. Materials and Methods: A hospital based cross-sectional study over a period of two years (October 2020 to September 2022). A total of 25,363 individual serum samples were screened for Hepatitis B surface antigen (HBsAg) and anti-Hepatitis C virus (anti-HCV) using ELISA Immunoassay. Other clinical and demographic details were also recorded and analysed. Results: A total of 25,363 individuals were screened, of which 14,711 (58%) were males. Among them 512 (2%), 1008 (4%), and 85 (0.33%) were found to be positive for HBsAg, anti-HCV, and co-infection respectively. Male gender showed a significantly higher prevalence rate of 75% (n = 512) for HBsAg and 83% (n = 1008) for anti-HCV among those testing positive. Age group 20–39 years had the highest prevalence rate of 58% (n = 512) and 74% (n = 1008) for HBsAg and anti-HCV respectively. Also, 72% of the individuals were from rural areas of Kashmir division. Conclusion: As evident, HBV prevalence continues to remain low for our region (2%), while the prevalence of HCV (4%) has markedly gone up over the years. A Safe and effective vaccine alongwith aggressive screening of suspected individuals has kept HBV prevalence low, while HCV prevalence is higher due to no vaccine, lack of knowledge and an unfortunate rise of injectable drug abusers in our society. Co-infections are also a cause of concern as they are associated with higher mortality. Determination of Transmission Dynamics Among Household Contacts of Drug Resistance TB Patients Farah Naaz 1, Devendra Singh Chauhan1, Mohd. Zeeshan2, Mohd. Farhan3, Kiran Katoch4, Abdul Mabood Khan1 1Department of Molecular and Microbiology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra 282 004 India. 2Dr. Bhim Rao Ambedkar University Agra, India. 3Jamia Millia Islamia University, Jamia Nagar, Okhla, New Delhi 110 025, India. 4Distinguished Scientist Chair of ICMR at IIHMR University, Jaipur 302011, Rajasthan, India Background: Drug-resistant tuberculosis (TB) continues to be a public health threat. The risk of contacts of people with MDR-TB for progression to active TB will be important for understanding the benefits of preventive treatment. Transmission of TB to the community occurs in the endless repetitive cycle of contact, infection, and development of active disease. The diagnosis and control of tuberculosis (TB) is very significant problem in global health. Objectives: This study aimed to determine the transmission dynamics among household contacts of drug resistance TB patients who might develop TB. Methods: This study conducted in ICMR-National JALMA Institute for Leprosy and Other Mycobacterial Diseases Agra, Total 168 samples were collected from Kanpur Nagar, Uttar Pradesh, in which 47 were MDR Patients and 121 their household contacts out of which 79 (47%) were males and 89 (52.97%) were females. Total 168 samples were screened by sputum smear microscopy, Nucleic Acid Amplification (Xpert® MTB/Rif) Test (NAAT), BACTEC MGIT 960 culture to determine the active TB disease in household contacts. Results: In this study 47 MDR patients were enrolled with their 121 household contacts, in 47 MDR patients 24 (51.06%) AFB smear positive and 23 (48.93%) AFB smear negative, 44 (93.61%) CBNAAT positive and 3 (6.38%) CBNAAT negative, 39 (82.97%) MGIT positive and 8 (17.02%) MGIT negative. In 121 household contacts 16 (13.22%) AFB smear positive and 105 (86.77%) AFB smear negative, 16 (13.22%) CBNAAT positive and 105 (86.77%) CBNAAT negative, 26 (21.48%) MGIT positive and 95 (78.51%) MGIT negative. In 121 household contacts 5 (4.1%) were found Rifampicin resistant in CBNAAT and 1 (0.8%) was found negative in every examination. Conclusions: The households of MDR-TB patients are at risk for infection of MDR-TB. To reduce transmission, MDR-TB patients should be diagnosed earlier and promptly treated in an effective manner, meanwhile, the close family contacts should be screened for TB infection. Detection of Cytomegalovirus by real time PCR in patients attending a tertiary care hospital - Single Centre Experience Bashir Fomda1, Insha Altaf 1, Sofia Zaffar1, Tufail Ahmed1, Irfan ul Haq1, Shiekh Imtiyaz1 1Department of Microbiology; Sher-i-Kashmir Institute of Medical Sciences, Soura-190011, J&K, India 1 Email:[e-mail] Background and Aims: Human cytomegalovirus (CMV) is another name of the human herpes virus 5, a highly host-specific virus of the Herpesviridae family. CMV poses an important public health problem as it may cause serious morbidity and mortality in congenitally infected newborns and immunocompromised patients, most notably transplant recipients and HIV-infected persons. The magnitude of this problem in India has not been adequately investigated and it still is a major health problem warranting strong preventive measures. Materials and Methods: Samples from 105 suspected CMV infected patients received by the department from December 2018 to October 2022 were processed for detection of CMV-DNA by real time PCR. All the samples were analyzed and there was no specific inclusion and exclusion criteria. The samples were processed immediately when received in the virology laboratory. In case of delay, the samples were stored at −70 °C before testing. Results: The prevalence of CMV infection in this study was 8% with highest contribution from patients admitted in the pediatric hospital and the kidney transplant unit. Underlying causes included congenital CMV infection followed by post renal transplant and renal failure. The overall mean age was 24.5 years (range 3–32 years). The highest percentage of positive cases was seen in age group of less than 1 years. The percentage positives showed maximum number of positive cases in 2019 followed by a dip in the number of cases in 2020. Conclusion: Awareness of diverse clinical manifestations of CMV infection and high index of suspicion is important for timely diagnosis. Surveillance of CMV infection is critical to avoid the ill effects of late diagnosis which can lead to progressive and irreversible sequels. Microbial Evaluation of Catheter Associated UTI in Patients Admitted to Surgical ICU of a Tertiary Care Hospital Bashir Fomda 1, Sofia Zaffar1, Mohammad Akbar Shah2, Amrit Pal Kour3* 1Department of Microbiology; 2Department of anesthesia; Sher-i-Kashmir Institute of Medical Sciences, Soura-190011, 3*Government Medical College, Baramulla, J&K, India 3* Email: [e-mail] Background: CAUTI is one of the most common nosocomial infection. Antimicrobial resistance pattern varies over time and place. Objectives: To find prevalence of CAUTI cases, identify and note the sensitivity pattern of associated microorganisms. Methods: A total of 280 urine samples were collected. Culture and antibiotic susceptibility patterns were done. Antifungal sensitivity of yeast isolates was done by VITEK2. Bio-data and clinical data was obtained. Results: Prevalence of CAUTI was found to be 27.5%. 78 microorganisms were isolated, (57 bacteria and 21 yeast). A. baumannii was the most common Gram-negative bacilli (4.2%) found followed by E. coli (3.57%), K. pneumoniae (2.86%) and P. aeruginosa (2.5%). Enterococcus spp. (5.7%) was most common Gram-positive cocci. Candida glabrata (2.8%), was predominant yeast found followed by Candida albicans (2.1%) and Candida tropicalis (1.42%). All bacterial pathogens recovered were resistant to most of antimicrobials. The Gram-negative isolates showed uniform sensitivity (100%) to polymyxin B. All Gram-positive isolates were sensitive to Linezolid. Yeasts were sensitive to most of antifungals tested. Increased duration of catheterization, imunocompromised were found to be significantly associated with CAUTI. Conclusion: Length of catherization was found to be an important risk factor. Females were affected more than males. Causative pathogens were mostly Gram-negative bacilli. Most of the bacterial pathogens were found to be Multi-drug resistant. Platelet secreted exosomes promote loss of vascular integrity during dengue infection Sayali Vedpathak 1,#, Archana Sharma1, Sonali Palkar2, Vidya Arankalle1, A C Mishra1, Shubham Shrivastava1,* 1Department of Communicable Diseases, Interactive Research School for Health Affairs, Bharati Vidyapeeth (Deemed to be University), Pune. 2Department of Pediatrics, Bharati Hospital and Research Centre, Bharati Vidyapeeth (Deemed to be University), Pune # Presenting author, *Corresponding author, [e-mail] Background: Thrombocytopenia and endothelial permeability are the distinguished features of dengue severity. Platelets are the major cell population which releases extracellular vesicles. Exosomes act as a mediator of intercellular communication in several diseases. But the linkage of platelets in endothelial dysfunction especially through exosomes in dengue infection is not clear. Objective: To investigate the role of platelet derived exosomes in endothelial dysfunction. Materials and Methods: Platelets were isolated from the platelet rich plasma of dengue patients and their activation was assessed by flow cytometric assay. Platelet secreted exosomes were isolated by ultracentrifugation method. For characterization of exosomes, electron microscopy, particle size analysis and western blot analysis were performed. Exosome uptake experiment was carried out to see the internalization of exosomes inside endothelial cells (HUVECs). To observe the effect of exosomes on endothelial cells, exosomes were added on HUVECs and expression of adherens and tight junctional proteins were examined by immunofluorescence assay and western blot. Results: We observed reduced expression of CD41/CD61 with higher levels of p-selectin in platelets of dengue patients in comparison to healthy individuals. Exosomal markers CD63 and CD9 were upregulated in exosomes of dengue patients. Lower expression of VE Cadherin, F-Actin and PECAM were observed in HUVECs after addition of exosomes isolated from dengue patients. Conclusions: Our study indicates altered platelet activity in dengue patients and highlights the novel mechanism of platelet-exosome mediated dengue severity due to damaged endothelium. Outbreak investigation of Acute febrile illness (AFI) from the foothills of Himalaya: solving the puzzle of Mystery Fever Shefali Dhingra 1, Vikrant Sharma1, Prakasini Satapathy1, Mannat Kang1, Ishani Bora1, Kanwalpreet Kaur1, Kapil Goyal2, Neeraj Arora3, Arun Aggarwal2 and R. K. Ratho1* Regional VRDL, Department of Virology1, Dept. of Community Medicine and School of Public Health2, PGIMER and Civil Hospital3, Panchkula *Corresponding author Background: In September 2022, Civil hospital, Panchkula flagged an outbreak of ‘Mystery fever’ in Pinjore, Haryana. There was an upsurge of AFI cases affecting different age groups. Most of the patients had fever > 38 °C, less than two weeks in duration, body aches, arthralgias, retro-orbital pain, rashes and thrombocytopenia. Objectives: The clinically suspected cases of AFI were investigated for Dengue (DENV), Chikungunya (CHKV), Japanese Encephalitis (JE), West Nile virus (WNV), Zika virus, Scrub typhus and Leptospira from the outbreak sample. Methods: A total of 58 Blood samples were collected and tested for Dengue NS1 antigen and the IgM antibodies for DENV, CHKV, JEV, Scrub typhus and Leptospira as per manufacturer’s instructions. The samples were subjected to DENV, WNV and Zika virus RT-PCR. Results: Of the 58 serum samples, DENV was positive in 77.58% (n = 45) of cases, followed by Leptospira (n = 2), CHKV (n = 2), JEV (n = 2) and Scrub typhus (n = 1). Serological test results of DENV showed 53.44% (31/58) positives for NS1 Ag ELISA and 41.3% (24/58) positives for DENV Anti-IgM ELISA. 44.82% (26/58) samples were positive for DENV by RT-PCR. Furthermore, DEN serotype-2 was commonly isolated. Co-infection with DENV and Leptospira in 2, DENV and JE virus in 1 and DENV and Scrub typhus in 1 patient were detected. One patient was CHKV RT-PCR positive. None of the samples tested positive for Zika virus and WNV viral RNA. Conclusion: Serological detection accompanied with molecular assay is very helpful in deciphering the outbreaks with AFI. Prevalence of intestinal parasitic infections in a tertiary care hospital in Kashmir India- a five year retrospective study Bushra Yousuf Introduction: Intestinal parasitic infections represent a grave public health problem especially in developing nations like India, leading to malnutrition, growth retardation, anaemia’s and vitamin deficiencies in early childhood. As such the burden of these intestinal parasitic infections in the society needs to be focussed at the right time which will in turn lead to enhanced health and improved economic conditions of the country. Aim: To find out the prevalence of intestinal parasitic infections in a tertiary care hospital in Kashmir. Materials and Methods: A retrospective study was carried out in the Parasitology division in the department of Microbiology of Government Medical College, Srinagar for a period of five years. Routine stool examination was studied to detect the parasitic infections among the patients attending various outpatient departments of our hospital. Results: A total of 2159 stool samples were examined in five years out of which 165 (7.6%) revealed the presence of parasites. The most common parasite identified was Ascaris lumbricoides (71.9%) followed by Giardia lamblia (16.4%). Conclusion: Intestinal infection due to Ascaris lumbricoides was the most common parasite identified in our study. The prevalence of these infections can still be higher as these parasites are excreted intermittently in stool samples. As such taking repeat samples from same patients suffering from intestinal parasitic infection is important followed by concentration methods, which together will help and enhance better retrieval of intestinal parasites reflecting the total burden of these infections in our community. Prevalence of brucellosis in cattle and cattle handlers in Bakshipora area of block Palapora, district Srinagar Dr. Anjum Asma Introduction: Brucellosis is an important re-emerging zoonosis with a worldwide distribution. It is still an uncontrolled serious public health problem in many developing countries including India. Brucellosis in India is yet a very common but often neglected disease. Routine serological surveillance along with high clinical suspicion and screening of family members of index cases would be essential in delineating the real magnitude of human brucellosis in endemic countries. Increased business and leisure travel to endemic countries have led to diagnostic challenge in non-endemic areas. Laboratory testing is indispensable for diagnosis. Advances in newer rapid, sensitive, and specific testing methodologies and alternate treatment strategies are urgently needed. A safe and effective vaccine in human is not yet available. Prevention is dependent upon increasing public awareness through health education programmes and safe livestock practices. Active co-operation between health and veterinary services should be promoted. Objectives: To find out the status of brucellosis in cattle and high risk human beings of Bakshipora, block palapora, district Srinagar. Materials and Methods: Serum samples from cattle and from human beings were obtained and processed through RBPT, STAT, and I-ELISA. Milk samples were subjected to MRT. Results: Cattle seropositivity by I-ELISA, RBPT and STAT were found to be 8.14%, 4.26% and 2.32% respectively. Milk sample analysis showed 3.44% positivity in MRT. In case of human beings the value obtained were 5.68% and 3.41% in RBPT and STAT respectively. The prevalence among veterinary officers, farmers, animal handlers, slaughter house workers and from patients with Pyrexia of Unknown Origin was 0.00, 9.09, 3.70, 12.5, 4.41 and 5% respectively. Conclusion: The current observation provides a baseline prevalence level of brucellosis in cattle and occupationally exposed human beings of Bakshipora. A broader investigation including small ruminants is required to elucidate the actual prevalence of the disease in the state. Study of Hepatitis B Virus Infection and its Genotypes in a Tertiary Care Hospital Mathura Uttar Pradesh Anju Rani, Shama Tomar, Bichitrananda Swain KD Medical College Hospital and Research Center Mathura Background: It has been estimated that HBV genome evolves at an error rate of ~ 10–3 to 10–6 nucleotide substitutions/site/year, which is approximately 100 times higher than that of other DNA viruses 27. The accumulation of these mutations over a long period of time results in a large amount of genetic diversity. It is important to note that the rates of disease progression, clinical and treatment outcomes may different for each HBV genotype. Aims and Objective: To determine HBsAg Seropositivity and genotype detection among rural population attending inpatients and outpatients at tertiary care center Mathura (U.P). Material and Method: A total of 3600 patients attending outdoor and indoor were screened for HBsAg by ICT manufactured by J Mitra & Co Pvt Ltd. and ELISA (Robonik). HBV genotyping was studied by PCR direct sequencing. Statistical analysis was performed using SPSS 13.0 software. Results: Seropositivity of HBsAg was 456/3600 (12.6%). Seropositivity was seen more in males 287/1844 (15.56%) as compared to females 169/1756 (9.62%). Among patients screened for HbsAg, we observed seropositivity of 8.28% (4/169) among women of childbearing age with age range 16 to 45 years. Genotype D and A were found, most prevalent was genotype D. Conclusion: To understanding of actual epidemiology of HBsAg in rural population especially women of childbearing age. Poor sanitation, lack of awareness and alcohol consumption are common reason for its widespread. Therefore, we should make efforts to prevent HBV infection by focusing on associated risk factors. Keywords: Hepatitis B, Childbearing age, Seropositivity, Rural population, HBV DNA. Medical Virology (Poster Presentation) Preclinical Animal Model of Influenza to Support R&D ShikhaSaxena, Bharat Lohiya, Preeti Vishwakarma, Ritika Khatri, Varun Kumar, Jolly Thomas, Gazala Siddiqui, Amit Kumar, Satish Kumar, Sweety Samal* *Laboratory of Influenza and respiratory viruses, Infection and Immunology, Translational Health Science and Technology Institute, NCR Biotechcluster, Faridabad, 121001 Background and Objectives: Influenza (flu) is a contagious respiratory illness caused by influenza viruses that infect the nose, throat, and lungs. Influenza viruses are negative strand RNA viruses and belong to the family of Orthomyx oviridae. Influenza viruses cause over 500,000 deaths world wide each year. H1N1 (Influenza like illness) was first reported in Mexico in 2009 and later its spread to United States and Canada. About 2 lakhs ases and 2000 deaths were reported due to this virus pandemic from 178 countries. The main objective of this study is to understand the Influenza A pathogenesis by using mouse model. Material and Methods: Influenza virus (Cal/09H1N1) was grown in MDCK cells and embryonated eggs (10–12 days old), the virus was titrated in 96 wellplate, the titer was calculated with Reed and Muench (1938) by CPE and HA based method. The titrated virus was serially diluted and intranasally (IN) inoculated in BALB/cmice for the calcul ation of MLD50. After this the animals (n = 5) were challenged with 10MLD 50 of the Cal/09 virus, the mice were observed from 0–14 days for the body weight change and clinical score. The mice were perfused on 3- and 5-days post infection for viral load by real time PCR and lung virustitre. Bioinformatic analysis was conducted to understand the specific mutation incurred before and after the 2009 pandemic strains. Results and Conclusion: Cal/09 (HINI) virus was successfully adapted in BALB/c mice after passaging two times in lungs. Virus grown in egg was more lethal and higher titre than the cell culture adapted virus. Validation of Commercial Kits Using Standard ICMR Protocol Varsha Potdar, Kavita Lole, Alagarasu K, Veena Vipat, Supriya Hundekari, Rashmi Gunjikar ICMR National Institute of Virology Pune Background: SARS-CoV-2 highlighted worldwide, the need of enhance testing capacity. Government of India, under Atmanirbhar Bharat provided platform to private/public companies to develop and manufacture diagnostic reagents /kits for SARS CoV 2 testing. Objective: Performance evaluation of commercial kits. Handholding of private/public companies to improve the kits quality for its diagnostic accuracy to use for Covid 19 diagnosis Material and Method : The SOP for the validation of diagnostic kits were prepared and approved by ICMR technical committee. The ICMR NIV single tube assay was used as gold slandered. The panels of known positives and negatives were prepared. Validation of commercially developed RT-PCRs, RNA extraction kits and virus transport medium were undertaken. The sensitivity and specificity of the kit were calculated and reported as per ICMR’s acceptance. Results: Real time RT-PCR kits evaluation: Total 165 kits were evaluated, which includes 12 LAMP assay. Among domestic kits, 31 kits were satisfactory while 83 were not satisfactory. Among the imported kits, 25 kits were satisfactory while 26 were not satisfactory. RNA extraction kits evaluation:- Total 157 kits were evaluated, Among domestic kits, 57 kits were satisfactory while 53 were not satisfactory. Among the imported kits, 31 kits were satisfactory and 17 were not satisfactory. VTM kits evaluated = Total 89 kits were evaluated among which nine kits were imported while 80 kits were of domestic origin. Performance of 10 kits was not satisfactory. Conclusions: Kit validation is important to access the quality of commercial kits and to enhanced the testing capacity exponentially in country. Funding: ICMR DHR. , attitude and practices among medical students regarding hand hygiene during the Covid-19 pandemic Shazia Jamsheed 1, Tansila Rashid2, Samina Farhat3, Muzaffar Ahmed Pukhta4 1Postgraduate Scholar, Department of Pharmacology, Government Medical College, Srinagar. 2Postgraduate Scholar, Department of Pharmacology, Government Medical College, Srinagar. 3Professor and Head, Department of Pharmacology, Government Medical College, Srinagar. 4Associate Professor, Department of Pharmacology, Government Medical College, Srinagar Background: Hand hygiene has been long been acknowledged as one of the most simple and cost-effective method to prevent the spread of many infections. Hand hygiene is defined as the cleaning of hands to reduce microbial load. Hand hygiene can be performed either by hand washing with soap and water or by using alcohol based hand rubs. The ongoing pandemic has further stressed the importance of hand hygiene in prevention of infections including the COVID 19 infection. In fact, hand hygiene along with use of face mask and social distancing are recommended as the first-line interventions in the prevention of COVID 19. WHO has come forward with certain recommendations pertaining to hand hygiene practices to be followed in this regard. The Government of India has also organized various campaigns for the general public regarding the importance of hand hygiene and the correct techniques to be followed in doing so. Our study aims to evaluate the knowledge, attitude and practices among medical students regarding hand hygiene since non-compliance on their part to adhere to such practices could lead to further transmission of infections. Methods: This study will be questionnaire based and is being conducted among the students of Government Medical College, Srinagar to evaluate their knowledge, attitude and practice regarding hand hygiene. Role of Vitamin C in the treatment of COVID-19: A comprehensive review study Dr. Mahish Mehraj 1, Prof. (Dr.) Rehana Tabassum2, Prof. (Dr.) Samina Farhat3 Department of Pharmacology, Govt. Medical College, Srinagar, Jammu & Kashmir Introduction: COVID-19 is now deemed as the global health burden. As of 24 july 2022, over 567million confirmed cases and over 6.3 million deaths have been reported. Symptoms include fever, cough, fatigue, body ache and shortness of breath, acute respiratory distress syndrome (ARDS). Vitamin C (ascorbic acid) is a water-soluble vitamin that plays a major role as antioxidant and as co-factor of various biosynthetic pathways in the immune system. Objectives: In this study we aimed to summarize the current evidence regarding the use of vitamin C in the prevention or treatment of patients with SARS-CoV2 infection, based on available publications between January 2020 and July 2022. Material and Methods: In this comprehensive literature review two electronic databases (PubMed and EMBASE) were searched from 1 January 2020 to 1 July 2022. Results: Numerous researchers and clinicians hypothesised that ascorbic acid could help prevent SARS-CoV-2 infection by boosting immune response and reducing the severity of the viral-mediated inflammatory response. Preliminary observational studies indicate low vitamin C status in critically ill patients with COVID-19. Conclusion: While some studies have found no correlation between vitamin intake and mortality, others have discovered that this vitamin c is beneficial in lowering the death rate. In summary, vitamin C possesses positive impacts on curing of infection and this may play a protective role in the current COVID-19 pandemic through boosting the immune system. Antibody response to COVID-19 vaccines used in India: An observational cohort study among healthcare workers Shubham Kadlag*, Prajakta Rane, Urmi Majumdar, Archana Munje, Ruta Kulkarni, Sonali Palkar, Rahul Patil, Jitendra Oswal, Sanjay Lalwani, AkhileshChandra Mishra, Vidya Arankalle # *Presenting author # Corresponding author – [e-mail] Background: COVID-19 pandemic witnessed rapid development and use of several vaccines. In India, a country-wide immunization was initiated in January 2021. COVISHIELD, the chimpanzee adenoviral vectored vaccine with full length SARS-COV-2 spike insert and COVAXIN, the whole virus, inactivated vaccine, were used. Objective: The present study was aimed at assessment and comparison of antibody response to COVISHIELD and COVAXIN. Materials and Methods: Blood samples were collected pre-vaccination, 1 month post-1/post-2 doses and 6 months post-dose-2, from healthcare workers receiving COVISHIELD and COVAXIN vaccines. The samples were tested for IgG-anti-SARS-CoV-2 (ELISA) and neutralizing antibodies (Nab, PRNT50). Results: In pre-vaccination-antibody negative COVISHIELD recipients (pre-negatives, n = 120), % Nab seroconversion increased from 55.1% post-dose-1 to 95.6% post-dose-2, that were independent of age/gender/BMI. Presence of co-morbidities reduced Nab titers (p = 0.004). In pre-positives (n = 67), Nab titers increased to 40.7 fold from 75 (IQR 29–129) before vaccination to 3050 (IQR 1282–3998, p < 0.001) post-first-dose, but declined to 1740 (IQR 911–3116, p = 0.037) post-2nd-dose. Nab response in pre-positives was independent of age/gender/BMI/co-morbidities. Post-dose-2 seroconversion (50%, p < 0.001) and Nab titers (6.75, 2.5–24.8, p < 0.001) in COVAXIN recipients were lower than COVISHIELD. Diminished Nab titers were observed at 6 months post-dose-2 for both vaccines. Conclusion: This first-time, systematic, real-world assessment revealed generation of higher neutralizing antibody titers by COVISHIELD. Relation of dose interval and decline in Nab titers post-2nd-dose in pre-positives need further assessment. Diminished Nab titers at 6 months emphasize early booster. Effect of COVID-19 Pandemic on the Diagnosis of Breast Lesions Compared with Breast Lesions in the Previous Five Years Saniya Nisar1, Rohi Wani2, Sheema Sheikh2, Josepheen Shahmiri3, Abdul Maajed Jehangeer4, Misbah Rashid5, Salma Gul5 1Post-graduate student, PG Department of Pathology, Govt Medical College Srinagar, 2Associate professor, PG Department of Pathology, Govt Medical College Srinagar, 3Demonstrator, PG Department of Pathology, Govt Medical College Srinagar, 4Consultant surgeon, SDH, Kreeri, J&K Health Services, 5Senior resident, PG Department of Pathology, Govt Medical College Srinagar Background: Covid-19 pandemic caused havoc in both people, the health care system and more so in patients with malignancies. Breast malignancies being one of the most common and relatively curable malignancies got hit a lot due to the impact of the Covid-19 pandemic. The multistep impact of pandemic delayed the diagnosis as also the treatment of this multidisciplinary approach disease. Objective: To determine the effect of the Covid-19 pandemic on the diagnosis of breast lesions. Methods: This is a descriptive type of histopathological study in which we have collected and studied the data of Breast lesions over the Five years (Jan 2015-Dec 2019) and compared it with the data of Breast lesions in the Covid-19 year (the year 2020). Results: The number of cases of breast specimens received for histopathology per year declined from an average of 224/year to 124/year in the Covid-19 year of 2020. Conclusion: The decrease in the number of cases alludes to the downfall in the number of patients coming for diagnosis and in turn getting treatment. This study also highlights the importance of anticipation of various cases which will cluster shortly. Antibody Subclass Responses to SARS-CoV-2 in severe and non-severe COVID-19 Patients Prajakta Rane1#, Harshad P. Patil1, Shubham Shrivastava1, Sonali Palkar2, Sanjay Lalwani2, Akhileshchandra Mishra1, Vidya Arankalle1 1Department of Communicable Diseases, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune, India. 2Department of Pediatrics, Bharati Vidyapeeth (Deemed to be University) Medical College, Pune, India Background: SARS-CoV-2 is a highly contagious respiratory virus responsible for COVID-19 pandemic. To understand the role of antibodies in neutralization, our study quantified circulating levels of IgA/IgG and IgG subtypes induced at different days post onset of symptoms, in severe and non severe patients. Objectives: To quantify circulating levels of IgA, IgG and IgG subclass in severe and non severe patients induced at different days post onset of symptoms. Material and Methods: Serum or plasma samples collected from 79 COVID-19 patients were used. Indirect SARS-CoV-2 specific IgA, IgG, and IgG subclass specific ELISAs were performed. Antibody titers between severe and non severe patients were compared at different times post onset of clinical symptoms. Titers in ELISA were correlated to neutralizing antibody titers. Results and Conclusion: Over 75% patients were positive for IgA and IgG antibodies in the first week. The ELISA titers did not differ during the first week of infection. However, patients with severe disease exhibited raised titers. Neutralizing antibody titers correlated with the ELISA titers in mild presentation but not in severe disease. IgA and IgG1 antibodies correlated stronger with neutralizing antibodies. The findings highlighted that IgA together with IgG play an important in SARS-CoV-2 neutralization. Symptom Profile and Risk Factors Associated with Long Covid Syndrome – A Prospective Observational Study at a Tertiary Care Hospital in Kashmir Background: Covid-19 disease initially started as respiratory illness but later was found to involve almost all organ systems. Earlier the focus of the research was more on preventing transmission of the disease and mortality but with time focus has shifted to addressing the impact of the disease on quality of life and managing the Long Covid Syndrome. Aim: To study symptom profile in Post-Covid patients and risk factors associated with Long Covid Syndrome. Materials and Methods: This observational, single-centre prospective study was conducted on Covid-19 patients who presented to post-covid clinic, during the months of July 2021 to December 2021, at Chest Diseases Hospital, Srinagar. Patients who had microbiologically confirmed Covid-19 Disease were included in the study. Patients were evaluated for Long Covid symptoms. Hospital stay, disease severity and co-morbidities of the patients were also taken into account. Results: In total of 720 patients, 388 were females and 332 were males. 622 patients had post-Covid symptoms, out of which 516 patients had received hospital care and 106 were treated on outpatient basis. Fatigue, shortness of breath, cough, headache and sleep disturbances were the most common complaints in patients who presented to our post-Covid clinic. It was also observed that female sex, prolonged hospital stay and older age were associated with Long Covid symptoms. Patient with severe disease were also at higher risk of having Long Covid symptoms. Conclusion: In our study, we concluded that Long Covid symptoms can be disabling for patients and have huge impact on quality of life of patients. Further studies are needed to understand the pathophysiology of Long Covid Syndrome and explore therapeutic options for the same. Covid 19 presenting as stroke and MI Its now a well known fact that covid 19 causes coagulopathy that has been associated with the inflammatory phase of coronavirus disease (COVID-19) and might be involved in this concurrency. Here we present a case of a 55y old female with no underlying comorbidity presented with the chief complaints of mild slurry speech and weakness over the right side of the body from last 8 h. Noncontrast brain computed tomography (CT) scan showed early signs of ischemia in left middle cerebral artery (MCA) territory, and a CT angiogram demonstrated a carotid atheromatous plaque with a superficial thrombus causing 40% stenosis in the left proximal internal carotid artery (ICA), however no intracranial artery occlusion was found. On ecg patient had ST segment depression in and depression in v5 and v6 leads with transthoracic echocardiogram showed lateral wall hypokinesia of the left ventricle, with qualitative troponin-T positive. There were no respiratory or other symptoms compatible with COVID-19 infection or chest pain. Chest CT ruled out inflammatory/infectious signs in the lung parenchyma, and Rapid antigen testing for covid 19 was negative on admission however RT-PCR for SARS-CoV-2 was positive. Patient was initially loaded with dual anti platelets and lmw heparin and was subsequently managed with aspirin 150 mg, clopidogrel 75 mg and atorvastatin 40 mg with resolution of the chest pain and slurry speech. IL-6 as a sensitive indicator of severity in COVID-19 patients-an observational study Background: Patients with COVID-19 can develop a cytokine storm, with a mortality rate of up to 45%. Because IL-6 is a relevant cytokine, early identification of patients at-risk can help reduce mortality. Objective: To determine whether serum IL-6 levels on admission can predict the outcome in terms of all-cause mortality in hospitalized COVID-19 patients. Materials and Methods: This observational, single-center retrospective cohort study was conducted in patients admitted with COVID-19 disease at Chest Disease Hospital, Srinagar from May 2021 to October 2021. Investigations like IL-6, HS-CRP and D-Dimer, were collected, once after hospitalization (median 1.53 days). Multivariable logistic and linear regressions and survival analysis were performed depending on outcomes, primary end point being all-cause mortality, secondary outcomes being, need for HFNC, NIV or IMV. Results: 198 patients, 55.5% males, median age was 67 years. Mean IL-6 levels in patients who were discharged were 77.4 pg/ml while those who died had 132.56 pg/ml, corresponding with the severity of the disease. Need for HFNC 25.3% vs 13.9% (P = 0.44), NIV 17.3% vs 4.1% (P = 0.002) and IMV 2.7% vs 1.6% (P = 0.614) was also higher in patients with high levels of IL-6. Conclusion: Baseline IL-6 greater than 90 pg/mL is a sensitive indicator of progression to severe disease in COVID-19 patients manifesting in terms of higher mortality, need of HFNC, NIV and IMV. Hence early identification of patients at risk may result in early intervention and hence reduce mortality. A case of mild COVID-19 presenting as massive pulmonary embolism Covid19, a novel coronavirus rapidly spread throughout the world, resulting in a global pandemic. The virus was designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the illness it caused coronavirus disease 2019 (COVID-19). The spectrum of COVID-19 in adults ranges from asymptomatic infection to mild respiratory tract symptoms to severe pneumonia with acute respiratory distress syndrome (ARDS) and multiorgan dysfunction. Here we present a case of 26 year old male with no known underlying comorbidity, started with complaints of fever, body aches, generalised weakness, anosmia, for 4 days, He visited a general practitioner at periphery where his vitals were found to be stable maintaining saturation of 96% at room air, was send home on acetaminophen 650, 2 days later in the morning patient developed sudden onset breathlessness & was rushed to GMC SRINAGAR, on preliminary examination He was having vitals of Bp = 70mmhg systol, Pulse 140b/m, Tem: 98 feh, R/R 26 with normal CXR, Ecg S/o sinus tachycardia, CTPA was done which showed PTE, Patient was thrombolysed on further investigations patient tested positive for COVID 19, with high antibody titers for covid & High inflammatory markers in the form of Il6, D-dimmer, Crp, Esr, Ferritin etc. Performance of multiplex bead based assay for detection of neutralizing antibodies in SARS CoV-2 infected and vaccinated individuals Rashmi Gunjikar, Vaishali Bhatt, Ojas Kaduskar, Gururaj Rao Deshpande, *Gajanan N. Sapkal, Manjusha Gopale, Prasad Gomade Diagnostic Virology Group, ICMR- National Institute of Virology, Maharashtra, India Background: After the pandemic of SARS COV2 it is evolving and causing a threat and concern worldwide. Studies on variants are crucial in understanding the change in virulence and transmissibility of the virus and further vaccine efficacy. Plaque reduction neutralization test (PRNT) is the gold standard to detect neutralizing antibodies. Consequently, it is essential to explore other neutralization platforms which give promising results with quick turnaround time and safety compared to live neutralization assay. Objectives: To compare and evaluate the neutralizing antibody responses of a multiplex bead-based assay (MBA), using the SARS-CoV-2 Variants Neutralizing Antibody Human 5-Plex ProcartaPlex™ Panel, with PRNT and further evaluate it to estimate the neutralizing responses in infected and vaccinated individuals. Materials and Methods: Confirmed RT PCR covid positive and serum samples from vaccinated individuals (Covaxin and Covishield) were assesssed for the presence of neutralizing antibodies using Multiplex bead assay Human 5-Plex ProcartaPlex™ kit. Results: The sensitivity and specificity of MBA was less as compared to PRNT. In our study, we observed that qualitative assay was more comparable than quantitative assay. Conclusion: This study demonstrated the utility of MBA for simultaneous measurement of neutralizing antibodies against multiple SARS-CoV-2 strains in serum. The qualitative assay performance was imparted excellent with high sensitivity and specificity but variation in quantitative titres was observed compared to live neutralization assay titres. Persistence of serum anti-SARS-CoV-2 IgA response in infection and vaccination Rashi Srivastava, Aparna Rakhe, Asha S Salunke, Gururaj Rao Deshpande, *Gajanan N. Sapkal, Bhagyashree Ghasolia, Keshav sharma, Ashwini Singh, Afsha Sheikh Diagnostic Virology Group, ICMR- National Institute of Virology, Maharashtra, India Background: SARS-CoV-2 evokes vigorous humoral immune responses which includes production of virus-specific antibodies of the immunoglobulin IgM, IgG & IgA isotypes. Seroconversion & production of detectable antibodies usually occurs within 20 days of symptom onset, while the kinetics of their production is variable. IgA is the major antibody class in mucosal membranes which plays an important role in SARS-CoV-2 infections. It’s response in the early stage of the disease seems to be more pronounced than IgM. Objectives: To detect the presence of serum IgA antibody response against Spike Receptor Binding Domain & Nucleoprotein of SARS-CoV-2 in naturally infected individuals as well as vaccinated individuals. Materials and Methods: Confirmed RT-PCR Covid positive serum samples were tested by in-house developed SRBD IgA ELISA & N protein IgA ELISA of SARS-CoV-2. The subjects were classified according to the post onset of disease date. Serum samples of vaccinated individuals (Covishield & Covaxin) were assessed to compare IgA response. Results: Our results suggest a linear trend in the level of IgA antibody response POD 8 onwards in natural infection. In vaccinated individuals Covaxin groups exhibits a prominent increase in the IgA response in comparison to Covisheld. Conclusion: IgA might play an important role in assessing the immune status of SARS-CoV-2 infected patients. This study suggests that IgA antibody act as a promising immunological marker for vaccine study. A rare case of fever with lymphadenopathy Muzamil Majeed Kumar Background: Young female with no underlying co morbidities with two month history of fever and cervical lymphadenopathy after a thorough investigations for infective causes was found to have a rare disorder which was a sequelae of a preceding viral etiology. The diagnosis was confirmed by lymph node biopsy. Objective: The purpose of the study was to establish a specific cause for the mentioned symptoms. Materials and methods: Viral serology, Lymph node biopsy, Blood and urine cultures, baseline lab investigations, CT Neck/Chest/Abdomen. Results and Conclusion: The diagnosis was established as case of Kikuchi disease with a preceding viral etiology. It was confirmed with lymph node biopsy with positive antibody titre for HSV1. Untargeted temporal analysis of serum metabolome in acute HEV infected patients. Shaheen Khan1, Yashwant Kumar3, Charu Sharma2, Sonu Kumar Gupta3, Amit Goel4, Rakesh Aggarwal4, Naga Suresh Veerapu1 1Department of Life Sciences, Shiv Nadar University, NH91, Tehsil Dadri, Gautam Buddha Nagar, UP201314, India. 2Department of Mathematics, Shiv Nadar University, NH91, Tehsil Dadri, Gautam Buddha Nagar, UP201314, India. 3THSTI, NCR Biotech Science Cluster, 3rd Milestone, Faridabad, Haryana, India. 4Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, UP226014, India Background: Hepatitis E is an enterically transmitted infection caused by the hepatitis E virus. It’s a multifaceted disease that causes a wide range of symptoms. Like any other virus, HEV is also reported to disrupt metabolic pathways in the host organism but a detailed analysis of metabolites and lipids in temporal space has not been explored yet. Objective: We investigated the changes in metabolome and lipidome in HEV-infected men and compared to gender and age-matched healthy controls. Materials and Methods: The changes in metabolites and lipids levels were determined in an untargeted manner using LC-MS coupled with mass spectrometry from 65 sera sequentially collected from 14 HEV infected and 25 samples from 5 healthy control subjects. Results: Total 167 metabolites and 228 lipids were found to be differentially regulated in acute HEV infected patients in comparison with healthy controls. The carbohydrate metabolism showed the most number of dysregulated metabolites such as glucose, and fructose 1–6-bisphosphate of glycolysis pathways and ribose-5-phosphate and ribulose-5-phosphate of pentose phosphate pathway showed upregulation in HEV patients. The end product of purine metabolism i.e. uric acid was downregulated while end product of pyrimidine metabolism, β-aminoisobutyrate showed upregulation. The level of kynurenate and homogentisate showed downregulation which contrast with previous studies of RNA viruses. The level of amino acids such as alanine, valine, arginine and glutamate were also found to be dysregulated in HEV infected group. Significant upregulation was observed in acylcarnitine (6/6) which is reported to be associated with viral infection along with decreased phosphatidylcholines, OxTGs and TGs indicates towards perturbations in energy metabolism. The acylcarnitines, sphingomyelins, phosphotidylcholines (PC), phosphotidylethanolamines (PE), lysoPC and lysoPE showed a decreased trend in the sera of HEV group. Conclusions: Acute HEV infected subjects differential metabolomic and lipidomic profile over a period of 15 days. Metabolites involved in carbohydrate and nucleotide metabolism showed increased profile. HEV infected patients also showed increased in bile acid biosynthesis metabolites which indicates liver function alteration during HEV infection. We have also observed downregulation of majority of glycerophospholipids and upregulation of acylcarnitines. Japanese Encephalitis Virus infected AG129 mice lacking both α/β and γ interferon receptors shows exorbitant susceptibility and extorsive vascular permeability Gazala Siddqui Senior Project Associate, Translational Health Science and Technology Institute, Faridabad, Haryana Introduction: JEV is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family. The clinical symptoms of JEV mostly range from asymptomatic to acute febrile illness, however, in severe infection, it can cause virus-induced encephalitis, mainly in the children and elderly population. Background: The JEV infection rate is high in rural and densely populated areas in semi-urban and urban cities of Asian countries with an estimated 68,000 clinical cases every year, although symptomatic Japanese encephalitis is rare, the case-fatality rate among those with encephalitis can be as high as 30%. Permanent neurologic or psychiatric sequelae can occur in 30%–50% of those with encephalitis. Objectives: To study the disease severity of JEV Virus into AG129 aged and adult mice. Materials and Methods: In this study, AG129 mice were injected with high titre JEV Indian clinical isolate by both intraperitoneal and intradermal route and it induces fatal encephalitis in both young and aged old mice. Results and Conclusions: JEV virus inoculation by both I.P and I.D route in AG129 mice manifests severe disease, total lethality and changes in hematological parameters. Lack of innate responses in AG129 mice resulted in high vascular permeability in JEV infected mice. IFN α/β and γ receptors knock out AG129 mouse has intact humoral and cellular responses upon JEV infection and could be used as a pre-clinical model for rapid testing of novel antiviral drugs or vaccines indicates towards perturbations in energy metabolism. The acylcarnitines, sphingomyelins, phosphotidylcholines (PC), phosphotidylethanolamines (PE), lysoPC and lysoPE showed a decreased trend in the sera of HEV group. Conclusions: Acute HEV infected subjects differential metabolomic and lipidomic profile over a period of 15 days. Metabolites involved in carbohydrate and nucleotide metabolism showed increased profile. HEV infected patients also showed increased in bile acid biosynthesis metabolites which indicates liver function alteration during HEV infection. We have also observed downregulation of majority of glycerophospholipids and upregulation of acylcarnitines. Trend Analysis of Animal Bite Cases: A Retrospective Study at Anti-Rabies Clinic of a Tertiary Care Hospital of Kashmir Dr. Abdul Hamid Dar Senior Resident, Department of Community Medicine (SPM) Government Medical College Srinagar (9419009773) [e-mail] Background: Rabies which is 100 fetal but preventable is caused by bite of rabid animals particularly dogs. Animal bites cases are major public health problem in India and also in the UT of Jammu & Kashmir. Objectives: To study the trend and seasonal Variation of animal bite cases attending the Anti Rabies clinic (from 2009 to 2022), run by Department of Community Medicine, Government Medical College Srinagar at SMHS Hospital. Materials and Methods: The retrospective Cross sectional study conducted at Anti-Rabies Clinic of SMHS Hospital, a tertiary care associated Hospital of Government Medical College Srinagar. Data was collected from the record of Animal bite register at Anti rabies clinic after proper permission from the incharge of the clinic. Data was entered and analyzed on Excel soft ware. Results: More than 70,000 number of animal bites cases were reported at the Anti-Rabies clinic from the year 2009 to 2022. there has been a rise of cases from the year 2009 to 2018 with a slight decrease in the year 2019–20. About 97% of all animal bites were dog bites. Majority of the cases (> 60%) were category of three (3) exposure. One year analysis for seasonal variation shows that incidence was more in March and May–June. Conclusion: the present study showed that animal bites cases were rising with little decrease in the year 2019–20 as may be due to lesser animal human interaction due to Covid-19 restrictions. Keeping in view the present trend it is expected that the Animal bite cases will show rising trend in coming years. Multidisciplinary approach is advocated to control the increasing trend. Development of a serological assay to detect IgM and IgG antibodies against hepatitis A virus 3C protease Supriya Hundekar , Nital Ganorkar, Kavita Lole Indian Council of Medical Research-National Institute of Virology, Pune Background: Change in endemicity from highly endemic to intermediate state of Hepatitis A infection is observed in developing countries. To identify target groups for vaccination in such areas, one needs a serologic test that enables discrimination between natural infection and vaccination. Objectives: To develop recombinant HAV non-structural protein 3C (protease) based ELISA to detect HAV anti-3C protein IgG and IgM antibodies. Material and Methods: Non-structural 3C protease of HAV was expressed with N-terminal 6His-tag using bacterial expression system. Purified recombinant protein (r3C) was used for ELISA standardization using a panel of known anti-HAV IgG and IgM antibody positive and negative samples. Cross reactivity of the test was checked by testing samples positive for other hepatitis viruses. To assess longevity of anti-3C antibodies after natural infection, anti-HAV IgG positive serum samples from 3 different age groups were tested. Results: HAV anti-3C IgG ELISA showed sensitivity of 81.8% and specificity of 99.94%. HAV anti-3C IgM ELISA showed sensitivity of 90% and specificity of 97.7%. All samples tested for checking cross-reactivity tested negative indicating high specificity of the test. Detection of 40% positivity in 6–10 year age group, 16.67% in 15–25 and 26.67% in 40 + year age group for anti-3C IgG antibodies indicated waning of antibodies over the period. Of the 30 samples from vaccinated individuals, 27 individuals tested negative and 3 individuals tested positive for the anti-3C antibodies. Conclusion: HAV r3C protein can be used as antigen to detect anti-HAV IgM and IgG antibodies. However, use of anti-3C IgG ELISA could be limited to differentiate between individuals having natural infection and vaccination as anti-3C antibodies wane over the period after natural infection. A Case of Febrile Illness with EBV Dr Inayat ullah Pall A 55 year Male underlying Rheumatoid Arthritis on DMARDS for last 4 years, presented with long-term febrile illness for more than 15 days with no localizing signs or symptoms. Initial work up of baseline investigations, septic screen and imaging studies were unremarkable. Patient was initially managed with antibiotics, but didn’t respond. On further evaluation, Viral Serology was sent, and EBV Serology was Positive. Patient was managed conservatively and he became free of symptoms within few days and got discharged. Vaccination Practices Regarding Hepatitis B Among Health Care Personnel Of A Tertiary Care Hospital In Kashmir - A Cross Sectional Study Basina Gulzar Senior Resident GMC Baramulla Background : Vaccination is one of the most successful public health interventions that has saved millions of lives so far. Due to the occupational exposure, health care workers have an increased risk of contracting Hepatitis B. Objectives: To assess the vaccination practices regarding hepatitis B among healthcare personnel (HCP). To study the factors associated with the vaccination practices regarding hepatitis B of these HCP. Methods: This cross sectional hospital based study was conducted for a period of 1 year at Sher-i-Kashmir Institute of Medical Sciences (SKIMS) among 450 HCP including doctors, nursing staff, laboratory staff and others and the required sample was drawn from each category on the basis of Probability Proportionate to Size technique. Information was collected from the participants by using a predesigned, pretested structured and validated questionnaire. Results : It was found that only 34.9% were vaccinated against hepatitis B. The coverage was highest among doctors 55.5% followed by technicians 25.6%, nurses 23.6%. The main reasons for not receiving this vaccine were: taking all necessary precautions (49.1%), hospital does not provide the vaccine (20.8%), not aware about hepatitis B vaccine (20.5%). Conclusion: The study revealed that the hepatitis B vaccination coverage of these healthcare personnel was quite low in spite of the importance of the vaccine for healthcare personnel who are always at risk of getting exposed to the virus during their duties. Surveillance of Dengue and Chikungunya Viruses in Wild Caught Aedes Mosquitoes Collected Concurrently with Lab Confirmed Human Cases in Punjab, India Kanwalpreet 1, Taruna Kaura2, Subharata Sarkar1, Vikrant Sharma1, NazatInder Singh3, Seema Devi3, Abhishek Mewara2, Rakesh Sehgal2, R. K. Ratho1 and Gagandeep Singh Grover3 1Department of Virology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012. 2Department of Medical Parasitology, Postgraduate Institute of Medical Education and Research, Sector 12, Chandigarh 160012. 3Department of Health & Family Welfare, Punjab, Sector, 34-A, Chandigarh- 160022 Background: The three arboviral diseases Dengue, Zika and Chikungunya are significantly important in relation to human health. These arboviral infections are transmitted mainly by two species of genera Aedes viz. Ae. aegypti and Ae. albopictus. Due to rapid urbanization and prevalence of these vector species, a continuous upsurge in the cases of arboviral infections has been reported from different districts of the state Punjab, India since year 2013. These mosquitoes were collected from high, moderate and low risk zones of the different districts of Punjab Objectives: To standardize, detect and determine the infection of three arboviruses i.e. Dengue, Chikungunya and Zika in the Aedes mosquitoes in close proximity of the confirmed patients’ infections. Methods: Both immature stages and adults of Aedes were collected from in and around patients’ house from August to November, 2021. After identification of species of Aedes, the mosquito pools were subjected to RT-PCR for arboviral detection. Results: A total of 184 Aedes aegypti mosquitoes i.e. 46 pools were tested for the presence of these three arboviruses. Out of 46 pool samples, 12 pool samples were positive for only DENV, 9 pool samples were positive for only CHIKV and 5 pool samples were positive for both DENV and CHIKV (co-infection) while none of the samples was found positive for ZIKV. Thus, detection of DENV and CHIKV in Aedes mosquitoes from the habitation of patients proves that the viruses are maintained in these arthropod vectors. Conclusion: Detection of DENV and CHIKV RNA from the wild caught Aedes mosquitoes serves as an indicator for sensing dengue/chikungunya transmission within the community. A Descriptive Study on Drug Prescribing Pattern in Hypertensive Patients in a Tertiary Care Teaching Hospital Dr. Syed Sajad Hussain, Prof. (Dr.) Samina Farhat, Dr. Chowdhary Zubair Abbas Department of Pharmacology, Government Medical College, Srinagar Background: Hypertension is highly prevalent and the goal of antihypertensive therapy is to abolish the risks associated with blood pressure (BP) elevation without adversely affecting quality of life. Drug selection is based on efficacy in lowering BP and in reducing cardiovascular (CV) end points including stroke, myocardial infarction, and heart failure. Not many studies are conducted in this part of world regarding drug utilization of antihypertensive drugs and hence this study was planned. Materials and Methods: A descriptive cross-sectional study was conducted for a period 6 months in outpatient department of a Tertiary care centre of Government Medical College, Srinagar, Jammu and Kashmir. The prescriptions containing antihypertensive drugs were collected from the patients attending the outpatient department. Results: During the study period a total of 230 prescriptions were collected, out of which a 196 were included for the final analysis. Mean age was found to be 62.42 ± 7.77 years. In majority of cases (44.89%), a combination of two drugs was prescribed and among the two drug combination, Angiotensin Receptor Blockers (ARBs) and Calcium Channel Blockers (CCBs) were used most commonly (40.90%). Angiotensin receptor blockers were used as single drug in most number of patients (41.66%). Proton pump inhibitors were the most common (35.71%) co-prescribed drug, followed by Anti platelet drugs (27.55%), Anti diabetics (16.32%) and Statins (16.32). Conclusions: Present study represents the current prescribing trend for antihypertensive agents. It implies that ARBs are the leading group of antihypertensive agents both when used singly and in combination. Further studies focused on the rationale for choice of drugs based on demographic data, economic status, associated conditions and complications would give additional insights into prescribing patterns in hypertension in India. Keywords: Anti-hypertensive drugs, drug utilization, prescribing pattern. Microcystin-LR modulates oxidative stress markers and the palliative role of Co10 in the kidney, heart and brain of murine model Roshni Rajpoot, Raj Kumar Koiri Biochemistry Laboratory, Department of Zoology, Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar-470003, Madhya Pradesh, India Email: [e-mail] Abstract : Microcystins are a group of cyclic heptapeptide toxins produced by cyanobacterial bloom. More than 100 microcystin analogues have been identified, among which microcystin-LR (MC-LR) is the most abundant and toxic variant. Present study was designed to reveal whether potential human carcinogen microcystin-LR could imbalance the oxidative status of kidney, heart and brain of mice and also to explore the amelioratory effect of coenzyme Q10. Balb/c mice were randomly divided to 3 groups with 6 mice in each group. The animals of normal control group (N) received water and normal diet ad libitum and (MC-LR as well as MC-LR + CoQ10) group received MC-LR (10 μg/kg bw/day, ip) for 14 days. After two weeks of MC-LR treatment, mice of (MC-LR + CoQ10) received coenzyme Q10 (10 mg/kg bw, im) for 14 days. In microcystin-LR treated mice as compared to control, significant increase in the level of lipid peroxidation, hydrogen peroxide, protein carbonylation was observed with a concomitant decrease in the level of glutathione. These parameters thereby suggest microcystin-LR induced toxicity via modulation of oxidative pathway. In conclusion, coenzyme Q10 alleviated MCLR-induced tissue toxicities by mitigating oxidative stress markers. Keywords: Microcystin-LR (MC-LR), Coenzyme Q10, Lipid peroxidation, Hydrogen peroxide, Protein carbonylation, Glutathione. Clinical effectiveness of Natrum Phosphoricum and Natrum Sulphuricum on motor coordination and antioxidant parameters in moderate hepatic encephalopathy caused by chronic alcoholism and paracetamol. Debabrata Dash, Raj Kumar Koiri Biochemistry Laboratory, Department of Zoology Dr. Harisingh Gour Vishwavidyalaya, Sagar - 470003 (M.P.) Email: [e-mail] The seventh most common factor in global death and disability is alcohol usage. A non-steroidal anti-inflammatory medicine called paracetamol is frequently used to alleviate pain and fever. According to recent findings, taking paracetamol and alcohol together increases the risk of liver damage, which may result in chronic liver cirrhosis. Liver Cirrhosis causes hepatic encephalopathy, which impairs brain functions. Schussler, who invented biochemical remedies, claimed that a precise balance of biochemical salts must exist in our body for overall health and wellbeing. Disease may be caused by a deficiency in the necessary amount, which can be treated by exogenous administration of the deficient mineral salts in modest doses to restore the necessary balance. Among the twelve biochemical tissue salts, Natrum Phosphoricum and Natrum Sulphuricum are generated from potassium and sulphate, respectively. Although it has been noted that both of these salts boost brain functions under some pathological circumstances, the claim has never been supported by scientific evidence. In the current study, an effort was made to analyze motor coordination by behavioral studies as well as different antioxidant and glycolytic enzyme level in moderate hepatic encephalopathy caused by persistent drinking and intake of paracetamol simultaneously as well as to assess the therapeutic efficacy of Natrum Phosphoricum and Natrum Sulphuricum. Results suggest that Natrum Sulph and Natrum Phos improved motor co-ordination and antioxidant parameters which were impaired during moderate hepatic encephalopathy. Keywords: Natrum Phosphoricum, Natrum Sulphuricum, motor co-ordination, liver cirrhosis, hepatic encephalopathy and antioxidant enzymes. Prevalence and Spectrum of Opportunistic Infections among People Living with HIV (PLHIV) attending Anti Retroviral Therapy (ART) Centre, Government Medical College (GMC), Jammu in Northern India Dr Heena Nazir Senior Resident Community Medicine, SKIMS Medical College and Hospital Bemina Senior Resident Background: Most of the morbidity and mortality in AIDS cases result from Opportunistic Infections. Identification of such pathogens is very important for clinicians and health planners to tackle the AIDS epidemic in more effective manner. Objectives: To find out the prevalence and spectrum of Opportunistic Infections among PLHIV on ART attending GMC, Jammu. Materials and Methods: Hospital-based, cross-sectional study conducted on OPD patients attending ART Centre. The patients were enrolled in the study based on inclusion and exclusion criteria. They were interviewed and the information was collected about socio-demographic profile, risk behavior, treatment adherence. Clinical details and treatment records were also studied. Results: A total of 200 HIV positive patients with OIs were interviewed and about 210 opportunistic infections were found among them as some of them had more than one OIs. The mean age of the study participants was 40.25 years with a Standard Deviation of 11.14 years. The prevalence of OIs was found to be 7% (210/3000). The commonest OI encountered was Tuberculosis (TB) which included pulmonary TB and Extra pulmonary TB (49%) followed by Candidiasis (24.8%) and Diarrhea (11.4%). Variables which had a statistically significant association with OIs were gender, residence, risk behavior (p < 0.05). Conclusions: Almost 7% of the HIV positive patients were diagnosed with OIs. Tuberculosis was the most frequent OI among the PLHIV. This necessitates for accurate and timely diagnosis, to ensure proper care and treatment. Keywords: HIV, AIDS, opportunistic infections (OIs), anti retroviral therapy (ART). Emergence of Non-Albicans Candida Species in Neonatal Candidemia Dr Munaza Aman SKIMS, Srinagar Background: Candidemia is an important cause of morbidity and mortality in Neonatal Intensive Care Unit. Incidence of neonatal candidemia is progressively increasing due to a marked improvement in survival of sick neonates. Aim: To identify the prevalence and epidemiology of neonatal candidemia in NICU of tertiary care hospital. Methods: A prospective study conducted, over a period of 18 months. Samples for blood culture received in Department of Microbiology SKIMS were processed on Bac T Alert 3D and VITEK 2 Compact system for identification and antimicrobial susceptibility testing. Results: Out of 1200 samples received, a total of 178 (88.11%) yielded clinically significant growth. Among these, 52 (29%) demonstrated Candidemia [EOS = 20 (20.4%); LOS = 32 (52.5%)]. All of them were non albicans Candida and showed preponderance towards late onset sepsis. Most found isolate was C. Krusie, 100% resistant to fluconazole. 90% non albicans Candida isolates were sensitive to Amphoteracin B except for C. pelliculosa which showed lesser sensitivity (11%) to Amphoteracin B and (50%) to Voriconazole. Conclusions: Candidemia in neonates is an ominous prognostic sign and is an important entity in our region. The present study highlights the mycological shift of Candida species in neonatal candidemia with a preponderance of non albicans Candida species. So measures should be taken to reduce risk factors wherever possible to reduce morbidity, mortality, and antifungal drug pressure especially in extreme age groups who have weak or waning immune system. Keywords: Neonatal septicemia, late onset sepsis, non albican Candida. Comparative susceptibility studies of two Indian Zika Virus strains in Aedes Aegypti Asha S Salunke , Mangesh D Gokhale, Gajanan N Sapkal*, Sachin D Dhaigude, Gururaj Rao Deshpande, Heena Shaman, Chetan A Patil, Shankar M Vidhate, Kirti A Khutwad, Sanskriti Saka, Devendra T Mourya Diagnostic Virology Group, ICMR- National Institute of Virology, Maharashtra, India Background: Zika virus (ZIKV), a mosquito-borne virus belonging to the genus Flavivirus of the family Flaviviridae, is transmitted to humans by Aedes mosquito species. It was first identified in Uganda in 1947. The Zika footprints in India have been detected since 2017. Three cases of ZIKV infection were reported from Bapunagar area, Ahmadabad (Gujarat), Krishnagiri district (Tamil Nadu) and in 2018 from Jaipur, (Rajasthan). Objectives: Elucidating the pathogenicity levels of the two Indian ZIKV strains by comparative approach using immunocompromised mice model. Materials and Methods: MR-766 (African strain) and Indian isolate (DVG D-18-065) was used for infection at two different infection threshold status. The low pathogenic (VERO CCL81 passaged) and high pathogenic strain was established from the low pathogenic strain (DVG D-18-065 MP-5) by consecutive passaging in mice (AG129 strain) at successive infection status. Comparative susceptibility studies and vertical transmission studies were conducted on Aedes aegypti mosquito and suckling mice with two ZIKV strains. Results: Both the ZIKV isolates [at low and high pathogenic status] were competent to infect the vector mosquito through parenteral route. While infection by oral route showed only high pathogenic virus was competent to infect the mosquito indicating the importance of the infection threshold and the midgut barrier system within the vector. Successful vertical transmission also seen in high pathogenic strain. Conclusion: The virus stains probably become virulent due to continued passage cycle between human and urban Aedes mosquitoes and mutation in the virus, resulting into higher susceptibility in the vector mosquito requiring low-threshold to pick-up infection for transability and higher rate of transmissions starts, thus, virus appears in the outbreak form. The confirmation of the vertical transmission of the virus has epidemiological significance. Changing trend of prevalence, genetic diversity and mixed infections of enteric viruses associated with acute gastroenteritis in pediatric patients in Pune, India: A four years study (2017–2021) Pune, Western India Madhuri S. Joshi 1, Nutan A. Chavan1, Manohar S. Shinde1, Vijay R. Kalrao2, Ram K. Dhongade3, Ashish R. Bavdekar4, Mallika Lavania1 and Varanasi Gopalkrishna1 1Enteric Viruses Group, ICMR- National Institute of Virology1, 20A Dr. Ambedkar Road, Pune 411001. 2Bharati Vidyapeeth Medical College and Hospital2, Pune. 3Sant Dnyaneshwar Medical Foundation & Research Centre’s Shaishav Clinic3, Pune. 4King Edward Memorial Hospital4, Pune Email address of presenting author: [e-mail] Background: In India, infections due to Rotavirus A (RVA) are reported to be responsible for an estimated 11.37 million episodes, 78,000 deaths, and expenditure of Indian Rupee (INR) 10.37 billion per year in children < 5 years of age. Few research have examined the various viral agents associated for Acute-Gastroenteritis (AGE) in India. On this background, the data on the prevalence of RVA and non-RVA enteric viral agents circulating in acute gastroenteritis (AGE) cases in the region is needed. The current study assessed alterations in the genetic diversity and prevalence trends of five enteric viruses contributing to severe diarrhoea in Pune children over a 4-year period. Material and methods: The study includes a total of 416 fecal specimens collected from gastroenteritis patients hospitalized in Pune city between 2017 and 2021 of which 250 were males and 166 females. Samples were individually tested for rotavirus-A, norovirus, astrovirus, and adenovirus using EIA/RT-PCR and genetically characterized by phylogenetic analysis. Phylogenetic analysis was used to define the genetic makeup of the samples after they had been individually tested for rotavirus-A, norovirus, astrovirus, adenovirus, and sapovirus using EIA/RT-PCR. Results: Rotavirus was the most predominant (29.6%) virus followed by norovirus (14.2%), adenovirus (8.2%), and astrovirus (5.5%). G3P[8] rotavirus strain was found most predominant (62.29%). GII (P16-GII.4) genogroup of norovirus were detected. Both type-1 and 8 astroviruses and HAdV-41 were detected during this period. Conclusion: The need for ongoing enteric virus surveillance to assess the effects of diarrhoeal vaccine(s) given in India is highlighted by a shift in prevalence pattern and increased variety from 2017 to 2021. Conserved serine and proline residues in SARS-CoV-2 spike cleavage sites modulate fusogenicity and infectivity Ritika Khatri, Bharat Lohiya, Vikas Maithil, Abhishek Goswami, Sweety Samal* *Respiratory and Influenza Virus group, Infection and Immunity, Translational Health Science & Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, 121001, India [e-mail] Background and Objectives: The spike (S) protein of SARS-CoV-2 virus binds to the host cell receptor which facilitates the virus entry. This interaction is primed by host cell proteases like furin and TMPRSS2 acting at S1/S2 and S2’ cleavage sites, respectively. Both the cleavage sites have Serine and Proline residues conversed in all the coronaviruses. It has been speculated that mutations at these conserved residues may provide a gain-of-function, easing the SARS-CoV-2 entry into the host cell and cell-to-cell spread, thus modulating the virulence and pathogenicity. Unravelling the effects of these conserved residues in the S protein cleavage site in virus entry and transmission might facilitate development of novel therapeutics. Materials and Methods: This study employed a lentivirus based pseudovirus (PSV) system, where P and S residues at S1/S2 site of Spike gene, present in an expression vector, were mutated to Alanine (Fig A). We then assessed the expression of the SARS-CoV-2-S variants in HEK293T cells and tested the infectivity and fusogenicity of mutant PSV and spike, respectively in the presence or absence of S1/S2 and S2’ protease inhibitors. Results and Conclusions: Conserved Serine residue mutation (S2SA) at S2’ cleavage site resulted in complete loss of spike cleavage by furin and cathepsins (Fig B). TMPRSS2 protease treatment was not able to rescue loss of spike cleavage and fusogenicity (Fig C & D). S2SA mutant showed no significant response against E-64d and TMPRSS2 inhibitor. Serine at S2’ site in spike protein provides an ideal site to be further evaluated for the therapeutic purpose against SARS-CoV-2. Epitope Prediction From Envelope Protein of Zika Virus for Vaccine Construction Using Immunoinformatics Approach Ishfaq Ahmad Khan 1, Preeti Sharma1, Parvez Singh Slathia1 1School of Biotechnology, Shri Mata Vaishno Devi University, Katra, J&K, India Introduction: The Zika virus (ZIKV) epidemic originated in Brazil in 2015 wherein nearly 1.3 million individuals got affected, and the infection rapidly spread toall over world. The development of safe and effective vaccine for ZIKVis a highly desired healthcare goal. Objective: The current study was basedon immunoinformatics approach to predict antigenic epitopes ofthe immunogenic envelope (E) proteinof ZIKV. The E protein of ZIKV is a receptor binding protein that mediates the fusion with host cells and is capable of eliciting antibody response. Materials and Methods: Tc cell epitope prediction was carried out by NetMHC and NetCTL servers both of which are artificial neural network-based servers. Th cell epitopes were predicted by NetMHCII server. For B cell epitopes tools available at IEDB analysis resources were used. Analysis of the predicted epitopes for toxicity and allergenicity was done by ToxinPred and AlgPred, respectively. The T cell epitopes were also used for docking studies with respective MHC molecules. Results: The epitopes predicted by these servers if found allergenic or toxic were removed from the final cohort of the selected epitopes. The molecular docking analyses of the predicted epitopes indicated the stability of the selected epitope, when bound to selected HLA-molecules. Only those T cell epitopes were considered that showed binding with the MHC molecules. Conclusion: The epitopes predicted in this study have the potential to be used in vaccine designing for future. Utilising an immunoinformatic-based vaccine design strategy could help in accelerating vaccine studies while lowering the risk of failure. Keywords: E protein CD8 + T cells, CD4 + T cell, Human leukocyte antigen (HLA), Molecular docking. Isolation and genomic characterization of Cell fusing agent virus from Aedes aegypti mosquitoes from Assam, India Abhranil Gangopadhayya, Onkar Ghuge, Ashwini Ramdasi, Asmita Kamble, Surendra Kumar, Sreelakshmi PR, Sudeep Balan, SS Cherian, KS Lole Indian Council of Medical Research-National Institute of Virology, Pune Background: India has a diverse population of haematophagous arthropods, the virus populations inhabiting which are hitherto poorly characterized, including that of Aedes aegypti, a nearly ubiquitous vector for major human pathogens. During our study on metagenomic analysis of Aedes mosquito viromes from different parts of India, we identified a wide range of insect-specific viruses (ISVs). Of these, some ISVshave been reported to influence replication of human pathogenic viruses in mosquitoes. Cell Fusing Agent Virus (CFAV), belonging to family Flaviviridae is one of such ISVs. It was detected in mosquitoes from Assam and West Bengal. Objectives: To isolate Cell fusing agent virus (CFAV) from Ae. aegypti mosquitoes, perform genetic characterization of the virus and study growth kinetics of the virus in insect cells and mosquitoes. Materials and Methods: ISV isolation, cytopathic effect (CPE) production, and growth kinetics studies were done using the C6/36 Aedes albopictus cell line. WGS was done using a multiplexPCR tiling approach. Phylogenetic analyses were done using MAFFT and MEGA 5.2 software. Results and Conclusion: Mosquito cell line, C6/36was inoculatedwithmosquito lysate containing CFAV and passaged blindly. CPE and characteristic cell fusion pattern was seen in infected cells after three passages. Analysis of CFAV WGSshowed highest homology (~ 96.66%) with 2014Australian isolate while lowest homology (~ 94.28%) was seen with 2015 Cambodian isolate. It would be insightful to know whether CFAV has any effect on the replication and transmission of clinically important arboviruses such as dengue, chikungunya and zika. The utility of molecular diagnostics for pathogen identification in CSF samples-A retrospective cohort study in the Tertiary care hospital Kashmir North India Raihana Ali 1, Bashir Ahmed Fomda1 Department of Microbiology 1, Sher-i-Kashmir Institute of Medical Sciences, Soura, Srinagar Background: Central nervous system (CNS) infections are significant cause of morbidity and mortality. Identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires both clinical and epidemiological information. Objectives: This hospital-based study aimed to determine the etiology of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis. Materials and Methods: We did a 14 month (30th August 2018 to 15th February 2020) retrospective study of both adults (> 16 years) and Children (0-15 months) of age group whose CSF samples were received for molecular testing. Clinical, laboratory and treatment details were extracted by screening and reviewing of medical records, discharge summaries, laboratory and pharmacy records of patients from MRD section at SKIMS. Results: 209 analysable patients were enrolled, majority of patients belong to adults 57.41% and 42.58% belong to pediatric age group (0–15 years). PCR confirmed acute viral meningitis in 24.72% (22/89) children. Enteroviruses (EV) was demonstrated in 22.47% (20/89) and HSV2 in 1.12% (1/89) cases. In adults PCR was positive for HSV (n = 5) patients and (n = 1) was positive for both HSV1 and HSV2. Conclusion: Study findings show that enterovirus is the main etiological agent of CNS infections among children in this cohort. Molecular diagnostics for pathogen identification can easily be implemented in routine diagnostics may improve health outcomes of CNS infection. Gene expression signatures of Hepatitis E virus infected cells and drug repurposing by identifying inverse drug-disease relationships Neha Bhise 1, Diya Roy2, Prudhvi3, Sarah Cherian, Kavita Lole ICMR-National Institute of Virology, 20/A, Dr. Ambedkar Road, Pune - 411001 Background: Two drugs, ribavirin and pegylated IFN-α arethe present offline options for the treatment ofindividuals infected with hepatitis E virus (HEV) requiring antiviral therapy. However, the associated severe side effects especiallyin pregnant women necessitates the development of new anti-HEV drugs. Objectives: To study the host response due to HEV infection for understanding the pathobiology and use reverse gene expression signatures for the prediction of repurposed drugs against HEV. Materials and Methods: Huh-7-derived clonal S10-3 cellswere transfected with HEV genotype 1 full genome replicon (pSK-HEV-2) RNA. Total RNA extraction was performed and only the mRNAwas processed further for RNASeq analysis. Differentially expressed genes (DEGs) wereidentified using R software. Further, gene ontology and pathway enrichment were performed by ShinyGo 0.76.2gene-set enrichment tool. DEGs were then subjected to ConnectivityMap (cMap) analysis for finding drugs based on inverse genomic signature approach. Results and Conclusions: A total no. of 5033 DEGs were identified as signatures of HEV infection, among which 3100 genes wereupregulated and 1933 genes were downregulated. CMap analysis of these DEGs enlisted ACE inhibitors, mTOR inhibitors, PPAR antagonistsas potential drugs against HEV with good scores. GO and pathway analysis revealed enrichment of majorly metabolic processes, viral translation/gene expression, ER functions, intracellular molecular transport, proteasomal and PPAR signalling pathways. These results indicate that HEV influences host cell gene expression to favour its replication. Also, by activating metabolic pathways, ER functions, PPAR and proteasomal pathways, host cells try to overcome stressful conditions generated due to HEV infection. Metagenomics analysis of viromes of Aedes aegypti mosquitoes in India Onkar Ghuge, Abhranil Gangopadhyayya, Ashwini Ramdasi, Asmita Kamble, Surendra Kumar, Sreelakshmi P R, Sudeep Balan, S C Cherian*, K S Lole* Indian Council of Medical Research-National Institute of Virology, Pune Background: India faces a high number of mosquito-borne illnesses each year. Aedes borne dengue and chikungunya viruses have emerged as a major public health concern. Mosquitoes are known to harbor a large number of insect specific viruses (ISV) in addition to viruses of public health importance. These ISVs are highly species specific and are non-pathogenic to humans or domestic animals. However, there is a potential threat of these ISVs evolving into human pathogens by genome alterations. Systematic investigation of mosquito viromes is thus vital to have insight into the threat to public health. Objectives: To employ a next-generation sequencing to identify the viromes of Aedes aegypti mosquitoes collected from different states of India viz., Punjab, Rajasthan, Maharashtra, Karnataka, West Bengal, Assam and New Delhi. Methods: Mosquitoes after identification were pooled and processed for viral RNA isolation. cDNA synthesis was done by using random anchored primers and amplified using sequence-independent, single-primer amplification method. Libraries were prepared using standard protocols and sequenced on the MinION platform from Oxford Nanopore Technologies (ONT). Contig assembly and sequence analyses for taxonomic classification were undertaken using Epi2Me by ONT. Results and Conclusion: The generated viral metagenomic data revealed that the viral community matched by the reads was diverse and varied in abundance among different locations. Most common viral species identified were Phasi-Charoen Like Virus [PCLV] and Cell Fusing Agent Virus [CFAV]. Other common virus families represented were: Baculoviridae, Rhabdoviridae, Genomoviridae and Bunyaviridae. Overall, this study provides information on diverse insect specific viruses present in Aedes aegypti mosquitoes in different states of India. It is crucial to understand whether these viruses have ability to interfere in the transmission of viruses of medical importance. Molecular Characterization of Hepatitis A Virus Strains Circulating In India During 2010–2020 Neeta C. Thorat, Ashwini Y. Ramdasi, Prudhvi Lal Bhukya, Supriya L. Hundekar, Anuradha R. Patil, Sunil D. Shelkande, Gajanan N. Sapkal, Kavita S. Lole Indian Council of Medical Research-National Institute of Virology, Pune Background: Monitoring of circulating viral strains in country is important for keeping track of introduction of new variants and genotypes. Objectives: The aim of this study was to characterize HAV strains from different parts of India over a period of past ten years. Material and Methods: Samples collected during hepatitis outbreak investigations or referred for diagnosis to ICMR-NIV from 18 different states of India (serum samples: 250 and stool samples: 8) were processed for HAV RNA detection by using 5’-Non-coding region (NCR) based RT-PCR for hepatitis A virus (HAV) RNA detection. The HAV RNA positive samples were further processed for HAV-VP3 gene amplification. Results: HAV RNA positivity was detected in 46/268 samples, while HAV-VP3 region could be amplified from 33 HAV positive samples. Phylogenetic analysis based on both 5’NCR and VP3 gene sequences showed presence of a single genotype IIIA virus in all Indian states. High percent nucleotide identity between sequences from different states of India, i.e. 99.97% for 5’NCR sequences and 99.92% for VP3 region sequences indicated circulation of highly homogeneous strains of HAV within India. Conclusion: A retrospective analysis of HAV strains across the country showed presence of a single HAV genotype IIIA circulating in India. In silico aided mutational analysis and structure guided stabilization of Human Respiratory Syncytial Virus Fusion protein Bharatendu Singh, Radha Kanta Ratho, Gursimran Kaur Mohi Background: Human Respiratory Syncytial Virus Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. RSV F goes through conformational changes leading to major loss of immunogenic epitopes as a result of change in three dimensional structure. Objective: Introduction of point mutation in hRSV fusion protein conferring stability in itsprefusion form. Methods: Sequence of RSV Genotype A2 was retrieved from NCBI and modelled using Tr Robetta server using homology modelling. Disulphide bonds were added to the 3D model via Disulphide by design 2. Beta turns in the protein were improved via substitution at the ‘i + 2’ positions of the sequence. Mutant structure was energy minimized in YASARA Energy minimization server. Stability analysis and comparison with wild type was done in FoldX. Results: A trimeric structure was obtained. Residue positions: 155:290, 443:466 were chosen for disulphide bond addition. 3 beta turns; 146–149, 238–241, 293–296 were chosen for sequence improvement. Final force generated following energy minimization was estimated to be −5 × 104 kj/mol. ΔG during pre and post mutagenesis were found to be −100.21 kcal/mol & −200.51 kcal/mol. Conclusions: Thus in silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. Human papilloma virus (HPV) in sinonasal papillomas and squamous cell carcinomas: A PCR based study of 60 cases Dr Farhat Abbas Senior Resident, Department of Pathology, GMC, Srinagar Kashmir Introduction: A variety of non-neoplastic and neoplastic conditions involve the nasal cavity and paranasal sinuses which are commonly encountered in clinical practice. The nose and paranasal sinuses are exposed to a variety of infections, chemically irritating, antigenically stimulating, traumatic and undoubtedly many other influences and consequences of these multifaceted deleterious exposures result in the formation of tumour-like and truly neoplastic conditions. Aims and Objectives: To study histopathological pattern of sinonasal lesions and association of Human Papilloma Virus (HPV) with sinonasalpapillomas and squamous cell carcinomas. Materials and Methods: The present study was a hospital based observational study conducted over a period of three years from January, 2010 to January, 2013 in the Department of Pathology, Government Medical College, Srinagar which included 181 cases of non-neoplastic and neoplastic lesions of nasal cavity and paranasal sinuses followed by study of association of HPV with sinonasalpapillomas and squamous cell carcinomas by polymerase chain reaction (PCR). Results: Out of total 181 cases, 114 were non-neoplastic and 67 were neoplastic. Out of 67 neoplastic lesions, 46 were benign and 21 were malignant. Nasal polyp was most common among non neoplastic lesions. Inverted sinonasal papilloma was most common benign lesion and squamous cell carcinoma was most common malignant lesion seen. Out of a total of 181 sinonasal lesions 101 (55.8%) were present in males and 80 (44.2%) were present in females with male to female ratio being 1.26:1. The age range of patients with sinonasal lesions varied from 5–75 years with a mean age being 34.67 (SD ± 16.58) years. HPV positivity was seen in 4 (13.33%) out of 30 cases of inverted papillomas, 3 out of 6 (50%) exophyticpapillomas tested positive for HPV. Out of 9 squamous cell carcinomas HPV positivity was seen in 2 (22.2%) cases. Conclusion: Categorizing the sinonasal lesions according to histopathological features into various types helps us to know the clinical presentation, clinical outcome and prognosis of the disease. Also low risk HPV types 6 and 11 show an association with sinonasalpapillomas and oncogenic HPV types 16 and 18 with squamous cell carcinomas. Prefusogenic-F based VLP as vaccine candidate for respiratory syncytial virus Ahmedali Mandviwala #1, ArchanaKulkarni-Munje2, Harshad Patil*1 1Department of Communicable Diseases, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune, India. 2National Immunogenicity & Biologics Evaluation Center (NIBEC), Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be University), Pune, India Background: Respiratory syncytial virus (RSV) infection is a major cause of severe respiratorydisease in infants and young children worldwide. There is no licensed vaccine and prophylactic treatment options are limited and expensive. RSV fusion (F) glycoprotein induces effective neutralizing antibodies and is a focus for vaccine development. Studies have shown that a partially cleaved, fusion-inactive protein, termed prefusogenicF (preFG) is significantly more stable and immunogenic than both the prefusion and postfusion F structures. Along with fusion protein, glycoprotein (G) also elicits neutralizing antibodies and matrix protein (M) plays a central role in viral assembly. Objectives: To develop RSV-VLPs and evaluate immunogenicity in mice after intramuscular immunization. Materials and Methods: RSV-VLP were developed using baclulovirus expression system. VLPs assembly and characterization was confirmed by electron microscopy and western blot. Immunization studies employing intramuscular route were conducted in mice. RSV protein specific ELISA and ELISpot were performed to evaluate immunogenicity of RSV-VLP. Results and Conclusion: Characterization of RSV-VLP revealed formation of spherical structures having size of 70-100 nm having preFg, G and M proteins. VLP-immunized miceinduced-F, G and M specific IgG antibodies. IgG subtype analysis revealed induction of both IgG2a and IgG1, with predominant IgG1 response, indicating a skewed Th2 response. Higher induction of both IL4 and IFNγsecreting cells was observed in VLP immunized mice. Overall, we found that the VLPs developed are highly immunogenic. Further studies need to be performed employing adjuvants to obtain skewed Th1 response. Angiomotins play a role in ESCRT mediated Nipah virus VLP budding Sabahat Gazal 1, Zifei Pei2, Greeshma Ray2, Phuong T. Schmitt2, Anthony P. Schmitt2,3, Sundus Gazal4, Deep Shikha1, G. A. Badroo1 1Division of Veterinary Microbiology and Immunology, F.V.Sc & AH, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, R.S.Pura, India. 2Department of Veterinary and Biomedical Sciences, and 3Center for Molecular Immunology and Infectious Disease, The Pennsylvania State University, University Park, PA, USA. 4Division of Veterinary Microbiology, College of Veterinary Sciences, Guru Angad Dev Veterinary & Animal Science University, Ludhiana, Punjab, India Nipah and Hendra viruses are highly virulent, zoonotic BSL-4 agents that are responsible for causing hundreds of human fatalities throughout Asia and Australia. Henipaviruses have been found to depend on ESCRT machinery to facilitate live virus budding. In this study, we have shown that henipavirus F VLP budding is ESCRT dependent and ubiquitin dependent. We have further shown that Nedd4L, a Nedd4 family ubiquitin ligase, enhances henipavirus F VLP budding and that the ablation of ubiquitin ligase activity of Nedd4L inhibits F VLP budding. These findings provide first indication that in case of henipaviruses, Nedd4L is recruited by a viral transmembrane glycoprotein to facilitate budding in an ESCRT dependent manner. For some paramyxoviruses that lack any of the conventional late domains, a host protein named angiomotin like 1 (AmotL1) has been found to play an important role in budding by acting as a bridge between the viral M protein and Nedd4 family ubiquitin ligases. As henipaviruses also lack any of the well characterized late domains andsince our studies have shown that henipavirus F VLP budding is ESCRT and ubiquitin dependent, we further determined the role of angiomotins in henipavirus F VLP budding. Angiomotin is a family of proteins that include angiomotin (Amot) and angiomotin like proteins 1 and 2 viz. AmotL1 and AmotL2. Using CRISPR/Cas9 mediated knockout of Amot or AmotL1, we have shown that knockout of angiomotins inhibit henipavirus F VLP budding but had no significant effect on M VLP budding. Double knockout of Amot and AmotL1 resulted in near complete inhibition of F VLP budding. Overexpression of mutant AmotL1 lacking LPTY and PPXY sequences lead to inhibition of Hendra virus F VLP budding. Co-localization studies showed that GFP fused to cytoplasmic tail of Hendra virus F co-localizes with AmotL1 and also Nedd4L. We propose that henipavirus F proteins instead of directly harboring PPXY sequences, bind to PPXY containing angiomotins and thereby recruit Nedd4L and thus ESCRT in a way similar to that observed with viruses harboring a PPXY late domain. Isolation, identification, and characterization of Bacteriophage against Stenotrophomonasmaltophilia Calmly M Koshy 1, S. Shobana1* Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu, India Presenting author: [e-mail]; *Corresponding author: [e-mail] Stenotrophomonas maltophilia is a Gram-negative, non-fermentative, aerobic, motile bacterium that causes acute respiratory tract infections (RTI), soft tissue infections, necrotizing otitis, keratitis, meningitis, arthritis, bacteremia, endocarditis, peritonitis, ophthalmic diseases, and osteochondritis. Due to its innate resistance to a broad array of antibiotics such as trimethoprim/sulfamethoxazole, β-lactams, macrolides, cephalosporins, fluoroquinolones, aminoglycosides, carbapenems, chloramphenicol, tetracyclines and polymyxin treatment of S. maltophilia infections is problematic. Phage therapy, the clinical application of bacteriophages to selectively destroy-bacteria, is one potential medical solution to the present antibiotic resistance crisis. We studied the morphological features, thermal stability, pH stability, one step growth curve and chloroform stability. Transmission electron microscopy revealed the morphology of the phage which we isolated from sewage sample has a head and tail structure which belongs to Myoviridaefamily. The biological properties testing indicated that phage is stable between 30 and 60 °C for both 30 and 60 min and pH 4–11. After treatment with different concentrations of chloroform (i.e., 0%, 1%, 2%, 5% and 10% (v/v)), the phage showed pfu of 1.5, 1.1, 1, 0.5 and 0.1 respectively, indicating that the phage isolated is sensitive to chloroform or contain lipids. It has short latent periods and normal burst sizes. The DNA was isolated and the band was observed at 10 kb. Aquatic Virology (Oral Presentation) Isolation and characterization of Infectious Pancreatic Necrotic Virus (IPNV) by using Fish Cell lines Keezia Khurshid 1*, Inain Jaies1, Feroz Ahmad Shah1, Syed Shariq N. Qadiri1, Imtiyaz Qayoom2, Bilal Ahmad Bhat3, Asifa Wali1, Adnan Abubakr4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil- 190006, Ganderbal, India. 2Division of Aquatic Environmental Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil- 190006, Ganderbal, India. 3Division of Social Sciences, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil-190006, Ganderbal, India. 4Division of Fisheries Resource Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil Cell culture techniques have started to replace the traditional research paths for efficient and precise research. In fisheries science researches have been initiated for development of cell lines and their application in different fields of fisheries sector particularly in virology. Cell lines of indigenous snow trout fishes have not been developed yet and ensuing this target can be a land mark in the development of vaccines, drug development, virus isolation and toxicological studies. In this perspective study was undertaken to establish primary cell culture system from heart of Schizothorax esocinus, an indigenous cold-water fish of Indian Himalayas. Utilizing multiple FBS concentrations and temperature ranges, L-15 medium was used to standardize the primary cell culture. The explant approach, which was utilized to prepare the tissue, was discovered to be more reliable than the enzymatic dissociation method. The developed primary cell culture system of heart consistently grew on FBS concentration of 15% and the temperature of 24 °C. Karyotyping was used to characterize newly established primary cell cultures. Chromosome number of the primary cell culture system was found to be 98 which is in compliance with the chromosome number of S. esocinus. The study opens a window for development of cell culture system from schizothoracids of Indian Himalayas. Keywords: Cell lines, virus isolation, Infectious Pancreatic Necrosis, Karyotyping, FBS Viral diseases in cold water aquaculture Inain Jaies 1*, Keezia Khurshid1, Feroz A. Shah1, Syed Shariq N. Qadiri1, Imtiyaz Qayoom2, Bilal A. Bhat3, Shabir A. Dar1 and Farooz A. Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquatic Environmental Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fishery Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] One of the biggest dangers to a thriving aquaculture industry is viral infestation. The ideal temperature for reproduction differs significantly between viruses from fish and animals. While viruses that infect warm water species do best at a temperature of 20–27 °C, those that infect cold water fish prefer a temperature of 10–20 °C. This succinct review is primarily concerned with the major viral pathogens that affect the growth of coldwater fish. The virus is extremely contagious and could result in a 100% fatality rate. The primary viral infections that have been observed to affect cold water fishes are explained, along with clinical symptoms and tables identifying their general characteristics and other, a diagnosis, a treatment plan, and a list of control measures. Viral infectious diseases (VHS), Infectious Haemopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV) are among the viruses that are typically referred to cause disease in cold waters. The development of biosecurity policies and implementation of biosecurity measures, creation of vaccines, use of antiviral medications and probiotics to treat viral infections, selective breeding of disease-resistant fish, use of improved diagnostic tools, disease surveillance, and promotion of good husbandry and management techniques are some of the proposed solutions to these issues. Keywords: Virus, Aquaculture, Cold water, Infectious, Temperature. Accumulation of antiviral treatments and plans to combat fish virus Kafeela Mukhtar1*, Irifa Fayaz1*, Kaiser Farooq Wani1*, Feroz A. Shah1, Shabir Ahmad Dar1, Anayitullah Chesti2, Bilal A. Bhat3 and Irfan Ahmad Khan4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquaculture, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fish Genetics and Biotechnology, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] The implementation of antiviral treatments and prophylaxis strategies in fish aquaculture needs to be established. One of the biggest production bottlenecks in fish aquaculture continues to be the lack of commercial vaccinations for the majority of viral illnesses affecting economically important fish species, as well as the complete lack of medicines to combat these diseases once they have appeared in a fish farm. Although anti-virals are not currently used on farms, numerous studies have been done to evaluate their potential application as therapies for a number of fish viruses. Some of these papers included in vivo tests to show the possible applicability of these treatments, even though most of them were based on in vitro methods. This review highlights the research on antiviral therapies for fish viruses that has been done both in vitro and in vivo. The efforts that are emphasized use metabolites, chemicals (some of which are used in medicine to treat human viruses), bioactive substances mostly obtained from plants and microorganisms, and developing DNA- and RNA-based therapies. Using this, antiviral methods can be developed for the aquaculture sector that is both specialized and broad-spectrum. Controlling viral diseases is a significant concern when taking into account the level of aquaculture species production. How farmers may prevent and treat viral infections, as well as how fish and shellfish combat viral diseases are both areas where knowledge is lacking. The unavailability of specific therapeutic medications and of commercial vaccinations is the main causes of the difficulties in controlling viral infections in aquaculture. Chemo immuno prophylaxis forms the basis of the overall disease resistance in aquaculture. Keywords: Viral diseases, Aquaculture, Diseases control, Vaccination, Antivirals. Role of medicinal plants against viral diseases in aquaculture Kaiser Farooq Wani1*, Irifa Fayaz1, Kafeela Mukhtar 1, Feroz A. Shah1, Asifa Wali1, Anayitullah Chesti2, Bilal A. Bhat3 and Feroz Ahmad Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquaculture, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fisheries Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India Corresponding email * :[e-mail] The most severe sort of infections affecting aquatic animals is viral diseases, which also pose a significant barrier to the growth of the aquaculture sector. Viral diseases are one of the largest challenges that aquaculture sector is facing currently. Viruses are the most serious type of disease causing agents which tend to be highly contagious, rapidly disseminating, have a wide host range, and a high mortality rate. Currently, the viral diseases in aquaculture are not considered to be curable but can be prevented by taking effective controlable measures. In this regard, medicinal plants have proven to be effective against number of viral diseases like white spot syndrome virus (WSSV), infectious pancreatic necrosis (IPNV), Infectious hematopoietic necrosis (IHN), viral hemoraghic septicaemia (VHS), spring veremia of carp (SVC), viral nervous necrosis (VNN) etc. Medicinal plants and their extracts containing active pharmaceutical ingredients have proven to be effective in the treatment and prevention of viral diseases in fishes. Most of the medicinal plants work against viruses by either interfering in the pathway essential for viral replication or by expression of genes critical for immune response. Medicinal plants contain numerous active ingredients such as phenolic substances, flavonoids, alkaloids, terpenoids, pigments, starch, steroids, and essential oils, which exert an immunostimulatory, growth promoting, anti-inflamatory and anti-viral effects on fishes. They mainly acts upon the immune system of an organism to provoke effective non-specific immune response, which additionally exert an anti-viral effect. Hence, medicinal plants can be an effective alternative to anti-viral drugs to reduce the toxicity, drug resistance and residue formation in aquaculture. Keywords: Medicinal plants, antiviral, viral diseases, virus, aquaculture, active ingredients. Clostridium perfringens from fresh water fish of Kashmir Himalaya and their aquatic environment: Toxinotyping and phylogenetic analysis Mehak Hafeez Division of Fish Biotechnology Faculty of Fisheres, SKUAST-K Background: Clostridium perfringens is an important indicator of faecal contamination in aquatic habitats. Aquatic resources of Kashmir are in a serious state of eutrophication-particularly cultural, which is more pronounced in lentic ecosystems that has witnessed an increase in foodborne pathogens. This bacterium is not known to cause any disease in fish but its importance in fish is primarily due to the health hazards to humans, as C. perfringens has been found to be the causative agent of many outbreaks of food poisoning associated with fish consumption. Objective: Investigate the occurrence of different toxinotypes of C. perfringens in commonly consumed fishes of Kashmir. Materials & Methods: Collection of 510 samples (210 water; 150 each of common carp and snow trout) from 3 different lacustrine habitats (Dal, Anchar and Nigeen Lakes). Isolation, identification and toxinotyping of C. perfringens isolates via biochemical and molecular techniques. Results: 210 water samples and 80 (26.66%) of 300 fish samples tested positive on 16S rRNA assay. The multiplex-PCR confirmed that all 290 isolates from water and fish were positive for Toxinotype A, as only cpa gene was amplified. Phylogenetic analysis of the amplified gene revealed 95%–98% homology with corresponding sequences reported from China, Egypt and India. Conclusion: Documentation of C. perfringens toxinotype A in ichthyofauna of Kashmiri Himalayan lakes, entailing that fish can likely act as transmission medium for C. perfringens food poisoning to humans via food. Role of Fish Viral Vaccines in Aquaculture Industry Irtifa Fayaz1, Kafeela Qureshi1, Kaiser Farooq1, Dr. Feroz Ahmad Shah2, Dr. Syed Shariq Nazir Qadiri3, Dr. Shabir Ahmad Dar3, Dr. Asifa Wali3 1Research Scholar, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Professor and HOD, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Assistant Professor, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India Corresponding email: - [e-mail] Fish viruses that impact species in aquaculture have been the focus of extensive research now-a-days. The staggering losses resulting from viruses highlight the requirement for the creation of efficient, affordable vaccinations for aquaculture. Aquaculture is increasingly relying on vaccination as a measure of disease control since it is considered to be a practical and affordable strategy. The majority of aquaculture virus vaccines now on the market are based on inactivated viruses or recombinant subunit proteins. The majority of fish virus vaccination on the market is based on a recombinant protein or an inactivated virus. There are currently no licensed live attenuated or DNA vaccines, although a DNA vaccine against IHN (Infectious hematopoietic necrosis disease) is being studied in controlled field trials in Canada. Additionally, the majority of commercial vaccines are developed for the high-value salmonid industry and include vaccinations for diseases like IPN, ISA, IHN, and pancreatic disease (PD). Moreover, there are commercially available vaccinations for diseases that infect fish other than salmonids, including KHVD, viral nervous necrosis (VNN), and SVC, which affect sea bass and sea bream as well as carp, European catfish, and rainbow trout (that infects common carp, koi, goldfish, ornamental catfish and sturgeon among many other species). The enormity of modern aquaculture, its reliance on intensive technologies, and the prevalence of viral diseases are emphasizing the view that aquaculture is dependent on vaccinations and that vaccines will play a bigger role in the industry. Keywords: Aquaculture, Salmonids, Vaccination, Viral diseases. Emerging fish viral diseases and their effects on the economy and environment Towseef Ahmad Tantry1*, Dr. Mohammad Lateef1, Feroz Ahmad Shah2, Syed Shariq Nazir Qadri2 1Department of Zoology, Central University of Kashmir, Ganderbal - 191 201, Kashmir, J&K. 2Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology, Kashmir, Rangil-191201, Ganderbal, Jammu and Kashmir, India * [e-mail] Aquaculture, also referred to as “underwater agriculture” is the fastest food-producing sector in the world, accounting for one-third of global food production. Viral diseases cause high economic losses in aquaculture. Emerging disease epizootics frequently cause substantial, often explosive, losses among populations of fish, resulting in large economic losses in commercial aquaculture and threats to valuable stocks of wild aquatic animals. Infectious haematopoietic necrosis, viral haemorrhagic septicemia, spring viraemia of carp, infectious salmon anaemia, koi herpesvirus disease, epizootic haematopoietic necrosis and other ranavirus diseases, red sea bream iridoviral disease, viral nervous necrosis and other nodavirus diseases, are the main emerging. Emerging illnesses in aquatic animals have had a significant negative impact on local livelihoods as well as the economies of many regions and countries. Numerous individual animals have higher commercial value than others (such as koi carp), and businesses that participate in intensive aquaculture may lose market share if their production schedules are disrupted. It is estimated that the development of ISAV in Scotland in 1998–1999 lost the industry $US 35 million and caused an ongoing annual loss to the industries in Canada and Norway of $US 25 million. Over $US 15 million was reportedly spent over the first three years of KHV's inception in Indonesia [10], with continued socioeconomic effects on low-income, small-holder farmers. Emerging aquatic animal illnesses have had both direct and indirect effects on the environment. Keywords: Diseases, fish, virus. A general overview of viral diseases that impact aquaculture: An Indian perspective Ashaq Sultan Dar1*, Fayaz Ahmad1, Feroz Ahmad Shah2, Syed Shariq Nazir Qadri2 1Advanced Research Laboratory, Department of Zoology, University of Kashmir, Srinagar - 190 006, Kashmir, J&K. 2Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology, Kashmir, Rangil-191201, Ganderbal, Jammu and Kashmir, India * [e-mail] The introduction of aquaculture has marked one of the major changes in the history of food production on a global scale. Population expansion, a rise in the demand for seafood, and a losing momentum of catch levels have all contributed to the rapid growth of aquatic animal husbandry into a large global industry. Aquaculture is important to the economy of our nation as harvests from natural waters have fallen or, at best, remained flat in the majority of water bodies due to overexploitation and habitat destruction. The common aquaculture products are fish and shrimp. Aquaculture has helped to alleviate pressure on the conservation of the natural products from our major water bodies like streams, rivers, lakes, and oceans and has been a driving factor of socio-economic development for the poor coastal and rural communities, particularly in Asian nations like those of India, China, Bangladesh, Myanmar, Indonesia, and others. Pathogenic infections in fish and shrimp, which can lower the production of aquaculture products, pose the biggest threat to the aquaculture industry. One of the most damaging pathogens among the different disease-causing organisms, such as bacteria, fungi, parasites, and so forth, is the virus. The deadliest illnesses in aquatic animals are caused by viruses, which include KHV, IPNV, SVC, CCV, IHNV, ISAV, and LCDV (in fish), as well as WSSV, YHV, IHHNV, and TSV (in shrimp). The main focus of this review paper is the development and traits of Indian aquaculture, the principal viral infections of fish and shrimp, and their effects. As aquaculture develops and meets the challenges of viral infections, it also takes into account the possibility of the emergence of viral diseases in aquatic animals in the future. Keywords: Aquaculture, Viral diseases. Antibacterial activity of certain folk medicinal plants from Kashmir Himalayas used to treat microbial infections Asifa Wali*, Feroz A. Shah, Shabir Ahmad Dar and Syed Shariq Nazir Qadiri *Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of AgriculturalSciences and Technology, Kashmir, Rangil Ganderbal (J&K) India The study was conducted to find out the activity of some medicinal plants against bacterial isolates. The bacterial isolates from fish samples were investigated for in-vitro drug sensitivity by standard disc diffusion technique (Bauer et al., 1966). Cultural examination of fish samples was done by method described by Quin et al. (2004) in which predominant isolates were Staph. (16.66%), Aeromonas hydrophilla (55.55%) and other isolates as 5.5% and were subjected to in-vitro antibacterial sensitivity test to selected herbal extracts and standard antibiotic (oxytetracycline). The four different aqueous concentrations of the herbs namely levandula stoeches (Kalwuth), Nepeta cataria (Gandh soi), Borago officinalis (Kahzaban), Adianthum capillus (Gavtheer), Fumaria indica (Shahter) collected from registered herbal shops prepared by standard procedure as 25 mg/ml, 50 mg/ml, 75 mg/ml and 100 mg/ml were used. The results indicated that levandula stoeches (Kalwuth), Nepeta cataria (Gandh soi), Borago officinalis (Kahzaban) against Aeromonas hydropilla exhibited maximum zone of inhibition 20.0 ± 1.21, 21.0 ± 0.19, 13.0 ± 0.37 17.0 ± 0.21; 18 ± 0.41, 12 ± 0.21, 13 ± 0.31, 15 ± 0.31 and 15 ± 0.33, 16.01 ± 0.19 14.09 ± 0.37, 13.31 ± 0.41 at 100 mg/ml respectively which was significantly low as compared to standard drug (oxytetracycline) at 30 µg concentration. Aqueous extract of Levandula stoeches against Aeromonas hydrophilla exhibited maximum zone of inhibition 18.0 ± 0.33 and 13.0 ± 0.141 at 100 mg/ml respectively. Fumaria indica (Shahter) has shown nil to non-significant bacterial growth inhibition activity. Keywords: Antibacterial activity, disc diffusion, fish pathogens, medicinal plants. Pathological alterations induced by Saprolegnia parasitica in the skin and gills of rainbow trout of Kashmir Himalayas, India: A scanning electron microscopic study Inain Jaies1 *, Feroz A. Shah1, Syed Shariq N. Qadiri1, Imtiyaz Qayoom2, Bilal A. Bhat3, Shabir A. Dar1 and Farooz A. Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquatic Environmental Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4 Division of Fishery Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] The aim of the present investigation was to study the morphological features of Saprolegnia parasitica and its associated pathology in the gills and skin of rainbow trout (Onchorynchus mykiss). After isolation of S. parasitica from infected trout specimens, its molecular identification was done using polymerase chain reaction (PCR). To observe the pathological changes in the S. parasitica infested organs, scanning electron microscopy technique was employed. The results revealed moderate to severe degeneration in the skin epidermis, disorganized and shrunken epithelium, and prominent abrasions at the infestation site. Whereas, gill lamellae were clubbed and fused together, and appeared swollen with necrosis in severely infected specimens. Also, the prominent feature observed in all the specimens was hypertrophied secondary gill lamellae with near or completely absent interlamellar spaces. The current findings are of value in deciphering the host–pathogen interaction of S. parasitica in the infected skin and gill tissue of rainbow trout. Keywords: Scanning electron microscopy, Gill, Skin, Saprolegnia parasitica, Rainbow trout. Aquatic Virology (Poster presentation) Role of Fish Cell Lines in Isolation and Characterization of Viruses Irtifa Fayaz1, Kaiser Farooq1, Kafeela Qureshi1, Dr. Feroz Ahmad Shah2, Dr. Syed Shariq Nazir Qadiri3 1Research Scholar, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Professor and HOD, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Assistant Professor, Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India Corresponding email: - [e-mail] Cell lines are an important biological tool that have revolutionized scientific research as they are useful for conducting research in the domains of physiology, virology, pharmacology, toxicology, oncology and transgenics. RTG-2 was the first fish cell line and was used in the beginning for virological studies. Numerous fish cell lines derived from fish tissues such as spleen, heart, swim bladder, ovary, fin, liver etc. have been developed to detect and isolate fish viruses. Since some pathogenic viruses are known to be organ or tissue-specific, the development of additional cell lines from various organs and tissues of a host species is crucial for the accurate monitoring of viral diseases. The cell lines are also useful for studying species specific responses to viral infection at the cellular level. Additionally, fish cell lines are making significant contributions to research in the fields of biomedicine, toxicology, evaluation of drug metabolism and cytotoxicity, gene function study, pathology, oncology, genetic regulation, and expression, as well as DNA replication and repair. To adequately understand viral pathophysiology and host immunity, as well as viral isolation, replication, and identification, fish cell lines are essential. Since viral infections cause significant mortality rates in both freshwater and marine fish, which seriously harm aquaculture, this is likely the principal application for fish cell lines. This review aims to educate new and veteran researchers about the value of fish cell lines in scientific research, particularly virology for research and development. Keywords: Biomedicine, Cell lines, Drug metabolism, Replication, Virology. Probiotics: An efficient therapy against viral pathogens in aquaculture Inain Jaies*, Keezia Khurshid and Feroz Ahmad Shah Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil- 190006, Ganderbal, India * Corresponding author: [e-mail] Aquaculture is one of the industries with the fastest global growth. India's extensive coastal territory provides a wealth of fishing opportunities. One of the biggest hurdles to aquaculture success is infectious disease. Viruses are among the most harmful pathogens in aquaculture, and they limit the production of many aquaculture farms by causing direct fish losses, indirect costs from decreased productivity and disease management expenses, and loss of export markets due to the enforcement of trade restrictions. Viral infections restrict aquaculture production, which has an impact on the development and prosperity of the world economy. In general, viral infections in aquaculture species do not have any recognised or efficient therapies. In particular, issues with latency and probable recurrence make temperature adjustments challenging. Long-term development of alternative therapies is therefore essential. Probiotics are among the most promising feed supplements for control or treatments of fish disease including viral infestations and also to enhance immunological responses, increase disease resistance and hence serve as an antibiotic substitute. The current study summarizes the efficiency of probiotic as an efficient substitute for the treatment of viral pathogens in aquaculture. Keywords: Probiotic, Viral infestation, Loss, Antibiotic substitute, Aquaculture. Remedial measures for viral diseases in cold water aquaculture Keezia Khurshid*, Inain Jaies and Feroz Ahmad Shah Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil- 190006, Ganderbal, India The cold water aquaculture industry is a vital resource for society and the economy because it is a fast expanding sector with many new entrants each year. Viruses, bacteria, fungus, protozoa, and metazoan parasites are only a few of the infectious agents that can cause fish disorders. Viral diseases are among them, the majority of which result in significant economic losses. Additionally, viral illnesses lower fish welfare by resulting in a number of situations that are harmful to fish welfare, including decreased feed intake, aberrant swimming behaviour, predation of sick fish, and unfavourable social interaction. Viral characteristics, the environment, reservoir host, sensitivity of the farmed species, transmission dynamics and viral pathogenecity play a role in the intensification of fish disease. The majority of these criteria are unknown for aquatic viruses, preventing the implementation of the best possible management measures. The development of global and national biosecurity policies, the implementation of biosecurity measures, the creation of vaccines, the use of antiviral medications and probiotics to treat viral infections, the selective breeding of disease-resistant fish, the improvement of diagnostic tools, disease surveillance, and the promotion of good husbandry and management techniques are some of the proposed solutions to these issues. To lessen the occurrence and effects of viral infections in aquaculture, several preventive and control measures are mentioned in this review. Keywords: Cold water, Disease, Environment, Prevention, Control. Viral Diseases in Aquaculture in India Kaiser Farooq Wani1*, Irifa Fayaz1, Kafeela Mukhtar 1, Feroz A. Shah1, Asifa Wali1, Anayitullah Chesti2, Bilal A. Bhat3 and Feroz Ahmad Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquaculture, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fisheries Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India Corresponding email * :[e-mail] With the intensification in aquaculture, viral infections are causing significant loss to the overall fish production whether in the wild or in captivity. Viruses have the innate ability to exist in various forms outside of their hosts, which gives them an advantage for simple transmission and translocation that increases the likelihood of infection. More than 125 different viruses have been found in fish all across the world and more viruses are being added to the list. However, India has received very few reports of viral infections affecting fishes. As compared to parasitic and bacterial diseases, fishes in Indian waters are less susceptible to viral infections. There has not yet been a single incident of a viral disease outbreak in freshwater aquaculture in India that has resulted in significant mortality. Cyprinid Herpesvirus-2 (CyHV-2), Koi Rana Virus (KIRV), Carp Edema Virus (CEV), Megalocytiviris, and Goldfish haematopoietic virus necrosis herpes are a few of the viral diseases that have been described in ornamental fish culture. There have been occasional instances of the Tilapia Lake Virus (TILV) showing up in some caged and tank-grown tilapia populations. However, the dominating species, Indian Native Species of Carps (IMC), is unaffected by any of the viruses mentioned above, which is a promising feature for the Indian aquaculture industry. Freshwater aquaculture industry is unaffected by fish virus epidemics, contrary to reports from other parts of the world particularly marine or brackish water shrimp farming. Viral disease outbreaks have not been a source for concern in fish culture in India, except White Spot Disease, which has caused chaos on the shrimp aquaculture industry. The possible reason for this may be that the India has the Indigenous Variety of Indian Major Carps (IMCS) that are resistant to the fish viral diseases that are common in other Asian countries, or that the cultural environment is not one that encourages the viral pathogens to flourish and produce disease. Keywords: Aquaculture, viral diseases, Indian aquaculture. Emerging Viral Diseases and their Impact Kafeela Mukhtar1*, Irtifa Fayaz1*, Kaiser Farooq Wani1*, Feroz A. Shah1, Shabir Ahmad Dar1, Anayitullah Chesti2, Bilal A. Bhat3 and Irfan Ahmad Khan4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil- 190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquaculture, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fish Genetics and Biotechnology, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] Global aquaculture expansion and the transmission of viruses linked to it have led to the emergence of viral diseases that have resulted in significant economic loss. This ongoing emergence of viral diseases can also be caused by other virus factors for instance animal host factors, external conditions or anthropogenic factors, pathogen exchange between farmed and wild fish populations and high farm stocking densities. Viral hemorrhagic septicemia virus, infectious haematopoietic necrosis virus, infectious salmon anaemia virus, piscine orthoreovirus, and Tilapia lake virus are a few examples of emerging viruses in aquaculture. A loss of up to 20% of biomass throughout a production cycle potentially represents the overall economic impact of virus infections in aquaculture. Additionally, these viruses may have negative consequences on aquaculture product export and production. Only a small proportion of major fish viral diseases may be prevented with effective vaccines, thus the recommended course of action is to eradicate the disease using necessary techniques Additionally, the diversification of farmed species, the introduction of species into new farming regions, and the rising practice of poly-culture or the alternate cropping of different fish among small-holder farmers in some countries are all risk factors for disease onset. However, there is a growing understanding of the significance of newly emerging diseases in aquatic animals, and it is likely that increased regulatory oversight of aquaculture as well as the development of better diagnostic tools and surveillance strategies will help to reduce the risks of new diseases emerging in the future. This review highlights the primary viral infections of finfish and their effects, as well as the emerging viruses in aquaculture and also considers future disease emergence in aquaculture as this sector is continuously expanding. Keywords: Aquaculture, Emerging viral diseases, Finfish. Viral Hemorrhagic Septicemia: A Pathogen Creating Global Havoc Inain Jaies1*, Feroz A. Shah1, Syed Shariq N. Qadiri1, Imtiyaz Qayoom2, Bilal A. Bhat3, Shabir A. Dar1 and Farooz A. Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquatic Environmental Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fishery Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] Fish are susceptible to the deadly, extremely contagious disease known as viral hemorrhagic septicemia (VHS), which is brought on by the virus named Viral Hemorrhagic Septicaemia Virus (VHSV). A rhabdovirus from the family Rhabdoviridae and genus Novirhabdovirus serves as the aetiological agent. The first time VHSV pathogen was isolated from rainbow trout (Oncorhynchus mykiss) was in Denmark in 1962, and the second time was again in Denmark in 1965. It thereafter appeared in numerous more European nations. In many temperate regions of the Northern Hemisphere, VHSV is probably endemic in fish populations. In the Northern Hemisphere, including North America, Asia, and Europe, the VHSV virus has so far been isolated from about 80 different fish species. Exophthalmia, hyperactivity, irregular swimming, pale gills, body darkening, and skin and gill haemorrhages are some of the external symptoms of infection. The liver and kidneys are stained and enlarged from the inside. It is thought that the kidneys are a primary target of the virus since, upon histological investigation; they show severe necrosis. It results in significant economic losses for the fish farming industry since it causes substantial mortality in many freshwater and marine fish species. VHSV is sensitive to ultraviolet light, iodophores, chloroform, and formalin. Although inactivated virus vaccination is effective, commercial vaccine development is not feasible due to the high cost of virus growth. A recombinant vaccine created by cloning the viral gene for the capsid protein into Escherichia coli has recently made advances. This study will provide a deep insight on early warning signs, effects and the best control measures for this deadly pathogen. Keywords: Rhabdovirus, Denmark, VHSV, Capsid, Necrosis. Infectious Pancreatic Necrosis: A deadly fish pathogen Inain Jaies1*, Feroz A. Shah1, Syed Shariq N. Qadiri1, Imtiyaz Qayoom2, Bilal A. Bhat3, Shabir A. Dar1 and Farooz A. Bhat4 1Division of Aquatic Animal Health Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 2Division of Aquatic Environmental Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 3Division of Social Sciences, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India. 4Division of Fishery Resource Management, Faculty of Fisheries, SKUAST-K, Rangil-190006, Ganderbal, Jammu and Kashmir, India *Corresponding email: [e-mail] Due to losses in salmonid fish, notably extremely young rainbow trout (Oncorhynchus mykiss) fry and Atlantic salmon (Salmo salar), infectious pancreatic necrosis (IPN) is a disease of major concern in aquaculture, especially among salmonid farmers. The IPN virus (IPNV) belongs to the family Birnaviridae and is a bi-segmented double-stranded RNA (dsRNA) virus that codes for 5 viral proteins. Clinical symptoms include anorexia, stomach enlargement, irregular swimming, skin discoloration, and faeces trailing from the vent. Internal damage (viral necrosis) to the pancreas and thick mucus in the intestines are frequently found after necropsy. The destruction of exocrine pancreatic acinar tissue, whether focal or widespread, is the only consistent histopathological hallmark of this illness. Infected cells burst, releasing zymogen granules, which results in necrotic alterations. By limiting conditions that encourage physiological stress (e.g., high density, inappropriate feeding protocols, poor hygiene), the prevalence of acute IPN can so be decreased. In addition to suggesting effective preventive strategies, this review gives a brief explanation of the disease, host defences, and the molecular structure and function of the virus and its viral components. Keywords: IPNV, Rainbow trout, Aciner tissue, Salmon, Host defence. Emerging and Re-emerging Viral Diseases in Animals Keezia Khurshid Division of Aquatic Animal Health Management, Faculty of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Rangil- 190006, Ganderbal, India India, being a country of extreme geo-climatic diversity, faces a constant threat of emerging and re-emerging viral infections in livestock of both zoonotic and non-zoonotic importance. Infectious diseases remain as the major causes of animal morbidity and mortality leading to significant economic loss in livestock sector in India. The country has experienced the outbreaks and epidemics of many infectious diseases recent being the Lumpy Skin Infection. Several pathogens have emerged in livestock, and some are becoming increasingly important. African swine fever virus (ASFV), swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV), bluetongue virus (BTV), lumpy skin disease virus (LSDV), and H5N8 avian influenza cause diseases of economic importance in livestock. Some emerging viruses, such as porcine hepatitis E virus (swine HEV), porcine endogenous retrovirus (PERV), Hendra virus (HeV), or porcine sapovirus (porcine SaV), are potential public health threats. Emergence of new serotypes and variant forms of viruses as in the case of blue tongue virus, avian infectious bronchitis virus, Newcastle disease virus adds additional level of complexity. There is a need for strengthening disease surveillance in the country focusing on the epidemiology and disease burden. The important challenges faced in the control and prevention of emerging and re-emerging infectious diseases range from understanding the impact of factors that are necessary for the emergence, to development of strengthened surveillance systems. Keywords: Disease surveillance, Emerging, Zoonosis, Livestock. Plant Virology (Oral) Versatile applications of CRISPR-Cas technology in plant virology Anirban Roy, Sunil Kumar Mukherjee and Bikash Mandal Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi Email: [e-mail] Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) is a prokaryotic adaptive immune system which has been reprogrammed for its adaptability into a precise, simple, and efficient gene editing technology. This emerging technology is being utilized to harness benefits in the field of biological sciences. In the field of plant virology, the technology is being exploited to engineer plant virus resistance by directly targeting single or multiple genes (or non-coding region) in the viral DNA or RNA, as well as targeting host factors essential for virus establishment. In addition, recombinant polymerase amplification (RPA) or loop mediated isothermal amplification (LAMP) assisted CRISPR/Cas technology allowed rapid, sensitive, specific on-site detection of plant viruses. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells and such virus-induced genome editing (VIGE) has emerged as a powerful method to understand functional role of any host gene in a simplified process without a transgenic route. To target DNA virus, we have developed a multiplexed CRISPR-Cas9 based strategy to inhibit chilli leaf curl virus (ChiLCV), which showed transient delivery of the CRISPR-Cas9 modules targeting multiple genes of the virus can reduce viral accumulation without escape mutant formation and limit symptom development. In case of RNA viruses, we have targeted VPg gene of papaya ringspot virus (PRSV) using a CRISPR-Cas13a module that showed negligible virus accumulation and no symptoms in the treated plants. In a collaborative effort, we have targeted eIF4E (iso) gene of papaya plant to impart resistance against PRSV. On the diagnosis front we have developed a RPA assisted CRISPR-Cas12a based DNA endonuclease targeted CRISPR trans reporter (DETECTR) system that could efficiently detect ChiLCV from crude leaf extract within 45 min. For functional characterization of host genes, we have developed a V2 gene deleted croton yellow vein mosaic virus based vector that can deliver a gRNA module against phytoene desaturase gene of host that resulted into photo bleaching phenotype. CRISPR-Cas technology thus can be applied in multiple domains in the field of plant virology. Decrypting Viruses: lessons from structural studies Dr. Sangita Venkataraman Assistant professor (UGC-FRP), Department of Biotechnology, Anna University, Chennai – 600025 Structural investigations of the viral proteins help uncover vital details regarding their modes of entry, replication, assembly, movement, and transmission. The data is invaluable in understanding the molecular function of individual proteins and their coordination in facilitating the viral life cycle. In silico reconstruction of the capsid of banana bunchy top virus aided the understanding of the quaternary organisation of subunits in the capsid and the location of the aphid binding EAG motif that is crucial in vector-mediated transmission. The coat protein (CP) structure of sesbania mosaic virus (SeMV) revealed key regions that are responsible for its assembly. The data was used to develop genetically engineered SeMV-based viral nanoparticles and identify possible interacting partners within the host. The human adenovirus structure identified the positions of minor and major proteins in the capsid and unearthed the mechanism of interactions of the fibre knob with its receptor. The structural data of the RNA dependent RNA polymerase from different animal viruses indicated the possibility of using NADPH-based derivatives as broad-spectrum inhibitors and helped in understanding the molecular phylogeny of animal viruses. Therefore, structural studies in virology are indispensable to not only providing a molecular view of viral biology but also combating the viral pathogens. Emergence of whitefly transmitted geminiviruses: a major threat to cucurbits production Mohammad Ansar and Vikash Kumar Department of Plant Pathology, Bihar Agricultural University, Sabour-813 210, Bhagalpur, Bihar Correspondence: [e-mail] Whitefly transmitted geminiviruses are a serious problem and limiting factor for agricultural crops throughout the world. They are mostly prevalent in the tropical and subtropical regions infecting dicot plants including cucurbits. A diverse range of symptoms like curling, mosaic, mottling and puckering were observed on different cucurbit plants in Bihar state. Primarily it seems the infection of Cucumber mosaic virus and Potato virus Y, but in PCR assay it was found negative. Symptomatic plant DNA samples depicted the clear amplification with whitefly transmitted geminivirus specific primers in PCR. Subsequently, full-length genome of virus was amplified through Rolling Circle Amplification (RCA) and sequenced. Both the DNA-A and DNA-B with a genome organization typical of Old World bipartite begomoviruses observed. Both parts having nonanucleotide and conserved inverted repeat sequences likely to form a stem-loop. The genomic sequence of DNA-A and DNA-B shared more than 91% identity with Tomato leaf curl New Delhi virus (ToLCNDV). Moreover, a few cucurbit plants also indicated the presence of a mono partite Begomovirus species e.g., Tomato leaf curl Joydebpur virus (ToLCJV) along with betasatellite. The nucleotide identity of ToLCJV (DNA-A) ranges between 91–96 with available sequences in the NCBI database. In PCR assay various cucurbits e.g., bottle gourd, pumpkin, sponge gourd ridge gourd, bitter gourd, watermelon were screened with both virus specific primers among them ToLCNDV was found most common. Moreover, the virus also confirmed in tomato, chilli and a seasonal weed nightshade. Additionally, intergenic region of different cucurbits, tomato and nightshade produced similar nucleotide upon sequencing. Whitefly-mediated transmission successfully attempted from tomato to cucurbits and nightshade plants. After developing evident symptoms, it inversely transmitted to tomato plants which produced typical symptom of virus. The information generated on sequence data of virus from different hosts and vector transmission, the present investigation explored that tomato and nightshade serve as viral inoculum and developing epidemics in various cucurbits. It also seems an adaptation of viruses is taking place from solanaceous to cucurbitaceous plants therefore a sustainable approach must be developed to manage the crops. Biological and molecular characterization of begomoviruses infecting cucurbits in south India R. Rajeshwari1 and M. Krishna Reddy2 1Dept. of Plant Pathology, College of Horticulture (University of Horticulture Sciences, Bagalkot), UHS Campus, GKVK, Bengaluru 560 065, Karnataka, India. 2Division of Plant Protection, Indian Institute of Horticultural Sciences, Hessarghatta, Karnataka, India Corresponding Author: Dr. Rajeshwari, R., Assistant Professor, College of Horticulture, UHS Campus, GKVK, Bengaluru 560 065, Karnataka, E-mail: [e-mail], Phone: + 91- 9880606319 Background: Cucurbits are grown in diverse climatic conditions throughout India. Yellow mosaic and leaf curl disease on cucurbits was observed in epidemic form in Southern parts of India and have been implicated as the major factor contributing to low yields. Begomoviruses vectored by whitefly, Bemisiatabacicaused yellow mosaic disease in cucurbits. The frequency of spread of begomovirus is enhancing posing serious threat to cultivation of cucurbits. Hence, the present study was conducted with the following objectives. Objectives: Survey, collection and identification of cucurbit begomoviruses and its vector, B. tabaci, development of diagnostic techniques, biological and molecular characterization of cucurbit begomovirus isolates and detection of secondaryendosymbionts associated with B. tabaci. Material and Methods: Field survey was conducted, nucleic acid assays of begomovirus strains and comparison of relative transmission rates were performed. Begomovirus DNA A and DNA B fragments were amplified by rolling circle amplification. Phylogenetic analysis using maximum parsimony approach and analysis of discrete recombinant through RDP analysis was performed. Presence of secondary B. tabaci endosymbionts in whitefly populations were determined. Results: The symptoms of yellowing, mosaic, leaf curl and crumpling symptoms were recorded from cucurbit growing districts of Karnataka, Andhra Pradesh, Tamil Nadu and Kerala. The yellow mosaic disease incidence on cucurbits in South India ranged from 10–100 per cent. Virus vector relationships revealed acquisition and inoculation access periods of 15 min and 10 min, respectively. The hosts of cucurbit begomovirus were restricted to ridge gourd, bitter gourd, pumpkin, cucumber, bottle gourd and snake gourd whereas tomato and tobacco were non hosts. The nucleotide sequence analysis revealed the association of Tomato leaf curl new delhi virus (ToLCNDV) and Squash leaf curl china virus (SLCCNV) isolates with 94% and 95% nucleotide identity, respectively with recombination sites. Species specific primers, ToLCNDV425F/ToLCNDV1435R and SLCCNVCPF/SLCCNVCPR for ToLCNDV and SLCCNV, respectively were designed for detection. Analysis of the nucleotide sequences showed the presence of secondary endosymbionts of B. tabaci like, Arsenophonus, Cardinium and Wolbachia in different populations of B. tabaci. Conclusion: Our study describes incidence and occurrence of new emerging recombined isolates of begomovirus on cucurbits in South India causing disease epidemics with broader host range and co-infection status by secondary symbionts in whiteflies. Keywords: begomovirus, cucurbits, endosymbionts, B. tabaci. Lateral flow immunoassay based detection of five flexuous filamentous virus particles Yogita Maheshwari1, Vijayanandraj Selvaraj1, Swati Bhuria1, Bikash Mandal2, Raymond Yokomi3 1Plant Molecular Virology Laboratory, CSIR - National Botanical Research Institute, Lucknow, Uttar Pradesh, India. 2Advanced Center for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India. 3Crop Diseases, Pests, and Genetics Research Unit, San Joaquin Valley Agricultural Sciences Center, USDA Agricultural Research Service, Parlier, CA Plant viruses are major constraints worldwide in various economically important crops. Rapid and highly sensitive diagnostic tools are required for accurate diagnosis of plant viruses, which helps in effective management, biosecurity to the imported and exported planting materials. Antigens and antibodies are key reagent for serodiagnosis of viruses. Traditional methods of diagnosis such as Enzyme linked Immunosorbent assay and reverse transcription polymerase chain reactions are cumbersome methods. The lateral flow immunoassay (LFIA) strips offered a cost-effective tool to detect viruses in the field or nursery by non-skilled personnel without laboratory equipment. We have successfully developed LFIA for five flexuous filamentous viruses (Large cardamom chirkey virus, Citrus tristeza virus, Papaya ringspot virus, Potato virus S and Potato virus Y) majorly infecting cardamom, citrus, papaya and potato crops worldwide. The LFIA detected virus within 10 min and showed sensitivity up to a 1:40 dilution of crude plant sap extract. The LFIA was free from false positive as no visible test line was developed with healthy samples. The immunostrip readily detected virus after six months of storage in a sealed bag with silica gel at room temperature. The results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. Molecular Detection of the Association of Pepper vain yellows virus and Cucurbit chlorotic yellows virus with Cumin and Lettuce hosts: Emerging Crinivirus and Polerovirus in India Ashwini Kumar1*, Shakshi Choudhary1, Yvonne A. Lyngdoh2, Girdhari L. Kumawat3, Virendra K. Baranwal1, Rakesh K. Jain1, Y.B. Basavaraj1 1Advance Center for Plant Virology, Division of Plant Pathology, and 2Division of Vegetable Science, ICAR-Indian Agricultural Research Institute, New Delhi, India. 3ICAR- AICRP on Spices, Department of Plant Breeding and Genetics, SKN College of Agriculture, Jobner, Rajasthan Background: In India, recently, the occurrence of crini- and poleroviruses have been recorded to infect some cucurbit hosts. Their occurrence was hypothesized in the other hosts as well. Objective: To investigate the possible occurrence of emerging crini- and polero-viruses in lettuce and cumin hosts. Material and Methods: Symptomatic cumin and lettuceplants collectedfrom Bikaner (Rajasthan) and New Delhi were subjected to leaf-dip transmission electron microscopy (TEM). To establish the association of said viruses, two-step RT-PCR was performed using generic primers (CriniPol-F/CriniPol-R for crinivirus and Pol‐G‐F/Pol‐G‐R for polerovirus). Further characterization was performed based on coat protein (CP) gene (~ 700 bp) as well as coat protein minor (CPm) gene (~ 1.4 kb) of crinivirus, and only CP gene (~ 621 bp) ofpolerovirus. Results and Conclusions: Isometric (~ 30 nm) and long flexuous (~ 850 nm) virions suggestive of crini- and polero-virus were found associated with lettuce and cumin respectively, in TEM. RT-PCR with generic primers yielded the amplicons of ~ 1100 bp and ~ 550 bp targeting of RdRp region of respective viruses. Cloning, sequencing and analyses suggested the association of pepper vain yellows virus (PeVYV) and cucurbit chlorotic yellows virus (CCYV) with cumin and lettuce, respectively. Further, the RT-PCR using specific primers yielded the amplicons of CP gene (~ 621 bp) of polerovirus, and CP (~ 700 bp) as well as CPm (~ 1400 bp) genes of crinivirus. The resultant sequences of CP from cumin sample (621 bp) shared 97% amino acid identity with the Indian chili isolates of PeVYV. While, the CP (700 bp) &CPm (1400 bp) sequences of lettuce sample shared upto 99% and 100% identities with the corresponding sequences of Indian cucumber and pumpkin isolates of CCYV. Subsequently, in the phylogenetic analysis the CP sequence from cumin sample got clustered with that of Indian chilli isolate of PeVYV, while the CP and CPm sequences from lettuce sample got clustered with that of Indian cucumber and pumpkin isolates. The results of this study report the emergence of PeVYV and CCYV in cumin and in lettuce hosts, respectively, for the first time in the world. Further, these findings ring the alarm to take up the necessary steps to contain these viruses to the other hosts/locations and to develop the strategies for their management. Quantitative evaluation of leaf curl disease in Ty-gene introgressed donor lines of tomato using a newly developed scoring scale and indexing of naturally occurring begomoviruses infecting them Firoz Mondal1, Shipra Saxena1, Zakir Hussain2, Sunil Kumar Mukherjee1, Bikash Mandal1 and Anirban Roy1* 1Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi. 2Division of Vegetable Science, Indian Agricultural Research Institute, New Delhi *Email: [e-mail] Tomato leaf curl disease is caused by a number ofbegomoviruses. Till date, six resistance loci (Ty-gene) have been identified in different wild relatives of Solanum lycopersicon and have been introgressed into cultivated species of tomato for their utilization as donor lines in resistance breeding programme. In this study, seventeen Ty-gene introgressed tomato donor lines were evaluated against tomato leaf curl disease under field condition using a disease scoring scale, which was developed by taking into account of both percent disease incidence and grade of severity of different categories of symptoms. Five genotypes, with Ty-2 and Ty-3 genes, showed highly resistance (HR) response under field conditions in two consecutive years. Amongst them, resistance behaviour of three HR genotypes was further confirmed under controlled conditions through viruliferous whitefly inoculation using tomato leaf curl New Delhi virus (ToLCNDV), the most predominant begomovirus of tomato crop in India. However, PCR based detection using eight begomovirus species specific primer sets and one genus specific primer set, revealed that ToLCNDV was present in only few genotypes in both the years and mixed infection of multiple begomoviruses was frequently observed in those genotypes. It was also interesting to notice that majority of the genotypes (thirteen) during the first year of evaluation showed presence of croton yellow vein mosaic virus (CYVMV) followed by tomato leaf curl Karnataka virus (ToLCKV) (twelve genotypes). In the second year also, ToLCKV was detected in all the five HR genotypes. Molecular characterization of the begomovirus that had amplified with ToLCKV specific primers was confirmed to be a strain of ToLCKV. Infectivity of the characterized ToLCKV strain was established on natural host (tomato) along with in the experimental host Nicotiana benthamianathrough agro-inoculation of the infectious construct developed in this study. Complete genome sequence of the other begomovirus that had amplified with CYVMV primers, revealed that this is a new species of begomovirus and tentatively named as Tomato leaf curl Ty Pusa virus. Its evolution was suspected to be due to an interspecific recombination between CYVMV and tobacco curly shoot virus (TbCSV). The study thus suggested that the introduction of Ty-gene(s) might have changed the population dynamics of the begomoviruses infecting those lines and such altered virus accumulation scenario may pose further challenges in resistance breeding in tomato. Next-Generation Sequencing based virome profiling enabled detection of a novel polerovirusand a new crinivirus in the naturally infected pumpkin plants Sudeep Adhikari1*, Y. B., Basavaraj1, Ashwini Kumar1, Bichhinna Maitri Raut2, Ram Mohan1, Shiksha Bhandari, Bikash Mandal1, Anirban Roy1 and Rakesh Kumar Jain1 1Advanced Centre for Plant Virology (ACPV), Division of Plant Pathology; and 2Division of Vegetable Sciences, Indian Agricultural Research Institute, New Delhi, India *E-mail address of presenting author: [e-mail] Cucurbitaceae, an important crop family, is affected, worldwide, by more than 200 diseases, of which, about 70 are incited by viruses causing enormous yield losses. In the recent years, novel, emerging and re-emerging viral infections have spiked due to the ever-changing climate as well as open global trade and travel, leading to the possibility of introducing such viruses in the newer locations. The traditional virus detection tools like ELISA, PCR, hybridization assays, etc., that depend on prior knowledge of virus genome sequences or specific antibodies, are able to detect only the known viruses, while, the novel and highly divergent pathogens are not easily detected. Very recently, the occurrence of a member each belonging to the genera Polerovirus and Crinivirus viz. cucurbit aphid-borne yellows virus (CABYV) and cucurbit chlorotic yellows virus (CCYV), known as the emerging plant viruses globally, were reported infecting few hosts in India through conventional methods. However, the occurrence of theother species of aforesaid genera cannot be ruled out. So, in this study, the Next-GenerationSequencing (NGS; deep sequencing) of mRNAome and sRNAome was performed for viromeprofiling to detect the possible association of hitherto undetected or novel virus(es) withnaturally infected field-grown pumpkin plants cultivated in the experimental fields of IndianAgricultural Research Institute, New Delhi, India. NGS data revealed the presence of threeDNA viruses (Begomovirus), three RNA viruses (Crinivirus and Polerovirus) and a viroid (genusPospiviroid). Further, in-silico genomic analysis transpired the presence of an aphid-transmitted polerovirus as a novel virus species in the world named tentatively as pumpkinyellows virus (PuYV). Besides, a known species of whitefly-transmitted crinivirus, cucurbityellow stunting disorder virus (CYSDV) was also detected, and was its first occurrence in India. While, the detected begomoviruses include previously documented squash leaf curl Chinavirus (SLCCV), tomato leaf curl Palampur virus (ToLCPalV) and an alpha satellite of tomato leafcurl New Delhi virus (ToLCNDV). The association of these viruses known, novel and newviruses was confirmed through PCR assays by designing virus species-specific primers in thepreserved parts of deep-sequenced source sample. These results were further validated forthe natural/biological association of aforesaid viruses through electron microscopy and PCRassays in another set of fresh field-grown pumpkin samples collected at different timeintervals to know the temporal variation in the occurrence of those viruses. Further, toexamine the occurrence of identified viruses in the other cucurbit hosts, the bitter gourdsamples were tested and found this as a new host for SLCCV in India. In addition, the biologicalassociation/infectivity was confirmed under glasshouse conditions through virus-transmission assays vectored by whiteflies (Bemisiatabacci) for begomo- and crini-viruses, and aphids (myzuspersicae) for the polerovirus. Presence of those viruses in the artificiallyfed viruliferous vector bodies and virus-transmitted symptomatic seedlings was confirmedthrough PCR assays. The results of this investigation suggest that the crop plants grown in India are harbouring several novel, hitherto undetected viruses and the NGS-based deepsequencing would be a better choice to detect them in a quick and economic way. Development and Field Validation of Robust Diagnostics for Emerging Virusesin Horticultural Crops in North East India Susheel Kumar Sharma1*, Nitika Gupta1, K. Sarad Devi2, Damini Diksha1, Nishant Sirivastva1, S.S. Roy2, and A. Ratankumar Singh2and V.K. Baranwal.1 1Advacnced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Institute, New Delhi 110012. 2ICAR Research Complex for NEH Region, Manipur Centre, Lamphelpat, Imphal-795004 *Corresponding Author: Susheel Kumar Sharma ([e-mail]) Viruseshave emerged as the major constraint in production and productivity of horticultural crops in North East region of India. Present studyfocused on development and validation of simplified and robust detection systems for major and emerging viruses infecting diverse horticultural crops in the region. Simplified multiplex diagnostics were developed for the simultaneous detection of chilli veinal mottle virus and cucumber mosaic virus infecting chilli in North East India. A simplified template preparation using crude sap extract of plants in isothermal recombinase polymerase amplification (RPA) was developed for the detection of potyviruses infecting passion fruit and chilli. Similarly the robust RPA assay for detection of citrus tristeza virus were developed using crude sap of infected citrus plant as template. Crude sap from infected citrus leaves extracted in simple buffers was best suited for the simplified detection of these virus and virus-like pathogens. Developed RPA assay could detection the target pathogens up to 10–7 of crude sap dilution and was as sensitive as bench mark PCR. These assays were validated using large number of field samples and found highly robust. Developed detection assays will have applications in routine indexing and production of virus-free planting materials in the region. Detection of four viruses infecting apple plants by DAS-ELISA and multiplex RT-PCR in India Mehraj D. Shah, Mushtaq Ahmad, Parvaiz A Sheikh, Sumiah Wani, Sumaira Hamid and B. A. Padder Plant Virology and Molecular Pathology Laboratory, Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, J&K, India [e-mail] Apple trees are well characterized natural hosts for economically important viruses worldwide including Jammu and Kashmir State of Indian subcontinent. The current study took the advantage of hybrid approach involving both serological (DAS-ELISA) and nucleic acid based molecular (multiplex RT-PCR with an internal control) detection methods to detect simultaneously four distinct apple viruses viz., apple mosaic virus (ApMV), apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV) and apple stem grooving virus (ASGV) from samples collected from top-tier apple growing districts of Jammu and Kashmir state. Survey was carried out from 2010 onwards for the collection of 2568 apple samples suspected to be infected with viruses from various districts. Out of these, 726 samples were found to be infected with four viruses singly or in combination and distributed throughout the different apple growing districts of the State. ApMV, ACLSV, ASPV and ASGV showed an incidence of 8.06, 5.92, 9.38, and 4.91, respectively. In most of the infections, two or more viruses were detected. The most frequent virus combinations were ApMV + ASPV with an incidence of 4.21 followed by ACLSV + ASPV (2.53), ApMV + ACLSV (2.38), ASPV + ASGV (1.87). The incidence of combination ApMV + ACLSV + ASPV + ASGV was found to be lowest (0.51). This work is the first extensive survey conducted for monitoring four distinct apple viruses which provides rigorous details regarding apple virus distribution and management in the Kashmir valley. Keywords: Apple, Viruses, ApMV, ACLSV, ASPV, ASGV, ELISA, Serology, RT-PCR. Recurrent emergence of cucumber mosaic virus in banana: Molecular characterization, detection, and proposed management strategies Selvarajan. R Principal Scientist, Molecular Virology Lab, ICAR-National Research centre for Banana, Thogamalai Road, Thayanur Post, Tiruchirapalli-620102, India *Corresponding Author: [e-mail] Cucumber mosaic virus (CMV) is one of the most significant constraints to banana production, and it has been found in most of banana-cultivating countries across the world. CMV causing infectious chlorosis disease in banana. CMV is the type member of the genus Cucumovirus and consists of three positive sense single stranded genomic RNAs It is transmitted primarily by infected planting material, mechanically by sap and secondarily spread is through aphid vectors in a non-persistent manner. In banana, infectious chlorosis caused by CMV has re-emerged in an epidemic proportion during July–August 2020, in Jalgaon, Maharashtra and Burhanpur, Madhya Pradesh. Subsequently in 2021, 2022 during the same seasons the same CMV disease incidence were recorded to be very high and almost 50–100 lakhs infected plants were uprooted by the farmers at the second or third month after planting. The incidence ranged from 11 to 100% in many plantations. The complete genome sequence of CMV infecting banana isolates from Jalagon and Burhanpur have been characterized and these isolates were grouped into subgroup IB. The farmers are adopting eradication of infected plants and spraying systemic insecticides to kill the aphid vector to contain the spread besides removal of weeds. In this talk the detection methods being adopted by the ATLs, other sensitive methods available for indexing and management strategies to eradicate the disease in the region are discussed in detail. Major and emerging transboundary diseases of tropical maize affecting livelihood and food security in Sub-Saharan Africa Suresh L.M., Yoseph Beyene, Manje Gowda, Dan Makumbi, Dagne Wegary, Thokozile Ndhlela, Tekelewold Adefris and B.M. Prasanna International Maize and Wheat Improvement Center (CIMMYT), ICRAF Campus, UN Avenue, Gigiri, PO Box 1041-00621, Nairobi, Kenya Maize (Zea mays L.) is the most important cereal crop in sub-Saharan Africa (SSA), covering over 35 million ha, largely in smallholder farming systems that produce over 70 million metric tons (MMT) of grain. Maize production in sub-Saharan Africa is affected by a wide array of diseases. Environmental conditions prevalent in the different agro-ecological zones are conducive to the growth and spread of pathogens. There are many fungal and viral diseases have been affecting the maize crop and its productivity. Diseases often reduce production and cause up to 100% yield loss under severe epidemics depending on environmental conditions. The key diseases in sub-Saharan Africa affecting the crop production are Turcicum leaf blight (TLB), Gray leaf spot (GLS), Common rust (CR), fusarium ear rot (FER) caused due to mixture of pathogens, Maize streak Virus (MSV), and Maize Lethal Necrosis disease (MLN). Maize Lethal Necrosis (MLN) disease first appeared in Kenya in 2011 and became a major threat to maize production in eastern Africa in subsequent years. In eastern Africa, MLN is caused mainly by synergistic interaction between two viruses, Maize Chlorotic Mottle Virus (MCMV) and Sugarcane Mosaic Virus (SCMV). MLN can cause up to 100% yield loss in susceptible maize varieties. The disease poses a complex challenge as the MLN-causing viruses are transmitted by insect vectors, and also through contamination of the seed, especially by MCMV. CIMMYT implemented a multipronged strategy in partnership with several international and national partners to tackle the MLN challenge. These efforts included: a) establishing a state-of-the-art MLN Screening Facility in partnership with Kenya Agriculture and Livestock Research Organization (KALRO) in Naivasha for identifying sources of resistance to MLN, MCMV and SCMV under artificial inoculation; b) accelerated breeding and deployment of MLN-tolerant/resistant maize varieties with other relevant traits preferred by African smallholders; c) optimizing MLN diagnostic protocols; c) strengthening capacities of national plant protection organizations (NPPOs) across sub-Saharan Africa on MLN diagnostics, monitoring and surveillance system; d) creating awareness among the maize seed sector institutions on SOPs for producing and exchanging MLN-free commercial seed; e) disseminating information on farming practices for minimizing MLN incidence; e) establishing an MLN Phystosanitary Community of Practice involving various stakeholders, including national plant protection organizations (NPPOs), seed companies, regional/sub-regional organizations, etc.; and f) probing the epidemiology of the disease, especially the factors underlying seed contamination by MCMV. These comprehensive efforts have led not only in preventing the further spread of MLN into other major maize-growing countries in sub-Saharan Africa, especially southern and West Africa, but also minimized the incidence of the disease in the MLN-endemic countries in eastern Africa. Poleroviruses: emerging plant RNA viruses in the horticultural crops in India Basavaraj Y.B.*1, Ashwini Kumar1, SudeepAdhikari1, Bichhinna Maitri Rout2, Baljeet Kaur1, Girdhari Lal Kumawat3, Malkhan Singh Gurjar1and Rakesh Kumar Jain1 1Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, India. 2Division of Vegetable Sciences, ICAR-Indian Agricultural Research Institute, New Delhi, India. 3Department of Breeding & Genetics, SKN College of Agriculture, SKN Agricultural University, Jobner-Jaipur, India The genus Polerovirus is currently ascribed to the family Solemoviridae. So far, 26 Polerovirus species infecting several important crops have been documented worldwide. Of all, the cucurbit-infecting ones are rapidly emerging at the global level, including in India. The first cucurbit-infecting polerovirus, cucurbit aphid-borne yellows virus (CABYV), was recorded in the world from France in 1988 infecting cucumber, melon, and zucchini squash plants. Subsequently, this has been documented worldwide infecting over 16 cucurbit crops and five weed species within a span of 30 years from its first documentation, indicating its rapid global distribution and hence have gained the status as an “emerging virus”. Globally, eight species have been documented to cause economically important diseases in cucurbit crops through very confusing symptoms with nutrient deficiencies. Besides, they cause severe yellowing, downward curling and leathery leaf symptoms thereby affecting chlorophyll synthesis and reduced yields followed by fruit abortion and significantly reduced fruit set. These viruses are mostly transmitted via aphid vector (green peach aphid: myzuspersicae) in a circulative, nonpropagative but persistent manner. In the recent past, the incidences of cucurbit infecting poleroviruses have been increasing worldwide in different hosts and geographic locations causing 10 to 100% crop losses in several cucurbits. The occurrence of these viruses was not known in India until the first report of CABYV in 2017 inbitter gourd, ivy gourd, and spiny gourd. Later, a novel polerovirus, pumpkin yellows virus (PuYV) has also been reported in 2021 to infect pumpkin plants in India. In the same year, the melon aphid-borne yellow virus (MABYV) was also documented infecting ivy gourd. So far, they are reported to infect five cucurbit species in the country. Besides, hitherto solanaceous crop-infecting polerovirus elsewhere in the world, pepper vein yellows virus (PeVYV), has also been found infecting cumin plants in India by inciting severe mosaic, stunting and leaf deformation symptoms. However, their impact on different crops has remained to be estimated in India. Currently, a total of eight Polerovirus species are known, globally, to infect cucurbitaceous hosts and three in India. Looking at the occurrence ofa few poleroviruses in India on the limited number of hosts, the possible occurrence of other Polerovirus species cannot be ruled out on several other crop species. As these viruses are known globally as emerging plant viruses and causing devastating symptoms and yield losses, the available scientific data necessitates our preparedness to take up the research works for holistic virome profiling through advanced Next Generation Sequencing (NGS), development of simple, rapid & specific diagnostics as well as the strategies to protect vulnerable crops before being hit by these viruses. MAMP molecules of B. velezensis VB7 reprogramme immune response for the management of GBNV in tomato Nakkeeran S1*., Vanthana, M1., Malathi V G1., and Renukadevi P.1 1Professor (Plant Pathology), Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641003, India Email: *[e-mail] The present investigation insights the reprogramming of plant defense systems upon interaction with MAMP (Microbe Associated Molecular Pattern) genes viz flagellin (Flg), elongation factor (EF-Tu), cold shock protein and superoxide dismutase of Bacillus velezensis VB7 and groundnut bud necrosis virus (GBNV) in tomato (PKM1). The agrodrenched plants with MAMP genes, flagellin and elongation factor of B. velezensis VB7 induced the transient expression of flagellin and elongation factor. MAMP genes under 35S promoter in plant system using qPCR induced 1.45 to 1.99 fold expression of Ag-Ba.Flg and Ag-Ba.EF-Tu on 0 to 3rd day after treatment. On 9th day, the expression level declined upto 0.64–0.84 folds. The GBNV symptom severity in bioagent treated plants was reduced compared to the inoculated control. It directly reflected on the decrease on virus titre using DAC-ELISA compared to the inoculated control. Investigation on defense gene expression during tritrophic interaction of tomato plant, bioagents and GBNV, upregulated MAPKK1, WRKY33B, PR1, PAL, PPO genes, whereas gene regulation of LOX1, NPR1, JAR1, MYC2 and PDF1.2 was inconsistent. The synergistic action of bacterial cells of B. velezensis VB7 and B. licheniformis Soya 1 suppressed GBNV in cowpea compared to GBNV inoculated control. Further the field study revealed that combined application of B. velezensis VB7 and B. licheniformis Soya1 through seed treatment, soil and foliar application on to tomato hybrid, reduced the incidence of GBNV upto 10.54% compared to 28.43% in untreated control. Besides, it also improved plant growth and yield upto 21.13 tonnes/ha compared to 16.02 tonnes/ha in untreated control. Keywords: MAMP genes; Bacillus velezensis VB7; groundnut bud necrosis virus; qRT-PCR; plant growth promotion, local lesion host. Regulatory Roles of Specific MicroRNAs during Sri Lankan Cassava Mosaic Virus infection in Cassava (Manihot esculenta Crantz) Sumayya, M and T. Makeshkumar ICAR- Central Tuber Crops Research Institute, Thiruvananthapuram [e-mail]; [e-mail] Cassava is an important food security crop across globe and is susceptible to several begomoviruses that cause cassava mosaic disease (CMD). This study inquires about the gene regulatory roles of specific microRNAs in the host plant Manihot esculenta Crantz (Cassava) during Sri Lankan Cassava Mosaic Virus (SLCMV) infection. Micro-RNAs are endogenous, non-coding, 20–24 nucleotide long RNA molecules that can regulate gene expression by directing cleavage or translational inhibition of mRNAs in cells. MicroRNAs are generated from their pre-miRNAs by RNase III-like Dicer-like enzymes in plants and then associate with Argonaute (AGO) protein to inhibit gene expression at the level of transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS). These small RNAs have a remarkable role as negative regulators of genes involved in developmental processes in eukaryotes. Naturally, they target mRNAs either via mRNA cleavage or inhibition of protein synthesis. The viral infection leads to several developmental and physiological distortions in plants and hints at the regulatory roles of microRNA during such an infection. In our study, 158 conserved miRNAs belonging to 22 families were identified in leaf libraries of cassava line CMR123, a cassava mosaic disease (CMD) tolerant cassava variety and H226, a CMD susceptible variety using deep-sequencing data. The level of cassava microRNA mes-miR159 was the highest followed by mes-miR166, mes-miR9386, mes-miR395, and mes-miR167. microRNAs viz. mes-miR395, mes-miR482 were ascertained for their regulatory roles in the expression of different transcriptional factors like WRKY family and domains like Leucine-rich repeat receptors involved in the immune response against viruses. A differential expression analysis based on log2fold changes was done. MicroRNAs with log2fold change greater than 2 were significantly upregulated and downregulated if the log2fold change was less than −2. Expression of mes-miR395a showed significant variation between susceptible and tolerant varieties upon SLCMV infection. Effect of dsRNA derived from the various genes of tomato leaf curl New Delhi virus (ToLCNDV) in suppressing ToLCNDV infection in tomato Dipinte Gupta , Oinam Washington Singh, Anirban Roy and Bikash Mandal* Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-IARI, New Delhi- 110012, India (*Correspondence to Dr. Bikash Mandal: [e-mail]) Abstract: Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (family Geminiviridae) is one of the most devastating viruses in tomato cultivation. Direct application of dsRNA is an emerging approach for protecting plants from virus infection. In the present study, an attempt was made to evaluate the protective efficacy of dsRNAs derived from multiple genes of ToLCNDV: AC1, AC2, AC4, AV2 and ßC1 gene. dsRNA was prepared in bacterial system using L4440 vector and E. coli strain, HT115. Initially, the effectiveness of individual dsRNAsand cocktail (dsCK) in aqueous suspension conatining 0.01% Celite were assessed against ToLCNDV in tomato. The progression of symptom development and the viral load were assessed at 13, 21 and 60-days post virus inoculation (dpi) through whitefly. All the dsRNA treated plants were found to be symptomless as compared to the control plants that develop typical curling symptom at 13 dpi. However, symptom appeared in dsRNA treated plants by 21 dpi. To test the level of resistance, qPCR was performed by using ToLCNDV-CP specific primer and observed a significant reduction of viral load in all the dsRNA treated plant, and the maximum reduction of virus load was observed in dsCK treated plants upto 13 dpi. Further to improve the stability of naked dsRNAs and its effectiveness against ToLCNDV, dsCK was conjugated with mesoporous silica nanoparticle (MSNP). Significant reduction in viral load were observed in plants treated with dsCK (72 fold) and dsCK-MSNP (74 fold) as compared to the control plants at 13 dpi. Whereas, at 21 dpi, dsCK-MSNP and dsCK treated plants showed 33 and 2.5-fold reduction of virus load, respectively, which further reduced to 2.12 and 1.4-fold at 60 dpi. The result indicated that foliar application of various dsRNA delayed the symptom expression for a limited period of time and nanocarrier conjugated dsCK enhanced the time of suppression of virus load. Seed transmission nature of ToLCNDV and BgYMV in bitter gourd Renukadevi, P., Gomathi Devi, R., Sundravadana, S., Sankari, A and Lakshmi, S. Background: Bitter gourd (Momordica charantia L.), also known as bitter melon or bitter squash is a valuable vegetable crop grown for highly nutritional fruits. One of the principal diseases affecting bitter gourd that causes a significant yield loss is a yellow mosaic disease, which is causedby begomoviruses. The present communication describes the seed borne and seed transmission nature of two begomoviruses viz., tomato leaf curl New Delhi virus (TOLCNDV) and bitter gourd yellow mosaic virus (BgYMV) in bitter gourd. Objectives: To determine the extent of seed borne nature and transmission efficiency of tomato leaf curl New Delhi virus (TOLCNDV) and bitter gourd yellow mosaic virus (BgYMV). To assess the extent of seed transmission and symptom expression under microplot study. Materials and Methods: Seed borne nature of begomoviruses: Bitter gourd seeds of four major hybrids viz., H1 which was predominantly grown by the majority of the farmers followed by H2, H3 grown only in Pollachi of Coimbatore district and H4 were collected from the seed market at Coimbatore. Bitter gourd seeds were dissected into three different parts viz., seed coat, endosperm and embryo. The different parts of seeds of all four hybrids (for each hybrid 30 seeds) were used for the detection of begomoviruses through DAS—ELISA using ToLCNDV antiserum for the detection of begomoviruses. The embryo positive samples were again confirmed with PCR using ToLCNDV and BgYMV specific primers. Seed transmission nature of begomoviruses: The market collected seeds of H1, H2, H3 and H4 (100 seeds per hybrid) were sown for grow out test in insect proof net house. The leaf samples of all the plants were collected at 35DAS and PCR was performed with specific primers of ToLCNDV and BgYMV. The microplot was established as completely closed insect proof net house and study was conducted with 125 seeds of H1 hybrid seeds from market as well as forty four seeds of H1 hybrid from infected fields. The symptomatic and asymptomatic leaves were collected from microplot bitter gourd plants and checked for both ToLCNDV and BgYMV through PCR. Results and Conclusions: The ELISA results for seed parts from market seed revealed that among four hybrids, H2 recorded the highest number of positive samples in seed coat (10), endosperm (14) and embryo (17) followed by H1 with seed coat (7), endosperm (9) and embryo (8). The percentage of embryo infection was high in H2 (62.96%) followed by H1 (26.6%), H3 (20%) and H4 (10%) respectively. The PCR results of DAS-ELISA positive embryo samples revealed that the hybrids H1 and H2 were positive for both BgYMV and ToLCNDV. The PCR analysis of grow-out test results revealed that there was no seed transmission of BgYMV. Contrastingly 5% seed transmission in H1 and H2 and 3% seed transmission in H3 were obtained for ToLCNDV. The symptom expression was very mild in grow-out test plants due to diffuse sunlight inside insect proof cages than the open field bitter gourd plants. In the microplot study, seeds of H1 collected from market as well as from virus infected field expressed symptoms after 45DAS. Out of leaves collected from 125 grow-out test plants of market seeds, 7 plants were positive for BgYMV, of which 2 plants were symptomatic and 5 plants were asymptomatic; 64 plants were positive for ToLCNDV, of which 6 plants were symptomatic and 58 plants were asymptomatic and 7 plants were positive for both BgYMV and ToLCNDV. The microplot study of market seed and field collected seeds revealed that the percentage of seed transmission of ToLCNDV was about 51.2% and 11.1%, and for BgYMV, it was about 8.8% and 5.6% respectively. Among the two begomoviruses, ToLCNDV is predominantly seed borne as well seed transmitted than BgYMV. The seed borne nature and seed transmission efficiency vary depending upon the lot of hybrid seeds purchased from the market. This study strongly proved that the seed serves as a source of inoculum for the early transmission of begomoviruses by whiteflies. Recombinase Polymerase Amplification coupled CRISPR-Cas12a- based fluorescence assay for point-of-care detection of chilli leaf curl virus Venu Emmadi, Shipra Saxena, Parimal Sinha, Bikash Mandal, Sunil Kumar Mukherjee and Anirban Roy* Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi – 110012 Email: [e-mail] Chili leaf curl virus (ChiLCV) is one of the most destructive begomovirus leading to considerable economic losses. To develop effective management techniques for this disease, detection of the virus is the most crucial step. Recently, CRISPR-based Cas12a nuclease systems have been harnessed to develop highly accurate, sensitive and rapid detection platforms for point-of-care (POC) diagnostics of plant, human and animal diseases. Here we developed a DNA endonuclease targeted CRISPR trans reporter (DETECTR) system on the basis of AC1 gene sequence of Chili leaf curl virus (ChiLCV) that couples recombinase polymerase amplification (RPA) for sensitive, specific, point-of-care (POC) diagnostics of Chili leaf curl virus (ChiLCV) from crude leaf extract. When cloned viral genome is used as template, the RPA assay can amplify the virus as less as 3 fg of the cloned DNA that corresponds to 1.014X102 copies of the viral genome. Using total DNA as template isolated from 50 mg leaf tissue, RPA assay could detect viral DNA from as less as 1 pg within 5 min at room temperature. From crude leaf extract, the virus can be detected from 10 −3 times diluted crude extracts, made from 10 mg of leaf sample in 100 µl of 1 X TE, within 10 min at 320C. The RPA-assisted DETECTR system was able to specifically detect ChiLCV DNA either from infected plant DNA or from crude sap, but unable to detect cloned genome of tomato leaf curl New Delhi virus, the other commonly occurring begomovirus in chilli and another six begomoviruses, indicating its specificity. The entire method, from crude sap extraction to fluorescence-based detection, takes 45 min, making it field deployable, point-of-care (POC) detection tool for ChiLCV. The developed assay delivers an effective and robust detection platform to monitor the on-site detection of other plant viruses in the field. Identification of viral inducible promoter during geminivirus infection M. Malavika1#, Sneha Yogindran1,2, Supriya Chakraborty1 1Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, NewDelhi-110 067, India. 2Department of Biotechnology, Cochin University of Science and Technology (CUSAT) Cochin, Ernakulam district, Kerala 682022, India Background: Gene silencing in plants is one among the vital defence pathways employed bythe host against the invading viruses. Plants utilize both Transcriptional gene silencing (TGS) and Post-transcriptional gene silencing depending on the invading viral pathogens. RNAdependent RNA polymerases (RDRs) are involved in production of dsRNA molecules thatinvolve in gene silencing mechanisms. During Tomato leaf curl Gujarat virus (ToLCGV) infection, the tobacco (Nicotiana tabacum) plants show symptom recovery with an increase inlevel of RDR1. RDR1 has been studied previously in promoting symptom recovery in variouspathosystems but the role of their promoter in geminivirus infection has not yet beencharacterized. This presents an opportunity to understand and investigate further about viralproteins interfering with promoters of host genes during viral pathogenesis. Objective: To study the role of NtRDR1 promoter during Tomato leaf curl Gujarat virusinfection. Methods: To examine the role of NtRDR1 promoter in recovery induction, 1 kb and 1.9kbpromoter sequence of NtRDR1 has been cloned in GUS expressing vector in Nicotianabenthamiana. The GUS reporter gene assay has been carried out and the GUS expression uponviral stress (ToLCGV) qualitatively determines the role of RDR1 promoter during viralpathogenesis. Moreover, quantification of GUS activity has been performed by MUG assay. Results: The 1.9 kb promoter sequence of RDR1 responded with high GUS induction in bothqualitative and quantitative analysis. Plant Virology (Poster Presentation) First report of ‘Candidatus phytoplasma asteris’ (16SrI) from Cassia fistula showing symptoms of flat stem and witches’-broom in India Kirti Rawat1, A. K. Singh2, Manish R.3, Hemavati Ranebennur1* 1Division of Plant Pathology, Indian Agricultural Research Institute, PUSA campus, New Delhi-110012. 2Division of Plant Pathology, Faculty of Agriculture, SKUAST, Chatha, Jammu 180009. 3BEElab, School of Biology, IISER Thiruvananthapuram, Kerala- 695016 *Corresponding author: [e-mail] Corresponding author address: Division of Plant Pathology, Indian agricultural research institute, PUSA campus, New Delhi-110012 Cassia fistula Linn, also known as the “golden shower tree”, is a member of the Fabaceae and is widely used for its herbal properties. Typical flat stem symptoms were observed on a C. fistula plant during March 2022, in IISER (Indian Institute of Science Education and Research) campus, Thiruvananthapuram and during May in SKUAST (Sher-e-Kashmir University of Agricultural Sciences and Technology), Jammu. On continuous observation, it was perceived that the younger flowers were not developing properly (the buds did not open for > 5 days). PCR assays were done using universal primer pairs (P1/P7 followed by R16F2n/R2) specific to the phytoplasma 16S rRNA gene to evaluate the possibility of phytoplasma etiology. The predicted amplicon of 1.8 kb product of the 16S rRNA region was not obtained from symptomatic samples in the first round of PCR amplification using primer combination P1/P7. However, in nested PCR using the R16F2n/R2 primer combination an amplicon of size 1.25 kb was obtained in symptomatic leaf samples. No amplifications were observed in non-symptomatic plant samples either in first round or nested round of PCR assays with phytoplasma specific primer pairs. The 16S rDNA sequences of the phytoplasma strain of this study showed 100% sequence identity with the strains belonging to the aster yellows (AY) group (16SrI). Phylogenetic and virtual RFLP analysis of 16S rDNA sequences of the identified phytoplasma strain ornamental plant further confirmed their clustering and grouping with member strains of ‘aster yellows’ subgroup-B. Leafhoppers were also collected and were tested for phytoplasma using the same nested PCR primer sets and found positive for 16Sr RNA gene. To the best of our knowledge, this is the first report of the phytoplasma association of ‘Candidatus Phytoplasma asteris’ (16SrI-B) subgroup with Cassia fistula in the world. First report of ‘Candidatus phytoplasma asteris’ (16SrI) from Clarkia unguiculata L. showing symptoms of flat stem and witches’-broom in India Hemavati Ranebennur* and Kirti Rawat Division of Plant Pathology, Indian Agricultural Research Institute, Pusa Campus, New Delhi-110012 *Corresponding author: [e-mail] Clarkia unguiculata, an ornamental plant species in the gardens of Division of Plant Pathology New Delhi, showed the symptoms of flat stem and witches’ broom during the month of February, 2022. To determine if phytoplasma may be the cause of the disease, PCR experiments were carried out using universal primer pairs (P1/P7 followed by R16F2n/R2) that are specific to the phytoplasma 16Sr RNA gene. In the initial round of PCR amplification, employing primer pair P1/P7, the expected 1.8 kb amplicon of the 16S rRNA region was not produced from symptomatic samples. However, symptomatic leaf samples from nested PCR employing the R16F2n/R2 primer combination yielded an amplicon of size 1.25 kb. In first round or nested round PCR experiments using phytoplasma specific primer pairs, no amplifications were seen in non-symptomatic plant samples. When compared to reference strains from the NCBI, the 16S rDNA sequences of the phytoplasma strain used in this investigation revealed 100% sequence similarity to aster yellows group (16SrI). The 16S rDNA sequences of this study subjected to phylogenetic and virtual RFLP analysis to further confirm by clustering and grouping with other member strains of “aster yellows” subgroup-B. The same nested PCR primer sets were used to test leafhoppers for phytoplasma, and they were found to be positive for the 16Sr RNA gene. This is the first report of an association between Clarkia unguiculata and the 16SrI-B subgroup of “Candidatus Phytoplasma asteris.” Keywords: ornamental, aster yellows, Hishimonus, insect vector, protein translocase subunitA. Detection and Characterization of Virus(es) Infecting Major Stone Fruits in Kashmir Sumiah Wani1, Mehraj D. Shah1, Sajad Un Nabi2, Bilal A. Padder1, Aflaq Hamid1, Mohammad Maqbool Mir3, Shoukat Ara4 1Plant Virology and Molecular Pathology Laboratory, Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, 190025, Jammu and Kashmir, India. 2ICAR-Central Institute of Temperate Horticulture. 3Division of Fruit Science, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, 190025, Jammu and Kashmir India. 4Division of Environmental Science, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, 190025, Jammu and Kashmir India Corresponding author: Sumiah wani, E. mail: [e-mail], Phone no. 8899654788 Background: Like in other crops, stone fruit production is also hampered due to various abiotic and biotic stresses resulting in poor health of trees and low productivity. Viruses are one of the major constraints in the production of stone fruits. Several plant viruses infecting stone fruit crops are extremely infectious and have sometimes devastating effects on the host trees. Objectives: Keeping in consideration the importance of stone fruits, economic losses caused by viruses, and a wide research gap in viral diseases of stone fruits in Kashmir, this research was proposed with the following objectives:- To ascertain the status of viral diseases of stone fruits in Kashmir; To characterize the virus(es) using Serological and nucleic acid-based techniques. Materials and Methods: Survey for collection of diseased samples Serological confirmation through DAS-ELISA. RNA isolation of samples collected and cDNA synthesis. Universal coat protein-specific primers of major stone fruit viruses are used for amplification via RT-PCR for the identification of different viruses. Sequencing of the of representative isolates was done and the sequences obtained were blasted in the database to identify the query sequences. Results: Based on DAS ELISA and RT-PCR diagnosis, many of the economically important viruses were detected in stone fruits and the most widespread stone fruit viruses were Prunus necrotic ringspot virus (PNRSV) and Apple chlorotic leafspot virus (ACLSV). Mixed infections PNRSV, ACLSV, and Cherry virus A (CVA) were also detected. Conclusion: The study gave us insight about the prevalence and spread of stone fruit viruses in the fields of Kashmir valley. And laid the ground for further investigation to determine the extent of viral infection in particular for stone fruits and the possibility of other strains and other viruses in other fruit crops as well. Analysis of Viral Diversity in Common Bean using Metatranscriptomic Approach Shahjahan Rashid1 and Aflaq Hamid1 1Department of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Srinagar, India, 190025 Background: Common bean is infected by several viral species worldwide and results in enormous economic losses. For managing any viral disease identification is the first step. New sophisticated technologies like high throughput sequencing (HTS) has been developed which can screen and identify all the genetic material present in a sample. Objective: To identify viruses associated with common bean in Kashmir using high throughput sequencing. To develop diagnostic tool for simultaneous detection of identified viruses. Materials and Methods: Survey and collection of bean samples was done in Kashmir valley and were analyzed using HTS. Bioinformatic pipeline was used for virus identification. Reconfirmation of identified viruses was done by RT-PCR using designed primers and Sanger sequencing. Multiplex PCR (mPCR) protocol was standardization for identified viruses. Results: In this study, we identified that common bean was infected with three viruses viz., Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV) and Clover yellow vein virus (ClYVV). RT-PCR results confirmed presence of these viruses with mixed infections in different samples. BCMV was found most predominant virus. Recombination was found in BCMV and ClYVV but not in BCMNV. Phylogenetic and pairwise nucleotide analysis indicated foreign introduction of these viruses. mPCR developed was successful in simultaneous detection of all the viruses. Conclusion: We identified three viruses in which BCMNV and ClYVV are first reports. mPCR developed can be used for early detection of these viruses and will be also helpful in seed certification programs. Molecular characterization and rapid onsite detection of Potato virus S Vijayanandraj Selvaraj 1, Yogita Maheshwari1, Swati Bhuria1 Vipin Hallan2 and Bikash Mandal3 1Plant Molecular Virology Laboratory, CSIR - National Botanical Research Institute, Lucknow, Uttar Pradesh, India. 2Advanced Center for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India. 3Plant Virology Laboratory, Biotechnology division, CSIR - Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh Potato stem tubers is an economically important starchy vegetable crop cultivated worldwide. It is propagated vegetatively from seed tubers, due to this many viruses are being carried over from one year to next year through seed tubers. Potato virus S (PVS) belongs to the genus Carlavirus, family Betaflexiviridae causes low to moderate crop losses worldwide. In this study, complete genome of PVS from India was characterized first time and the genome was further utilized for development of rapid onsite detection of PVS. The complete genome of PVS Palampur isolate (MW331321) was characterized. The genome contained 8460 nucleotide (nt) long. The PVS palampur isolate showed 95.4–77.6% nucleotide identity with other 130 full length PVS genome. The PVS-Palampur showed close identity with Kenyan isolate (MN689443). Phylogenetic analysis revealed that the PVS isolates are found in six groups. Rapid on-site detection of PVS was achieved by lateral flow immunostrip using recombinant antibody. PVS coat protein (CP) gene expressed in Escherichia coli was used to generate polyclonal antibody (PAb) in rabbit. The PVS—PAb specifically detected PVS up to 1:102,400 dilution in ELISA. The PAb was further utilized for development of rapid and field-deployed lateral flow immunoassay (LFIA). The sensitivity of LFIA for detection of PVS was up to 1:50 dilution of infected plant sap. The diagnostic specificity of LFIA with ELISA was 100% and showed no cross reaction with other viruses infecting potato viz., PVY, and PVX. Further, the LFIA was successfully utilized for detection of virus with field samples. Detection and Characterization of Begomoviruses Affecting Chilli in Five Agroclimatic zones of Tamil Nadu Jayanthi P 1, Pradeep Kumar2, Anirban Roy2, Bikash Mandal2, Swapna Geetanjali A1* 1Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulattur, Tamil Nadu, India. 2Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New, India Begomoviruses (family Geminiviridae) are serious constraints in crop production in tropical and subtropical agriculture. Leaf curl disease has emerged as a serious problem in various states of India. In the present study, multiple begomoviruses were explored using begomovirus species-specific PCR in the key commercial chilli production locations in the thirteen districts of Tamil Nadu (Krishnagiri, Dharmapuri, Kanchipuram, Thiruvannamalai, Thiruvallur, Salem, Vellore, Thuthukudi, Viruthunagar, Coimbatore, Tenkasi, Trichy, and Karur). A significant incidence of the leaf curl disease was detected in these places during 2018–2022 and the symptoms of the collected samples showed yellowing, mottling, curled leaves, and plant stunting. Eight hundred and thirty-three samples were collected from all 13 districts. Total genomic DNA was extracted from these samples and analysed by PCR using species-specific primers of Tomato leaf curl Bangalore virus (ToLCBaV), Tomato leaf curl Gujarat virus (ToLCGuV), Tomato leaf curl Joydepur virus (ToLCJV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl New Delhi virus (ToLCNDV) and Chilli leaf curl virus (ChiLCV), universal primers for begomovirus DNA A and betasatellite (CLB F/CLB R). The PCR results revealed the presence of all six begomoviruses in the samples collected from Thiruvannamalai district and five begomoviruses from the samples of Krishnagiri district. A single chilli plant showed a minimum of five to six different species of begomoviruses. Among the six viruses, ChiLCV is the most prevalent virus discovered in all thirteen districts (45.7%) whereas ToLCNDV, ToLCGuV, ToLCPalV and ToLCJV were found in 17.8%, 16.6%, 3.6% and 1.5% respectively. Only 37% of the samples were tested positive for beta satellite. 24 PCR positive samples were subjected to Rolling Circle Amplification (RCA). The unit length betasatellite and begomoviral fragments of ~ 1.3 Kb and ~ 2.7 Kb were cloned in pUC19 followed by Sanger sequencing. A total number of 40 clones were generated, out of which 24 of them contained ~ 2.7 Kb DNA A genome and 16 clones contained ~ 1.3 Kb. Complete nucleotide sequence obtained for 2 clones (KG.T.CH18-krishnagiri and SA.E.20-salem) containing 2761 nt and 2728 nt showed 99.31% and 95.54% sequence identity with chilli leaf curl virus (MW760306) and chilli leaf curl virus Bhavanisagar (NC055130) respectively. Further characterisation of remaining clones will help us to identify the Genetic diversity of begomoviruses occurring in the major chilli growing areas of Tamil Nadu. Keywords: Begomoviruses, chilli leaf curl virus, PCR, Agroclimatic zones, Tamil Nadu. Molecular Characterization of Chilli leaf curl Ahmedabad virus, and Tomato leaf curl Bangladesh betasatellite and its infectivity studies in chilli Gnanaprakash Jeyaraj 1, Sravya G1, Swapna Geetanjali A1* 1Department of Genetic Engineering, School of Bio-Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu, Tamil Nadu, India Presenting author: [e-mail]; *Corresponding author: [e-mail] Chilli (Capsicum annuum L.) is one of India's commercially significant vegetable harvests. Leaf curl disease in chilli, caused by begomovirus, is a problematic issue. The causal infection is transmitted to the plants by white flies (Bemisia tabaci). Infected plants display typical symptoms such as curling of leaves, puckering, turned petioles and stunting. Infected samples were collected from Guntur, Andhra Pradesh and genomic DNA was isolated following CTAB method. PCR-based detection of begomovirus and betasatellite was done by using universal primers. The begomovirus detected gDNA was subjected to Rolling Circle Amplification (RCA) followed by Restriction Digestion with selected enzymes. The restricted fragments of length ~ 2.7 Kb representing the unit length of begomoviral DNA A genome was cloned in pUC19 and sequenced. For betasatellite characterization, the gDNA was amplified with universal primers and cloned in the pGEM-T Easy vector. Complete sequence characterization identified them as the Chilli leaf curl Ahmedabad Virus (ChiLCAV) and the Tomato leaf curl Bangladesh betasatellite (ToLCBB). In the pCAMBIA 1302 vector, the ChiLCAV and ToLCBB partial dimer constructs were developed. The Partial dimer constructs of ChiLCAV (pCAM-GuC1) and ToLCBB (pCAM-GS3) were mobilized separately into Agrobacterium strain LBA4404. The host chilli plant was agro-infiltrated with pCAM-GuC1 and pCAM-GS3 infectious constructs. At 28 days post infiltration (dpi), the chilli plants developed mild leaf curl symptoms in DNA-A (pCAM-GuC1) + DNA-Beta (pCAM-GS3) infiltrated plants, whereas the plants infiltrated with DNA-A (pCAM-GuC1) and DNA-Beta (pCAM-GS3) separately did not show any disease-like symptoms. The presence of begomovirus in the infiltrated plants with DNA-A + DNA-Beta and DNA-A alone have shown positive amplification with specific primers, whereas the infiltrated plants with DNA-Beta alone didn’t show any positive amplification by using specific primers. The present study showed that the ChiLCAV DNA-A can infect chilli host causing no symptoms and when associated with ToLCBB DNA-Beta it has developed mild symptoms in the chilli. Keywords: Chilli leaf curl Ahmedabad Virus, Tomato leaf curl Bangladesh betasatellite, infectivity studies. Interaction between Chilli leaf curl Ahmadabad virus and Tomato leaf Bangladesh betasatellite proteins: An Insilco approach Neha Angelin 1, Gnanaprakash Jeyaraj1, Swapna Geetanjali A1* 1Department of Genetic Engineering, School of Bio-Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu, Tamil Nadu, India Presenting author: [e-mail]; *Corresponding author: [e-mail] Plant viral diseases are a leading threat to the farmers creating economic loss. Among them the Begomoviruses which belong to the family Geminiviridae, has a wide host range and highly infectious. The Begomoviruses are known to have bipartite genome among many of its members, while others have a monopartite genome. Various studies are carried out on the plant virus interaction to understand the mechanism of infection and symptom development. These viruses are found to be associated with some satellite molecules which are molecular structures having circular genome coding for one or two proteins. They play a major role in infectivity. The satellite molecule cannot act alone in distressing the host. It needs a supportive element to drive its function for which they depend upon any viral genome. This association of satellite molecule and the virus can happen only if there is some interaction happening between their proteins. We speculate the same in case of Chilli leaf curl Ahmedabad virus (ChiLCAV) which is found to infect the host in association with Tomato leaf curl Bangladesh betasatellite. The Protein–Protein Interactions can be studied in order to know the interaction between the proteins of Beta-satellite and The ChiLCAV. The protein structures of both viral and satellite were derived through homology modelling. Here, Modeller 9.25 is used to predict the structure of all the proteins present. While, ClusPro (https://cluspro.org) is used to do the docking of Intra and inter-protein structures of the virus and satellite to know the protein–protein interaction. Doing this analysis is a prior step in knowing the actual mode of infection and how these associate molecules require support from the viral genome. This can also provide details on which viral protein the satellite proteins bind and how is it enhancing the function of that protein. Details of protein interactions between the beta-satellite protein and the viral protein can help scientist to target such protein to develop RNAi based resistance also CRISPR mediated regulation of viral genome against any viral diseases. Keywords: Chilli leaf curl Ahmedabad Virus, Tomato leaf curl Bangladesh betasatellite, Insilco approach. Incidence study for effective diagnostics of begomoviruses causing leaf curl disease in papaya Priyanka Bharti Background: Though India is agriculture based country and also largest producer of papaya in the world. The growing range of plant viruses are the major threat for the papaya growers throughout the globe. Infection with the range of begomoviruses in plants are responsible for having huge loss to million dollar industry and also to the economy. Due to its genetic variability and composition, effective diagnostics of the begomoviruses is a troublesome. Objectives: To detect the virus infection, degenerate primers were designed from the conserved region of DNA-A genome which can be used as detection tool for detecting the wide range of begomoviruses. Variable regions were also used to design the species-specific primers for detecting the specific isolate from specific locations. Materials and methods: Sequence retrieval of DNA-A sequences and MSA of retrieved sequence followed by phylogenetic analysis. After that Primers were designed based of different geographical regions and screened the designed primers. Results: Different samples of papaya infected plants showing leaf curl symptoms were collected from different locations of different states. The designed degenerate primers from the conserved regions results in the amplication of the all begomovirus positive samples. Whereas the infected samples collected from specific locations were screened through primers designed from the variable regions were give amplification from their respective primers only. Conclusions: The degenerate primers were used to detect the presence of all range of begomoviruses irrespective of their geographical location. While the species specific primers were capable of amplifying only those isolates which are from the specified geographic location. Species-specific primers were used for the identification of virus transmitted from one to another location and can also be used to determine the host range of papaya infecting begomoviruses. The transmission history of begomoviruses can also be tracked using these species-specific primers. Detection of tomato infecting begomoviruses in cucurbits in India Naveen Nayaka S1, Vikas Solanki2, Rakesh Kumar3, Anirban Roy1 and Bikash Mandal1* 1Advanced Centre for Plant Virology, ICAR-Indian Agricultural Research Institute, New, India. 2Department of Research and Development, Advanta Seeds, Kallakal, Hyderabad. 3Department of Biotechnology, JK Agri Genetics Ltd, Hyderabad *Correspondence to Dr. Bikash Mandal: [e-mail] Cucurbits are an important summer vegetable crops and they are affected by several biotic and abiotic factors. Among the various biotic factors, tomato infecting begomoviruses are emerging threat for the production of major cucurbits in India. PCR based species—specific primers of begomoviruses infecting tomato and chilli that were developed in our laboratory previously, were utilized to detect begomoviruses in cucurbits from different parts of India. During 2020 and 2021, leaf samples of cucurbit plants were collected from different locations of various states i.e., Chhattisgarh, Delhi, Haryana, Karnataka, Maharashtra, Telangana and Uttar Pradesh. The total plant DNA was isolated using CTAB method and the isolated plants DNA were subjected to PCR detection using begomovirus universal primers and the samples which were positive were tested for the half a dozen of specific begomovirus species i.e., Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Joydebpur virus (ToLCJoyV), Tomato leaf curl Gujrat virus (ToLCGV), Tomato leaf curl Bangalore virus (ToLCBV) and Chilli leaf curl virus (ChiLCV). The PCR results showed that out of 460 plant samples tested, 138 samples were positive with universal PCR, among them 45, 66, 3 and 2 samples were positive for ToLCNDV, ToLCPalV, ChiLCV and ToLCJoyV respectively. None of the samples however, were positive for ToLCGV and ToLCBV. Our study showed that the tomato infecting begomovirus, ToLCNDV and ToLCPalV prevalent in major cucurbits in India. Present study also showed that there was a differential distribution of begomoviruses in cucurbits in northern and southern India. In silico epitope prediction and experimental validation of the peptide specific antibodies for early and rapid detection of nucleopolyhedrovirus infecting silkworm, Bombyx mori L. Insha Shafi, Mudasir Gani, Aamir Shehzad, Moazur Rahman, Khalid Hussain Bhat, Mohd Jamal Dar, Mohd. Altaf Bhat, Sadiah Shafi, Parvaiz Ahmad Sofi, M. A. Mantoo Background: Bombyx mori nucleopolyhedrovirus (BmNPV) is the most devastating viral pathogen that selectively infects the domestic silkworm. The presumable most effective solution for the management of grasserie disease is to detect BmNPV at early stages of infection in order to initiate appropriate treatment against the disease and prevent further spread of the disease in silkworm rearing units. In our study, we determined the immunogenic region of the GP64 protein using in silico approach and generated the polyclonal antibodies against the peptide sequence corresponding to the immunogenic region of the full-length protein. The experimental validation of the most potent epitopic region for early and rapid detection of BmNPV was conducted through antigen–antibody interaction. Objectives: Epitope mapping of Bombyx mori nucleopolyhedrovirus (BmNPV) major envelope protein, GP64 Production and optimization of antibodies against GP64 and exploring their potential in the early and rapid detection of Bombyx mori nucleopolyhedrovirus Material and Methods: The multiple methods were used for the prediction of B—Cell and T—Cell epitopic regions of GP64 and later these methods were subjected to consensus method for the final epitope prediction of GP64 protein. The peptide sequence of 24 amino acids was synthesised, conjugated with KLH and the peptide specific antibodies were produced in mouse at Indian Institute of Integrative Medicine (IIIM), Council of Scientific and Industrial Research (CSIR), Kanal Road, Jammu. The sensitivity and specificity of the peptide specific antibodies were conducted using Western blotting. Results and Conclusion: The epitope prediction consensus method predicted a total of 12 potential epitopic regions with 08 more promising regions on the basis of antigenicity score. The most potential epitopic region was 24 amino acid sequence and the same was used for immunization studies in mouse model. The GP64 protein (64 KDa) of BmNPV was clearly detected at 10 µg concentration and at 1, 2, 3, 4, 5, 6 and 7 dpi by using the generated peptide specific antibodies through western blotting. The western blotting of the antibodies against the host proteins and common silkworm pathogens viz., Bacillus thuringiensis, Serratia marcescens, Streptococcus sp., Staphylococcus sp., and Nosema bombycis revealed no cross-reactivity and confirmed the specificity of the antibodies. The experimental validation revealed that the generated polyclonal antibodies are sensitive and specific to BmNPV GP64 protein and hence can potentially be used for the development of BmNPV diagnostic kit. Evidence of true seed transmissible nature of turnip mosaic virus in mustard species Pankhuri Singhal1, Virendra Kumar Baranwal1 Division of Plant Pathology, Indian Agricultural Research Institute New Delhi-110012 Mustard is a commercial oilseed crop worldwide infected by highly infectious turnip mosaic potyvirus (TuMV) in mixed infection with cucumber mosaic virus (CMV). Recently, widespread infection of TuMV and CMV was reported from brown, black and yellow mustard varieties in experimental field at Indian agricultural research institute (IARI, New Delhi). A low/nil incidence of aphids during the September sown crop infected with 100% incidence of TuMV but not CMV, indicated possibility of seed transmission. For this, the susceptibility of immature seeds obtained from field infected mustard plants to TuMV and CMV was tested via reverse transcriptase-polymerase chain reaction (RT-PCR) via coat protein gene-specific primers. The seeds of all the tested varieties were found to be associated with TuMV but not CMV. Further, the TuMV was found to be localized in embryo and cotyledon in seeds indicating the true seed-borne nature of TuMV. The seedlings from seeds of infected plants were grown in aphid free growth chamber/containment facility and the seedlings of all the 17 varieties were found associated with TuMV. In another experiment carried out in a growth chamber, out of 25 seedlings of Pusa Gold and Pusa Karshima, 21 and 18 were found infected by TuMV, respectively. The plants generated from the infected seeds were observed for symptom expression in aphid free growth chamber/containment facility and symptoms of TuMV infection i.e. leaf distortion and puckering was observed in the plants. Plant Virology (Poster) Suppressor activity analysis of Tomato leaf curl New Delhi virus genes and their subcellular localization Mehulee Sarkar, Bikash Mandal and Anirban Roy* Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi Corresponding Author email: [e-mail] Background: Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, is the most important begomoviruses infecting tomato in Indian subcontinent. Begomovirus is known to evade RNA silencing of host plants through viral suppressors of RNA silencing (VSRs). However, in case of ToLCNDV, limited information is available so far on its VSRs. Objectives: To evaluate suppressor activity of ToLCNDV genes To determine their subcellular localization Materials and Methods: Three putative suppressor protein encoding genes (AV2, AC2 and AC4) of ToLCNDV were amplified from a pure culture of the virus that was established in Nicotiana benthamiana through agroinoculation. These genes were cloned into a GFP tagged plant expressing binary vector pEarleygate103 and their suppressor activity was evaluated through a GUS reporter and GUS hairpin assay system. The subcellular localization of the suppressor genes was predicted using various bioinformatic analysis and further confirmed by confocal microscopy. Results: All the three proteins showed strong silencing suppression in a GUS reporter and GUS hairpin assay system with the AV2 protein being the most effective. Peptide 2.0 server predicted that all these proteins have more basic amino acid residues and AV2 protein has more hydrophobic amino acid residues. ScanProsite server predicted presence of different functional motifs amongst which presence of kinase motif was observed in all of them. Virus mPLoc server predicted their subcellular localization. Confocal microscopy results revealed that AV2 localizes in the host cell membrane and nucleus, AC2 in the nucleus and AC4 in the host cell membrane. Conclusion: AV2, AC2 and AC4 gene of ToLCNDV act as suppressors of RNA silencing AV2 localizes in the host cell membrane and nucleus, AC2 in the nucleus and AC4 in the host cell membrane. Such localization study will help understand the mechanism of their suppression activity. Cloning of SUPRESSOR OF GENE SILENCING 3 (SGS3) and RNA DEPENDENT RNA POLYMERASE 1 (RDR1) genes from resistant and susceptible cultivars of soybean Dharmappa Chavan1, Shipra Saxena1, Yeluru Mohan Babu2, Bikash Mandal1, Sunil Kumar Mukherjee1, and Anirban Roy1* 1,2Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, and New Delhi – 110012 Email: [e-mail] Yellow mosaic disease is a major constraint in soybean cultivation. In order to understand the genetics of resistance, composite interval mapping revealed two major quantitative trait loci (QTLs), located on chromosome 6 and 2. Annotation of these QTLs on soybean genome suggests presence of two candidate genes, namely RDR1 and SGS3 with these QTL corresponding DNA sequence. Function of these genes governing the resistance has not been carried out. Our main Objective was Cloning of SGS3 and RDR1 genes from resistant (SL-1024) and susceptible (JS-335) cultivar of soybean against yellow mosaic disease. To full fill this objective plant were grown in pots and maintained in growth chamber, leaf samples were collected for RNA extraction and total RNA was obtained using the Trizol technique. cDNA was prepared using the Revertaid™ first strand cDNA synthesis kit following the manufacturer's instructions. Primer designed for reverse transcriptase PCR (RT-PCR). Amplification of gene has done and directional cloning was done using pUC18 vector. Final outcome of our study was the amplicon size of the cDNAs of SGS3 and RDR1 genes irrespective of their plant type (resistant/susceptible) was found to be 1.9 kb and 3.3 kb, respectively. Both the amplified products from both the cultivars have been cloned using directional cloning method in pUC18 vector and clones are confirmed through restriction digestion and sequencing. Cloning of these two genes will help us to do further functional analysis of these genes and find out the function of these genes in silencing pathway involved in disease resistance mechanism. Molecular cloning and in-silico characterization of Pelota gene in solanum lycopersicum Yeluru Mohan Babu1, Shipra Saxena1, Dharmappa chavan1, Vijay Shree Gahlot, Sunil Kumar Mukherjee1, Bikash Mandal1 and Anirban Roy1* 1Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi *Email: [e-mail], [e-mail] Tomato leaf curl disease is caused by a number of begomoviruses with Tomato yellow leaf curl virus (TYLCV) being the most dominant one to tackle this Recently various resistance loci have been identified in tomato which have shown promising resistance to TYLCV. Amongst them only few are well characterized. However, functional characterization of Ty-5 loci which codes for pelo protein (ty-5) is not yet done. With respect to this, we have isolated ty-5 gene from susceptible variety (Pusa ruby) for functional characterization. All the available sequences of pelota were analysed through MEGA software, we were able to identify a single SNP change in 1st exon of Pelota gene T-to-G at 47th positions, resulting in a Valine-to-Glycine substitution While this mutation is not found in wild relatives of tomato, in-silico protein interaction studies through PHYRE 2.0 and string we found 3 ERF like domains which were interacting with 7 different host proteins like Guanine nucleotide-binding protein subunit beta-2-like 1 protein, 40S ribosomal protein S17 which were found to be associated with susceptibility of other tomato infecting viruses. For siRNA studies we found Domain one is suitable, thus we have amplified pelota gene from susceptible variety and cloned in DEntry TOPO vector and sequenced found same mutation in our sequence too. These findings are essential for further functional characterization of pelota gene and its involvement in imparting resistance against tomato infecting begomoviruses. A CRISPR/Cas13 toolkit for conferring resistance against RNA viruses in plants Shipra Saxena, Bikash Mandal, Sunil Kumar Mukherjee and Anirban Roy* Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi – 110012 Email: [e-mail] Global food security is highly threatened by plant viral diseases which hamper both crop yield and quality. In recent years, CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), the molecular immunity system of bacteria and archae, has been widely used in crop plants to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. In order to deploy CRISPR/Cas system to confer resistance against plant viruses, a toolkit enabling both stable and transient expression along with easier methods for the assembly of one or more gRNAs is an essential requisite. In the present study, a plant codon optimized LshCas13a has been cloned in pGWB402 binary vector. Also, a gRNA expression cassette carrying direct repeat for LshCas13a, poly A tail and PaqCI sites for cloning of spacer (target) sequences under AtU6 promoter has also been placed in binary vector pGWB402 harboring Cas13a. Further, a TRV RNA2 vector for gRNA expression cassette under PEBV promoter has also been developed. The toolkit is under validation using different RNA viruses. Thus, in the current study, a golden gate compatible CRISPR/Cas13 binary vector as well as a TRV module for systemic movement of gRNA has been developed. The developed vector system provides an efficient, inexpensive, time-saving, user-friendly and multifaceted toolkit for the generation of CRISPR/Cas13 constructs carrying one or more gRNAs for targeted mutations of multiple genes. In-planta expression of ToLCNDV-CP gene using cucumber green mottle mosaic virus as virus vector Pradeep Kumar1,2, Abdul Kader Jailani1, Anirban Roy1 and Bikash Mandal1* 1Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-IARI, New Delhi- 110012, India. 2Department of Plant Pathology, Narain College Shikohabad, Firozabad, Uttar Pradesh - 283135, India (Correspondence to Dr. Bikash Mandal: [e-mail]) In the present study, an attempt was made to express the full-length coat protein gene of Tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in Nicotiana benthamiana (NB) plant using cucumber green mottle mosaic virus (CGMMV) based virus vector. The full-length ToLCNDV-CP gene was amplified in PCR by using a gene-specific primer (BM-1017F/BM-1018R) and cloned in the backbone of CGMMV based virus vector using BamH1 and Xba1 restriction enzymes. The cloned ToLCNDV-CP construct was used to transform Agrobacterium tumefaciens GV2300 strain. The transformed agrobacterium with CGMMV virus carrying the ToLCNDV-CP gene was infiltrated on 20–28 days-old NB plants. The agroinfiltrated leaves were harvested at 5, 7, 9, 11 and 13 days post-infiltration (dpi). The expression of RNA transcripts was confirmed by RT-PCR using a gene-specific primer. The RT-PCR results showed the amplification of the expected size of 770 bp ToLCNDV-CP gene in the agroinfiltrated leaves. The protein extraction and SDS PAGE analysis were performed to confirm the protein band in the SDS PAGE gel. The SDS PAGE analysis results revealed the expression of 33 KDa size protein of the ToLCNDV-CP gene at 5, 7, 9, 11 and 13 dpi. However, the expression of the protein was higher in 28 days-old plants and the expression of the protein was maximum at 11 dpi. The present study demostrated the expression of a begomovirus protein in NB plant using a tobamovirus based vector. Induction of antiviral protection in host plants through topical application of dsRNA mixture derived from NSs and NP genes of groundnut bud necrosis virus Suryakant Manik, Oinam Washington Singh, Dipinte Gupta, Anirban Roy, and Bikash Mandal* Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India *Correspondence to Dr. Bikash Mandal: [e-mail] Groundnut bud necrosis virus (GBNV, genus Orthotospovirus, family Bunyaviridae), a tripartite ambisense RNA plant virus, is an economically most important plant virus infecting a wide variety of crops. Due to lack of resistant cultivars, management of this viral disease is most challenging aspect. DsRNA mediated induction of RNAi against virus has emerged as an alternative and novel approach. In the present study, two dsRNA molecules were prepared from NSs and NP genes, encoding silencing suppressor protein and nucleocapsid protein of GBNV, respectively by using the vector L4440 in the E. coli strain, HT115 (DE3). Experiments were conducted to test the effective dose of dsRNA and comparative analysis of the efficacy of dsNSs, dsNP and the combination of dsNSs + dsNP against the GBNV infection in cowpea and Nicotiana benthamiana. It was observed that 10 µg of dsRNA per plant was found to be effective in inducing resistance in the host plant. The disease severity data showed that the two individual dsRNAs as well as their combine treatment were able to reduce the GBNV infection significantly as compare to the control plants. qRT-PCR using the partial NP gene specific primer showed that the viral load was significantly reduced up to 4.2-fold, 16.2-fold and 47.9-fold in dsNSs, dsNP and dsNSs + dsNP treated plants, respectively. This study showed that dsNP was more effective than dsNSs, while the combined application of the two dsRNAs (dsNSs and dsNP) was the most potent treatment in protecting plants from of GBNV infection. Effect of salicylic acid and its analogues on seed germination and virus protection in plants Vijay Shree Gahlot, Oinam Washington Singh and Bikash Mandal* Advanced Centre for Plant Virology, ICAR-Indian Agricultural Research Institute, New Delhi, India (*Correspondence to Dr. Bikash Mandal: [e-mail]) Salicylic acid (SA) is an important phytohormone that regulates the plant growth and enhanced resistance against biotic and abiotic stresses. In this study, the role of SA and its analogues, also known as salicylates were tested for their effect on seed germination, growth of plant and virus resistance. Seeds of cucumber, chilli, tomato and mungbean were soaked in 1.0 mM of SA and its five analogues viz., 5-fluorosalicylic acid (FSA), 5-iodosalicylic acid (ISA), 5-methylsalicylic acid (MSA), 2-acetylsalicylic acid (ASA) and disprin (DP) for 24 h, dried with sterile paper and transferred to sterile petri dishes. After 10 days, the number of germinated seeds and root and shoot length were recorded. It was observed that the germination of cucumber and mungbean were increased by 50% and 28.57%, respectively by DP treatment, increased by 28.57% in mungbean and 33.33% in tomato by SA treatment, and 50% increase in tomato by FSA treatment. Treatment of ASA had no effect in germination in any of the above crops, while ISA and MSA were observed to have a detrimental effect in the germination of the tested crops. Further, out of the six chemicals, DP was tested for its effect on induction of resistance against chilli leaf curl virus in Nicotiana benthamiana. Leaf curl incidence was significantly reduced and in 60% of treated plants of N. benthamiana no leaf curl symptoms developed. The study indicated that disprin, salicylic acid and fluorosalicylic acid enhanced seed germination in cucumber, mungbean and tomato and disprin showed leaf curl disease protection in N. benthamiana plant. Roles of two distinct alphasatellites in yellow mosaic disease of Alcea rosea Manish Kumar1, Fauzia Zarreen1a, Supriya Chakraborty*1 1Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110 067, India Alphasatellites are small coding DNA satellites frequently associated with a begomovirus/betasatellite complex, where they are known to modulate virulence and symptom development. Two distinct alphasatellites, namely, Cotton leaf curl Multan alphasatellite (CLCuMuA), and Gossopium darwinii symptomless alphasatellite (GDarSLA) associated with Cotton leaf curl Multan Virus-India (CLCuMuV-IN) and Ludwigia leaf distortion betasatellite (LuLDB) were previously isolated from hollyhock (Alcea rosea) plants exhibiting yellow mosaic symptoms. Objective: This study provides evidence that alphasatellites have a role in symptom modulation and suppress helper virus replication without any discernible effect on the replication of the associated betasatellite. Material and Methods: Two distinct alphasatellites, namely, CLCuMuA, and GDarSLA associated with CLCuMuV-IN and LuLDB were previously isolated from hollyhock plants exhibiting yellow mosaic symptoms. Nicotiana benthamiana plants were co-agroinoculated with CLCuMuV and its associated alphasatellites and betasatellite molecules and samples were collected at 7, 14 and 21 days post inoculation (dpi). The viral DNA molecules were quantified in N. benthamiana plants by qPCR. Result and Conclusions: In this study, we show that alphasatellites CLCuMuA and GDarSLA attenuate and delay symptom development in N. benthamiana. The presence of either alphasatellites reduce the accumulation of the helper virus CLCuMuV-IN. However, the levels of the associated betasatellite, LuLDB, remains unchanged. These results suggest that the alphasatellites could contribute to the host defence and understanding their role in disease development is important for developing resistance strategies. Mutational analysis of DNA-B component reveals the role of coding and non-coding regions in leaf curl disease development Divya Singh, Dibyendu Ghosh#, Biju George, Manish Kumar and Supriya Chakraborty* Molecular Virology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India # Presenting author ([e-mail]) Background: Leaf curl disease of tomato (ToLCD) caused by begomoviruses, is one of the major limiting factors in the production of tomato throughout the world including India. Tomato leaf curl Gujarat virus (ToLCGV) and Tomato leaf curl New Delhi virus (ToLCNDV) are the two predominant begomovirus species that cause severe tomato leaf curl disease throughout the country. These two distinct species have earlier been reported to trans-complement each other where infection of ToLCNDV and ToLCGV together resulted in the supervirulent pseudo-recombination leading to extremely severe symptom development as well as higher accumulation level of DNA-A of ToLCNDV and DNA-B of ToLCGV. In the case of bipartite begomoviruses, DNA-B has been known to confer role in viral movement, host range, pathogenicity and symptom development. However, its role in governing pseudo-recombination is not known. Objectives: This study aims to decipher the role of (i) coding and (ii) non-coding region (NCR) of DNA-B component of ToLCGV in leaf curl disease development. Materials and Methods: Site directed mutagenesis was performed to incorporate mutations in the NCR of ToLCGV-DNA-B. Intergenic regions and ORFs were swapped between DNA-B components of ToLCGV and ToLCNDV to generate chimeric molecules. The confirmed agro clones (wild-type and mutants of both ToLCNDV and ToLCGV) were inoculated in N. benthamiana and tomato plants following the standard protocol. The severity of the symptoms was monitored. Geminiviral titre and transcripts were detected through southern and northern hybridization respectively. In addition, replication of chimeric molecules was also assessed in tobacco protoplasts. Result and Conclusions: The present study comprehensively demonstrates that untranslated region (UTR) of BC1 and BC1 ORF are necessary for leaf curl disease development and severe virus infection. Elucidating the occurrence, molecular characterization, and secondary metabolites profiling of Cannabis (Cannabis sativa L.) to Begomovirus infection in India Sujata Singh Yadav, Akanksha Singh, Birendra Kumar and Abdul Samad Division of Crop Production and Protection, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India Cannabis sativa L. (hemp/marijuana) is one of the most important medicinal plants of family and cultivated since antiquity as a source of fibre, food, and medicine. Cannabinoids, a class of compounds specific to Cannabis, are produced by stalked glandular trichomes and have a vast majority of their medicinal properties. It has broad array of pharmacological properties, such as an immunomodulatory, anti-oxidant, anti-inflammatory, antimicrobial, antistress, antitumor, neuroprotective, cardioprotective, antihypertensive, antidiabetic, anticancer, Alzheimer’s disease etc. In the last two decades, its medicinal demands have been increased significantly worldwide. During a survey in 2019, characteristic symptoms as severe yellow mosaic, curling, and stunting of the leaves were noticed in the experimental field at CSIR-CIMAP, Lucknow. The presence of whiteflies and typical symptoms suspected the association of Begomovirus. Disease incidence was estimated about 20 per cent on the basis of plant population. About 3 healthy and 10 infected plant samples were collected from the fields and run for PCR with Begomovirus coat protein specific primers (Hallan et al. 1998) for initial screening of begamovirus infection in the respective samples. For whole genome amplification, two sets of overlapping primers K1F/K1R and K2F/K2R (Kumar et al. 2011) and universal primers for the detection of DNA-B and alphasatellite components (Rojas et al. 1993; Bull et al. 2003) were used. The presence of the betasatellite was detected by primer pair β01/β02 (Briddon et al. 2002). PCR amplicon of 771 bp was obtained using coat protein specific primers in seven out of ten symptomatic samples, whereas no amplification was obtained in asymptomatic samples. CP-positive samples were used for further detection of genomic and satellite components of begomoviruses. Genomic DNA-A fragment was amplified in two parts of ~ 1200 bp and ~ 1700 bp via PCR. However, presence of DNA-B and alphasatellite componenents were not detected by PCR. Betasatellite of 1376 bp was detected using primers β01/β02. Mechanical inoculation by sap obtained from infected sample showed no local or systemic symptoms on C. sativa plants and other Nicotiana plants and all were also found negative in PCR tests. For morphological identification, virus purification was attempted and scattered geminate like particles (16–18 nm) were observed under Transmission Electron Microscope (TEM). Putative full-length (approximately 2.7 kb) amplicons were obtained, processed, sequenced and data were compared with the sequence database available in GenBank for best sequence identities and phylogenetic relationships. This study also analysed the abundance of the glandular trichomes as a major source of production of the Cannabinoids compounds. Quantification of secondary metabolites (CBD, THC) was done using High-Pressure Liquid Chromatography and metabolite content varied according to the severity of disease symptoms. The present study comprises the first report on unravelling the molecular identification and characterization of the Begomovirus on Cannabis and its effects on trachoma’s morphology and their production of cannabinoids compounds. Induction of plant resistance against Bean Common Mosaic Virus (BCMV) through exogenous application of double stranded RNA (dsRNA) Wani Farhana1, Shahjahan Rashid1, Gowhar Ali2, Aflaq Hamid1 1Department of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar J&K, India, 190025. 2Department of Genetics and Plant Breeding, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar J&K, India, 190025 Background: Viruses are the major constraint in the production of Common bean (Phaseolus vulgaris L). Bean common mosaic virus (BCMV), a member of the Potyviridae family, is the most widespread and causes " mosaic disease" in beans. Due to lack of resistant crop varieties managing BCMV is difficult, resulting in frequent disease outbreaks and significant crop losses. Objectives: In the present work, exogenous application of dsRNA molecules derived from the BCMV HC-Pro and BCMV CP genes were tested for their efficacy against BCMV in hosts, Tobacco (Nicotiana tabacum), Common Bean (Phaseolus vulgaris L), and Cowpea (Vigna unguiculata). Materials and Methods: Amplification/Cloning and synthesis of dsRNA from two RNA silencing suppressors i.e. HC-Pro and CP gene. Exogeneous application of dsRNAs on Tobacco (Nicotiana tabacum), Common Bean (Phaseolus vulgaris L.) and Cowpea (Vigna unguiculata) plants for protection against virus infection. Quantification of viral load in dsRNA treated plants and control through qPCR. Results: Plants treated topically with dsRNAs (dsRNA HC-Pro or dsRNA CP, or dsRNA HC-Pro plus CP) showed a delay in symptom expression. qPCR data shows a steady increase in BCMV expressionin BCMV-inoculated plants, whereas expression was significantly lower in dsRNA-treated plants. Conclusion: Exogenous application of dsRNA derived from viral sequences causes antiviral RNA interference (RNAi) and provides resistance against pathogenic viruses, according to the findings. Beneficial fungal root endophyte, Piriformospora indica confers tolerance to Banana bract mosaic virus under field condition with enhanced yield and fruit quality Sinijadas K*., Amitha Paul, Radhika N. S., Heera G. and Joy Michal Johnson Department of Plant Pathology, College of Agriculture (Kerala Agricultural University), Vellayani, Thiruvananthapuram, Kerala - 695 522 *Correspondence: [e-mail] Background: Banana bract mosaic virus (BBrMV) infection results in upto 100 per cent yield loss depending on the stage of infection. Tissue cultured banana plantlets are used to prevent the spread of the virus diseases through planting materials though the vector-borne diseases also infect the crop in the field. Beneficial fungal root endophyte, Piriformospora indica promotes plant growth and yield with enhanced tolerance to (a) biotic stress in crop plants. Objectives: The study was undertaken to evaluate P. indica-colonized TC plantlets and suckers against BBrMV infection and to decipher the molecular mechanisms involved in the P. indica-mediated tolerance to the disease. Materials and Methods: P. indica-colonized TC plantlets and suckers were evaluated against BBrMV on artificial inoculation using viruliferous aphids and also on natural incidence under field condition. The genes involved in the symptom development and P. indica-mediated tolerance to BBrMV were studied through RT-PCR. Results and Conclusion: P. indica helped in establishment of TC plantlets and suckers (Var. Nendran) in the field with enhanced growth promotion. P. indica-colonized plants reduced the symptoms produced by BBrMV. The endophytes reprogrammed the symptom development and defense genes (Chlorophyll Synthatase, Chlorophyllase, Pheophytin Pheophorbide Hydrolase, Phytoene Synthase, Catalase, Superoxide Dismutase and Ascorbic acid Oxidase) and also BBrMV specific genes—CP, HC-Pro, P3 to reduce the disease severity by 85.7 percent. The yield was increased by 32.8 percent in P. indica-colonized plants in addition to the enhanced fruit quality and shelf life. Thus, P. indica-colonized plants could tolerate BBrMV infection with enhanced yield in banana. Indexing of mother stock of Khasi mandarin against Citrus tristeza virus disease free and immune mother plant of Kashi mandarin (Citrus reticulata) tree from different orchards of Northeast India Halima Khatoon1*, Shaviya singh1, Elong marimuthu1, K.K. Biswas1 1Advanced Centre of Plant Virology, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi-110012 (Delhi) *: [e-mail] Citrus tristiza virus (CTV), is widely distributed around the world and has killed more than millions of tree all around the world. The virus is phloem limited and contain flexuous filamentous particle, the genome contains ssRNA, 19.3 kb in length comprising 12 ORFs encoding 19 putative proteins. It is predominantly transmitted by brown citrus aphid (Toxoptera citricida) in a semi persistent manner. Khasi mandarin (KM) (Citrus reticulata) is the most economically and widely cultivated citrus fruit in Northeast (NE) India, which is tremendously affected decline caused by CTV. The production and supply of CTV free and immune grafted KM planting materials are the important control measures. Thus, effort has been made to identify disease free/immune KM mother plants as source of scion. A survey was made and KM twig samples from healthy looking-best fruit quality trees from seven orchards of Assam were collected. Total 30 twigs were collected and grafted on rough lemon root stock and maintained in nursery. CTV infection was detected by direct antigen coated enzyme-linked immuno-sorbent assay (DAC-ELISA) using CTV specific antisera and PCR using specific primes targeting coat protein and 5’ORF1a gene. Most of the samples showed CTV infection in ELISA with considerable virus titre. The ELISA positive samples were confirmed by PCR. Of the KM trees tested, one plant was showing negative for CTV and seven plant showing positive for CTV. A phylogenetic tree was construct to study the variability and it was found that the mild plant were forming separate clade. These plants were considered as immune mother plant and will be used further for production grafted plating materials to manage the KM decline NE India. Virus infections varied the proximate and mineral contents and Consumers’ decision to purchase fluted pumpkin (Telferia occidentalis) leaves Arogundade, O.*1, Amao, I.O.1, Egbekunle, K.O.1, Aliyu, T.H.2, Atanda, H.B.1, and K.E Oke1 1Fruits Research Programme, National Horticultural Research Institute, Jericho Reservation Area, Idi-Ishin, Ibadan, Oyo State, Nigeria. 2Department of Crop Protection, University of Ilorin, Tanke Road, PMB1515, Ilorin, Kwara State, Nigeria *Corresponding author email: [e-mail] Background: Fluted pumpkin is an important leafy vegetable in Nigeria which contributes to dietary needs and household income. The consumption and marketability of the crop is under threat of virus infection which reduces the quality and quantity of yield and consumer purchase behavior. Objectives: This study was conducted to detect pathogens causing virus-like symptoms, determine their effect on food quality, and gauge consumer decision to purchase fluted pumpkin. Materials and Methods: Virus-like symptoms were observed on experimental fields and young leaves from diseased and healthy plants were collected and sampled for both virus indexing using ELISA and photochemical analysis. Also, respondents were purposively selected for physical field and pictorial assessment as basis for responses to structured questionnaire. Results: The symptomatic leaves reacted positively to anti-CMV and potyvirus antibodies. The values of moisture, protein, Total Titratable Acidity (TTA), and chlorophyll in healthy leaves were higher than those of the diseased leaves. Conversely, the disease leaves had significantly higher values for Ash, crude fibre, fat and carbohydrate with 58.82%, 38.4%, 14.29% and 16.39% respectively compared to healthy leaves. The values of Vitamin C and Sodium were higher in healthy leaves than those of infected leaves while values of Vitamin B, Iron, Potassium, Calcium and Manganese were higher in diseased samples. Most respondents (91%) were not willing to purchase severely diseases leaves. Conclusions: Results from this study revealed that plant virus infection altered the proximate, minerals and vitamin contents of fluted pumpkin leaves and decision to consume fluted pumpkin leaves. Veterinary Virology (Oral) African swine fever: Emergence, molecular epidemiology, associated risk factors and its impact on North-Eastern states of India Nagendra Nath Barman 1*, Lukumoni Buragohain1, Kuralayanapalya Puttahonnappa Suresh2 1College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India.2ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru, Karnataka Presenting author: Nagendra Nath Barman ([e-mail]) African swine fever (ASF) is a fatal hemorrhagic viral disease of domestic and wild pigs. ASF is caused by an enveloped dsDNA virus of the genus Asfivirus and family Asfarviridae. The size of the virus is about 200 nm and the genome of the virus is about 170–193 kbp and its genome contains more than 150 ORFs. It is an emergingand transboundary disease in India. African swine fever virus (ASFV) was detected for the first time in early 2020 after several outbreaks in neighboring Asian countries, particularly in China since 2018. Initially it was detected from Arunachal Pradesh and Assam states of India, however within two years it has spread to all the eight states of north-east (NE) India. Recently, the virus has disseminated to other states of India. North-Eastern Region (NER) is the heart of the Indian pig industry and the livelihood of many families is dependent on pig farming. NER shares about 47% (4.24 million) of the total pig population of India (20th Livestock Census) and the highest populated state is Assam followed by Meghalaya and Nagaland. However, the emergency of ASF in NER is one of the biggest setbacks for the piggery sector of India. Almost 1,00,000 pigs died and 22,000 culled due to ASFV infection in NER from the first outbreak up to June, 2022. Highest death was recorded in Mizoram followed by Assam and Arunachal Pradesh states of India. The positivity rate of ASFV in PCR or qPCR is about 37%. Till date, 24 genotypes of ASFV have been reported from different parts of the globe. However, most of these genotypes are confined to African countries. The commonly encountered genotypes in Asia and Europe are genotype-I and II and the most dominant one is genotype-II. Molecular analysis of ASFV detected in the NER based on phylogenetic markers (B646L and E183L gene) has revealed that the ASFV circulating in the NE region of India belongs to genotype II. Moreover, the analysis of representative whole genome sequences of ASFV shared the highest nucleotide identity with genotype-II Asian and European strains that were reported post 2007. The continuous outbreak of the virus in NER indicates association of certain risk factors that is actively disseminating the disease to each and every corner of the NER. The transmission of ASFV is associated with several risk factors and the risk factors that are identified to be significantly associated with spread of the virus in NER are free-range rearing system, lack of proper biosecurity measures in farms, presence of weekly market, slaughter point and river in close proximity to farms, swill feeding, panic selling of pigs and piglets during outbreaks, and lack of farmers’ awareness. Besides these, transmission of ASFV in wild boars of this region is another emerging threat to the piggery industry because infected wild-boar is a potent carrier. There is ampleof small-scale traditional farmers’in NER whose livelihood is highly dependent on pig farming. But, introduction of ASFV and its continuous spread in the pig population have disrupted the rural economy of this region. The total economic loss was estimated to be more thanRs. 50 crore (till June, 2022) which is devastating to the small-scale farmers of this region. The incessant outbreaks of ASFV in NER is not only a threat to the nation’s economy but it may handicap the piggery sector permanently. Since there is no effective vaccine against ASFV, therefore it is necessary to diagnose the disease as early as possible and all the necessary prevention and control measures must be adopted immediately. Based on the pig-rearing system followed in this region and identified risk factors that are associated with spread of the disease the authors proposed a novel concept viz., ‘Bio-Excon’ which mainly focuses on bio-exclusion and bio-containment of ASFV at village or individual farm-level along with adaptation of new policies and laws. Additionally, the concept of zoning, compartmentalization and other standard practices must be followed strictly for effective control of ASFV. Keywords: ASFV, Bio-Excon, Genotype-II, NER, Risk factors. Zooanthropogenic potential of SARS-CoV-2 virus: Implications for vaccine-mediated immunity Nagarajan, S., Tosh, C., Manoj Kumar, Sanyal, A. ICAR-National Institute of High Security Animal Diseases, Anand Nagar, Bhopal – 462022 MP The human pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that started in December, 2019 is still continuing in various parts of the world. The SARS-CoV-2 has evolved through sporadic mutations and recombination events and the emergence of alternate variants following adaptations in humans and human-to-animal transmission (zooanthraponosis) has raised concerns over the efficacy of vaccines against new variants. The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in bats and pangolins. A recent report of back-and-forth transmission of SARS-CoV-2 between humans and minks on mink farms in the Netherlands has sparked widespread interest in zooanthroponotic transmission of SARS-CoV-2 followed by re-emergence to infect human populations. The risk of animal to human transmission depends on virus-host interaction in susceptible species that may be short-term or long term risks. The short term risk might be due to infection to humans during the viremic stage in susceptible animals. The long term risk might be either due to persistence of the virus at population level or latency of infection leading to risk of evolution and re-emergence of the virus. Experimental studies have identified a range of animals that are susceptible and permissive to SARS-CoV-2 infection viz. cats, ferrets, hamsters, mink, non-human primates, tree shrews, raccoon dogs, fruit bats, and rabbits. The health impacts of SARS-CoV-2 infection in animals are unknown and it is likely that other susceptible species have not been discovered yet. Apart from farmed animals, stray cats and rodents have been identified as a potential opportunity for ongoing transmission in intense farming situations. Recognizing animal species that are most susceptible to infection is the first step in preventing ongoing transmission from humans. Minimizing the risk of zooanthraponosis requires multi-sectoral coordination that includes implementation of strict biosecurity measures such as controlled access to farms that house susceptible animals, bio-secure entry and exit protocols, disinfection protocols in farm, down time for animal transport vehicles and daily assessments of human handlers for exposure to SARS-CoV-2. Hence, active surveillance in animal species that are prioritized based on risk assessment need to be initiated in coordination with health and environment sectors for early identification of emerging and re-emerging variants of SARS-CoV-2 virus in animals. Development of nucleic acid-based diagnostics to detect African swine fever virus with special reference to polymerase spiral reaction (PSR) Lukumoni Buragohain1, Nagendra Nath Barman1, Arpita Bharali1, Sophia M. Gogoi1*, Suparna Sen1, Durlav Prasad Bora1, Sachin Kumar2 and Yashpal Singh Malik3 1College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. 2Indian Institute of Technology, Guwahati, Assam, India. 3College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India * Presenting Author : Sophia M. Gogoi, Email ID: [e-mail] Background: African swine fever (ASF) is a fatal haemorrhagic disease of pigs (domestic and wild) caused by dsDNA virus known as African swine fever virus (ASFV) belonging to the genus Asfivirus. In early 2020, African swine fever (ASF) emerged in India which caused huge economic damage to the piggery sector of North-Eastern Region (NER) of India. The disease entered very recently in India, hence there is no indigenous diagnostics available hitherto. As there is no commercial vaccine available, therefore early diagnosis is an integral part of prevention and control strategies. Objectives: This study aims to develop nucleic acid-based diagnostics for rapid and early detection of ASFV in clinical samples. Materials and Methods: Primers were designed for polymerase spiral reaction (PSR) and Real-Time PCR (qPCR) targeting highly conserved regions of B646L (p72) gene. Additionally a probe was also designed for Real-Time PCR targeting the same gene fragment. The PSR was standardized for optimum reaction conditions (time, temperature) and reagent concentrations. Similarly, qPCR was also optimized for reaction condition and concentration of primers and probe. The sensitivity and specificity of the PSR and qPCR was determined. And finally, both the assays were tested with clinical samples. Results and Conclusion: The PSR assay produced optimum result at 650 C for 40 min and the results could be visualized with naked eyes by adding SYBR Green I dye. A positive reaction exhibited bright green colour whereas the negative reaction remained orange. The qPCR was optimized at 600 C annealing temperature. Both the PSR and qPCR assays were specific for ASFV and none of them produced positive results against other swine pathogens. Compared to conventional PCR, the developed PSR and qPCR assays were more sensitive; however, the sensitivity of qPCR was higher than the PSR. The developed PSR assay is a rapid, cost-effective and simple method to detect ASFV genome with high sensitivity and specificity. It could be used for screening of ASFV suspected clinical samples in remote or rural areas with minimum laboratory set-up. Keywords: ASFV, Diagnostics, PSR, qPCR. Immunogenicity of Bacterially Expressed Recombinant H5HA1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus Clade 2.3.2.1a J. L. Hati Boruah, G. Venkatesh,* S. Nagarajan, D. Senthilkumar, M. Kumar and V.P. Singh ICAR -National Institute of High Security Animal Diseases, Anand Nagar, Bhopal -462 022 The HA1 portion of heamagglutinin protein of H5N1 (H5HA1) highly pathogenic avian influenza virus (HPAIV) belonging to Clade 2.3.2.1a was expressed in E. coli and affinity purified. The purified recombinant H5HA1 (rH5HA1) could be refolded by dialysis against buffers with decreasing concentrations of urea. The refolded rH5HA1 was tested by hemagglutination assay, Western blot and ELISA which showed that it maintained its biological property. Four-week-old specific pathogen free (SPF) chickens were immunized with water-in-oil emulsion of 50 µg of refolded rH5HA1 protein and Montanide ISA 71 VGA. Boosters were given at 21 and 35 days after primary immunization. The humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. All the immunized chickens survived the challenge with 106 EID50 of A/chicken/India/03CL488/2011 of H5N1 HPAIV clade 2.3.2.1a The HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge as evaluated by RT-qPCR. These results indicate that rH5HA1 was able to induce protective immune response in chickens and could be a potential vaccine candidate. Development of C147L gene-based qPCR for the detection of African swine fever virus. Shazia Y1, Choudhary S1, Choudhary RK1, Kumar S2, Barman NN3, Lukumoni B3, and Malik YS1# 1College of Animal Biotechnology Guru Angad Dev Veterinary and Animal Sciences University Firozpur Road, Ludhiana, 141,004, Punjab. 2Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati- 781,039, Assam, India. 3College of Veterinary Science, Assam Agricultural University, Khanapara Campus, Guwahati-781022, Assam, India # Correspondence: [e-mail] Background: India reported African swine fever (ASF) outbreaks in early 2020 from the northeastern region and devastatingly affecting pigs industry. Clinically, ASFV diagnosis is impossible due to its similarity with other porcine viruses. Thus, a laboratory confirmation test is needed to diagnose the disease. Objective: Aim of this study was to develop a qPCR-based test to detect viral nucleic acid for diagnosis. Materials and Methods: We targeted the C147L gene of ASFV. Sequences from NCBI retrived and aligned by multiple sequence alignment to locate conserved regions. Gene specific primers covering conserved regions of C147L were used to design primers. A synthetic construct was prepared in a plasmid cloning vector (pUC57). The E. coli competent cells were used for transformation, followed by plasmid isolation and restriction enzyme digestion to varifiy length of PCR product. Results: The C147L gene-specific primers showed successful amplifction of in qPCR-based real-time assay was developed using SYBR Green chemistry. The specificity of C147L amplification was tested with host-specific other viral DNA (PPV1, PPV2, and CSF) and showed no specific amplification, evidenced by melt curve analysis. The assay's sensitivity was tested using a serial tenfold dilution method showed a detection limit of 1.35 fg of DNA, with an efficiency rate 101%. Specificity of C147L based assay showed no cross reactivity with other procine viruses. Conclusions: These results demonstrate that C147L gene-based qPCR assay was specific and sensitive in detecting ASFV. Keywords: ASFV, C147L gene, qPCR, SYBR Green. Detection of Non-Structural Proteins in inactivated FMDV antigen lots using dot blot immunoassay Uzma Jabeen, Ranjitha H. B., Kailash Singh Bisht, B. P. Sreenivasa, Pratik M. Kulkarni, Aniket Sanyal, Bhanuprakash V., Dechamma H. J., Suresh H. Basagoudanavar ICAR-Indian Veterinary Research Institute, Hebbal, Bengaluru Background: Foot and Mouth Disease (FMD) is a highly transmissible disease of cloven-footed animals caused by FMD Virus (FMDV). Immunization with inactivated vaccines has been used as an effective strategy to control the disease in FMD endemic countries. Rigorous quality control measures are practiced in the production of FMD vaccine so as to eliminate the non-structural proteins (NSPs). Antibodies against structural proteins (SPs) are induced in both vaccinated and naturally infected animals, while antibodies against NSPs are expected only in animals naturally infected with FMDV. Vaccine antigens should be free from NSP antigens as, traces of NSPs in a formulated vaccine can hamper FMD serological surveillance. However, to the best of our knowledge, no quick and cost-effective assay has been developed so far to test the presence of NSPs at the point of production in the process of vaccine manufacturing. This study describes dot blot immunoassay as a simple evidential test for the same. Objective: To detect NSPs in inactivated FMDV antigen preparation. Materials and Methods: Recombinant C-terminal 3A protein was expressed in E. coli expression system and polyclonal serum was raised against this non-structural protein. Inactivated virus preparation was dot-blotted onto PVDF membrane and probed by the polyclonal serum. Sensitivity of the assay was determined by blotting with various dilutions of the recombinant protein. Result and Conclusion: Up to 50 ng of non-structural proteins can be detected by dot blot immunoassay. The assay is specific as it is non-reactive to structural proteins. Thus, it is a simple and sensitive semi-quantitative test which can be performed in the process of antigen preparation to ascertain the vaccine NSP free. Detection of Kyasanur Forest Disease Virus in different tick species from Southern India Bhimanagoud K*1&4, M Mudassar Chanda1, Kundave VR1, Balakrishnan N1, Stefanie Schäfer2, Sarah Burthe2, Abi Vanak3, Abhijitkumar N3, Suresh DK1, Santoshkumar P4, Mujeeb Rahman3, Darshan N4, Bethan Purse2, SL Hoti4 1ICAR-National Institute of Veterinary Epidemiology and Disease Informatics, Yelahanka, Bangalore-64, Karnataka, India. 2Centre for Ecology and Hydrology, United Kingdom. 3Ashoka Trust for Research in Ecology and the Environment, Bangalore, Karnataka, India. 4ICMR-National Institute of Traditional Medicine, Belagavi, Karnataka, India *Presenting author Background: Kyasanur forest disease (KFD) is a zoonotic tick-borne haemorrhagic fever, first reported in 1957 in the Western Ghats Forest, caused by KFD virus (genus Flavivirus: family Flaviviridae). The virus has been isolated in nature from man, monkeys, small mammals and ground nesting bird and several species of ticks. KFDV is transmitted to humans by the bite of infected ticks majorly Haemaphysalis species. Given extensive forest degradation and potential population changes in key vectors and reservoir species, this study aimed to better understand vector species involved in KFD transmission at the interface between agriculture, human habitation, and forest. Objectives: Collection of ticks from endemic areas of KFD Real-time PCR based detection of KFDV Materials and Methods: Tick samples were collected from different agro-forest habitats surrounding 30 villages in Karnataka state and Kerala state. Ticks were identified to species level, and 847 individual tick samples were assayed for the presence of KFDV using highly sensitive and specific Real-time PCR. Results and Conclusions: Kyasanur forest disease virus was detected in 16 tick samples. The KFD virus was detected in five, different tick species collected from forest, crop/plantation, and village habitat; Haemaphysalis spinigera (n = 5), Haemaphysalis bispinosa (n = 1), Haemaphysalis species (n = 2), Ixodes species (n = 5) and Rhipicephalus microplus (n = 2). The study indicated that the KFD virus is circulating in different species of ticks. Further work is needed on vector competence and host feeding of these species in order to understand the role of different ticks in the transmission of KFDV and to plan systematic vector control strategies. Role of Climatic Factors in Temporal Variation Inoccurrence of Japanese Encephalitis Cases in India Using Time Series Modelling Priyanka Kharkwal*1, Bethan V Purse2, Richard Hassel2, Bhimanagoud Kumbar1, Dhanya1, Nikesh1 and Md. Mudassar Chanda1 1ICAR- NIVEDI (National Institute of Veterinary Epidemiology and Disease Informatics), Ramagondanahalli, Yelahanka, Bengaluru-64, Karnataka, India. 2UK Centre for Ecology and Hydrology, United Kingdom *Presenting author Background and Objectives: Japanese Encephalitis (JE) is a vector borne viral zoonosis caused by the Japanese Encephalitis virus of the family Flaviviridae and genus Flavivirus which is transmitted from animals to humans through bite of infected mosquito (Culex tritaeniorhynchus). It is the leading cause of encephalitis and related deaths in India among children. India, for years has seen an increase in number of JE cases which might be affected by the changes in climatic conditions. The studies on association between climatic factors and JE cases becomes more important when there are only a limited studies worldwide. This study was also undertaken to develop forecasting models for JE in India using machine learning. Material and Methods: JE occurrence data from year 2009–2018 was analysed. Minimum, mean temperature and precipitation values was used to identify correlation and develop forecasting models for JE in India. Temporal models were used to predict JE cases in India. Auto Regressive Integrated Moving Average (ARIMA) models were used for predictive modelling in R software. Results: There was significant and a positive association of rainfall and minimum temperature with JE cases in India. Forecasting models were developed to forecast the occurrence of JE cases in India using climatic parameters. Conclusions: Further forecasting models needs to develop at state level for implementation of routine and timely vaccination. This would help authorities be bettered prepared to handle a predicted amount of cases within the given climatic zones in future and to deploy essential resources and manpower accordingly. Assessing the Incubation Period of Rabies Virus in Cattle, Buffaloes and Fatality in Exposed Livestock with Dog Bite Site Above the Neck Region in Maharashtra Ashok Bhosale1, Sambhaji Chavhan2, Swati Sakhare1, Mohini Kamble1, K Mallinath3, Baswaraj Awati3, Arun Kharate3, Gopal Bharkad2 and Ravaji Mugale2 Department of Veterinary Microbiology1, College of Veterinary & Animal Sciences, Udgir2, Maharashtra Animal and Fishery Sciences University, Nagpur, Maharashtra Veterinary College, Bidar, Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar, Karnataka3 Abstract: A total of 86 cases in cattle and buffaloes with dog bite site above the neck region were studied for the time of onset of symptoms of rabies and distance from the brain. Reported 100 percent fatality, even after post exposure vaccination alone, were confirmed using direct fluorescent test and RT-PCR using N gene amplification. The prediction of onset of time of symptom stands to be very accurate in cattle and buffaloes, in cases under study. The prediction of incubation varied in calf, heifers and adult buffaloes ranges from 15 to 20 days in dog bite site above the neck region. Intervention with local instillation of diluted rabies immunoglobulin @100 IU, at each site of bite within seven days after the dog bite, prevented the death of 100 percent in treatment group. The study concluded the need for use of rabies immunoglobulin (RIG) as local instillation and the dog bite wound above the neck region should be considered as Category III and should be treated with vaccine and RIG as a post exposure regime. Characterization of Elephant endotheliotropic herpesviruses circulating in Assam Sophia M Gogoi, Arpita Bharali, Madhusmita Dehingia, Durlav P Bora, Lukumoni Buragohain, Kushal K Sharma and Nagendra N Barman College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India *Presenting Author: Sophia M. Gogoi, Email ID: [e-mail] Background: The Asian elephant (Elephas maximus) population worldwide faces a significant threat due to Elephant endotheliotropic herpesvirus (EEHV) infections which are severe, acute and often causes fatality in young elephants. The causative agent, Elephant endotheliotropic herpesvirus (EEHV) is classified under the genus Proboscivirus of the Herpesviridae family and Betaherpesvirinae, subfamily. There are eight genotypes of EEHV which includes EEHV1A, EEHV1B, and EEHV2–7. EEHVs are capable of causing latent infections and can be shed intermittently by healthy elephants. Objectives: This study was envisaged to detect the presence of EEHV in the apparently healthy captive elephant population of Assam. Materials and Methods: Whole blood samples collected from apparently healthy captive elephants which were received at the Animal Disease Diagnostic and Vaccine Research Centre, College of Veterinary Science, Assam Agricultural University were selected for this study. Viral DNA was extracted from 115 blood samples and the presence of EEHV was confirmed by amplification of the U38 gene employing published primers. Representative 3 positive samples were outsourced for sequencing and the nucleotide sequences thus obtained were analysed using relevant softwares. The phylogenetic tree was constructed by the neighbour joining method using MEGA X. Results and Conclusion: Among the samples screened, 16 were found to be positive by PCR. BLAST analysis of the nucleotide sequences of the 3 selected positive samples revealed more than 98% identity with the other EEHV 1A sequences available in the NCBI website. Similarly, by close observation of the phylogenetic tree it was evident that the sequences obtained in this study clustered along with the other EEHV 1A sequences from the database while the other EEHV types formed separate clusters. Presence of the virus in healthy elephants highlights the importance of regular screening of elephant herds in this region over an extended duration in order to establish the latency and viral shedding pattern, which in turn will help in devising effective control strategies. Keywords: EEHV, diagnostics, captive elephant, latency. Characterization of Avipox viruses isolated from the duck population of Assam Sumi Chungkrang, Durlav P Bora *, Sophia M Gogoi, Sutopa Das, Deep Prakash Saikia and Nagendra N Barman College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India *Presenting Author: Durlav P Bora, Email ID: [e-mail] Background: Avipoxvirus (APV) is a slow spreading, highly contagious viral disease causing morbidity and mortality in both domesticated and free ranging birds which results in economic losses in poultry industry. APV in duck is endemic in Assam. A few studies have been conducted on APV in duck. Farmers of Assam rear duck without following much scientific managemental practice leading to increase the chance of APV infection in duck. Objective: The present study was undertaken to study the prevalence of Avipoxvirus in duck population of Assam, to isolate the virus in suitable system and to characterize the isolated virus by molecular techniques. Materials and Methods: Clinical samples in the form of tissue scab/pox lesion were collected from affected ducks from various farms and households of different districts of Assam.. The presence of the APV in the samples was detected by PCR amplification of P4b core gene. Representative positive samples were initially adapted in chorioallantoic membrane (CAM) of embryonated duck/chicken egg followed by adaptation in chicken embryo fibroblast (primary) as well as vero cell lines. Successful isolation of the virus was confirmed by observing virus specific CPE and PCR amplification of P4b core gene. The P4b core gene amplicons were sequenced and phylogenetic tree was constructed. Results and conclusions: Out of 61 samples collected from the infected ducks of Assam, 57 (95%) were found to be positive for APV. According to age group, highest number of positivity was recorded in 0–8 weeks (45/45) followed by 9–20 weeks (12/14). Among different age groups the highest morbidity was recorded at 0–8 weeks (85%) followed by 9–20 weeks (60.9%) and above 20 weeks (11.1%). The total morbidity 71.4% and cause specific mortality 14.06% was recorded in this study. A total of five isolates were selected for isolation in embryonated eggs, CEF/DEF and Vero cell line. Morphological changes such as oedematous thickening, swelling and typical pock lesion on CAM and characteristic CPE including rounding, vacuolation, shrinking and detachment of cells were observed. CPE like rounding and detachment of cells were observed from the 2nd passage and from the 8th passage in CAM adapted isolates and field isolates respectively in Vero cell line. Isolation of APV at each passage level was confirmed by conventional PCR targeting the P4b core gene. Molecular characterization of the isolated APV was done by cloning and sequence analysis revealed the sharing of P4b gene under present study with other APVs reported from different parts of the world at nucleotide level (nt) level. The detection of APV in ducks warrants the need for comprehensive studies to evaluate the virus pathogenicity in other species of poultry and to establish its implications on the poultry industry. Keywords: Avipox virus (APV), Duck, Isolation, P4b gene, Molecular characterization, Assam. Antimicrobial Efficacy of Aqueous Extract of Ginger (Zingiberofficinale) in Spent Hen Meat Mir Rovida*, Sarfaraz A. Wani, Sheikh Rafeh Ahmad, Asif H. Sofi and Tahir Nazir *Corresponding author email ID:[e-mail] Abstract: The present study was done to evaluate the efficacy of aqueous extract of ginger (Zingiberofficinale) as natural preservative in thigh meat obtained from spent hen of Commercial Layer Stock. The thigh pieces of spent hens treated with five treatment solutions viz. 0.2% papain as positive control (T1), naive control without any treatment (T2); 2% Ginger Extract (T3); 4% Ginger Extract (T4); and 6% Ginger Extract (T5) were kept for 24 h at 4 °C. One group of samples was then cooked for 35 min in 1.5% salt solution. To evaluate the storage stability of raw and cooked samples treated with 6% Ginger Extract (selected as optimum), the samples packed in LDPE bags at 4 ± 1 °C wereevaluated for microbiological and other quality parameters for 21 days. The total plate count showed significant increase from day 0 to day 21 in both raw and cooked samples. Coliform counts showed growth after day 7 and day 14 in raw and cooked samples, respectively, whereas, yeast and mold counts were observed on day 21 only. The values of microbial counts were within acceptable limits for all days of storage period in all the samples. Although, the sensory scores decreased with storage period for all attributes but remained acceptable throughout the period. It was concluded that ginger extract can be exploited for itsanti-microbiological ability in thigh meat of commercial layer stock without adversely affecting their quality under refrigerating storage conditions. Keywords: Commercial layer stock, ginger, thigh, microbiological, refrigerating storage, spent hen. Reverse Transcription Recombinase Polymerase Amplification integrated with CRISPR Technology and LFA for point-of-care detection of SARS-CoV-2 virus Bera B. C., Virmani Nitin and Anand Taruna ICAR- National Research Centre on Equines, Sirsa Road, Hisar, Haryana Corresponding author: Dr. B.C. Bera, Sr. Scientist; email: [e-mail] The field-deployable point-of-care diagnostic test for rapid detection of SARS-COV-2 is needed for implementation of the control measures. In this direction, recently developed CRISPR technology combined with isothermal recombinase polymerase amplification assay is a versatile highly sensitive detection platform for rapid diagnosis of infectious diseases. Here we report the development of RT-RPA-CRISPR based LFA assay for detection of SARS-CoV-2 targeting conserved RdRp and E genes. Various sets of primers and gRNAs were designed targeting conserved regions of the RdRp and E genes of different lineages of SARS-CoV-2 viruses. The isothermal RT-RPA based amplification reactions were standardized using in-vitro transcribed RNAs of the target regions. The optimum amplification was observed at 42 °C for 30 min as confirmed by visualization of the amplicons in agarose gel. Subsequently, CRISPR-CAS12 reaction was implemented for specific detection of amplicons. Different sets of gRNAs targeting RdRp and E genes were designed and synthesized by in-vitro transcription. The CRISP/CAS12-gRNA complex and single stranded fluorescence probe were added to the RT-RPA amplicons for cleavage of fluorescence probe in positive reaction. Subsequently, the cleaved probes were detected in pre-coated LFA strips. Upon probe cleavage reaction, the product was mixed with buffer and loaded into LFA strips. In positive reaction, test line showed strong band in test line and light band in control line. The standardized RT-RPA-CRISPR-LFA assay was tested for detection of SARS-CoV-2 using previously isolated RNAs from clinical cases of human SARS-CoV-2 infections. The developed assay successfully detected the positive cases. In conclusion, the developed assay could serve as versatile POC platform for rapid detection of SARS-CoV-2 nucleic acids in human as well as animals. Insights on the spread of Lumpy Skin Disease in Kashmir Division Anjum Andrabi1; A. Muhee2; M.A. Bhat3; Ishrat Shakeel1 and Arif Shikari4 1Dept of Animal Husbandry, Kashmir; 2Division of VEPM, FVSc & AH, Shuhama, 3Division of Vety. Microbiology & Immunology, FVSc & AH, Shuhama, 4 Technical Officer, AHD Background: Lumpy skin disease (LSD) is a viral disease caused by Lumpy skin disease virus which is genetically related to the goat pox and sheep pox virus family. It is an emerging threat to livestock worldwide. It is a highly contagious trans boundary disease of high economic importance that infects cattle and water buffalo mainly through vectors (blood-sucking insects) like mosquitoes, flies, ticks and also through saliva, contaminated water and food. In India, which has the world’s highest bovine population numbering 303 million, the first outbreak of LSD was reported from Odisha in August 2019 and the disease has since then spread to 21 states mostly during 2022 in a more severe form. Objectives: The study was conducted to study the pattern of spread of LSD in Kashmir Division. Material and Methods: The UT of Jammu and Kashmir shares borders with the states of Punjab and Himachal Pradesh and the first case of LSD was reported in Jammu division on 11th July 2022, largely because of transboundary trade and import of cattle from these states. The first case in Kashmir division was reported in remote Uri area (Baramulla) on 27th July 2022. All other districts reported their first cases in the month of August mostly along the cattle import and mandi routes. The Government of UT of J&K immediately notified the disease under Prevention & Control of Infectious and Contagious diseases in Animals Act, 2009 and took urgent steps to stop the spread of disease by employing measures like LSD vaccination campaigns, ban on import & movement of animals, fogging of infected zones, constitution of rapid response teams, public awareness besides, sampling & testing and proper carcass disposal. Results: The disease continues to spread in all the districts of the Kashmir Division with more than 13,000 cases reported till date with 900 mortalities. So far, over 1250 blood samples have been collected from animals with clinical signs of LSD from different districts of the Kashmir Division. The samples were initially sent to National Institute of High Security Animal Diseases (NIHSAD), Bhopal but later the testing facilities were developed at Institute of Animal Health and Biological Products (IAH&BP), Zakura Srinagar (Dept of Animal Husbandry). So far, 320 samples have been confirmed positive as per the OIE prescribed methods (PCR, Real-time PCR, nucleotide sequencing and virus isolation). For differential diagnosis, representative LSDV positive scab samples were tested by PCR and found negative for pseudo lumpy skin disease caused by BHV-2, buffalopox caused by BPXV, cowpox caused by CPXV, pseudocowpox caused by PCPV and bovine pappular stomatitis caused by BPSV. Following virus isolation, identifcation of LSDV was carried out by real-time PCR and DNA sequencing. Nucleotide sequence analyses of partial P32 and F genomic sequences showed highest genetic identity with globally circulating LSDV field strains and complete identity with the sequences obtained from clinical samples confirming the isolation. Conclusion: Till date more than 7000 animals have recovered from the disease in Kashmir with an equal number of active infections. More than 3.89 lac cattle have been vaccinated against LSD using Goat pox vaccine-Uttarkashi Strain, at a dose of 103.0 TCID50. The total cattle population of Kashmir division is 11 lacs (approximately) with more than 90% of the population being cross breds which makes the cattle population more susceptible to the occurrence of disease. Further, illegal transportation/movement of cattle, lack of quarantine, inadequate vector control and low levels/coverage of vaccination could be the possible factors contributing to the spread of the disease in the Kashmir division. Veterinary Virology (Oral) Unusual Equine Rotaviruses of Bat and Bovine Origin Reported From India: Implication in Vaccine Development Baldev R. Gulati* and Anubha Pathak Indian Council of Agricultural Research -National Research Centre on Equines, Hisar, Haryana Background: Rotaviruses are the most common viral agents associated with diarrhea in young animals and humans. Reassortment of segments of RVA strains from multiple species drives the diversity of strains among animals along with zoonotic transmissions between animals and humans. An annual prevalence of rotavirus in diarrheic foals in India has been reported to be ranging between 18 and 28%. However, the complete genomic constellation of ERVAs of India have not been studied. Objectives: Whole genome sequencing of four ERVA isolates (RVA/Horse-wt/IND/ERV2/2015/G6P[1], RVA/Horse-wt/IND/ERV4/2017, RVA/Horse-wt/IND/ERV6/2017 and RVA/Horse-wt/IND/ERV3/2003/G6P[1] was carried out to understand the diversity, inter-species transmission and reassortment of ERVAs of India. Materials and Methods: Isolates were confirmed using sandwich ELISA and RNA-PAGE and isolated by passaging in MA104 cells. Whole genome sequencing was done using Illumina platform and the GCs were inferred using RotaC2.0 annotation pipeline. The phylogenetic analysis was conducted using MEGA11 using appropriate models. Results and Conclusion: All the ERVAs revealed novel GCs, the isolates ERV2 and ERV3 revealed G6-P[1-]I2-R2-C2-M2-A3-N2-T6-E2-H3 GC which is identical to the NCDV strain of bovine and Ro8059, a human strains isolated from a bovine to human zoonotic case in Israel in 1995. Whereas a bat-like GC of ERV4 and ERV6 G3-P[3]-I8-R3-C3-M3-A9-N3-T3-E3-H6 identical to bat RVA MSLH14 isolated from China in 2012. The genotypes P[1], A3 and I8 of VP4, NSP1 and VP4 have been reported from equines for the first time. The phylogenetic analysis of individual genes revealed that most of the genes of ERV2 and ERV3 clustered with NCDV and RF strains of bovine RVA. Whereas the genes of ERV4 and ERV6 clustered with RVAs of Africa/China or with human strains of bat origin. These unusual findings highlight that interspecies transmission of rotaviral segments is an active phenomenon. The surveillance of animal rotaviruses is of utmost importance to identify new reassortants with possibility of zoonotic spillover and vaccine resistance, for development of control measures, including development of vaccine. Bluetongue: Current Status in Karnataka Divakar Hemadri and Md. Mudassar Chanda ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI) PB No. 6450, Yelahanka, Bengaluru-560064 Abstract: Bluetongue is a culicoides borne economically important disease of ruminants especially sheep. The disease, which was reported for the first time in the country during the early 1960s had later spread to various parts of the country. Currently, the disease is reported most frequently from the southern states. Since its first report in 1980s, the bluetongue is reported almost every year in the state of Karnataka, which ranks third in terms of sheep population in India. The cuastive agent, bluetongue virus, which belongs to the genus Orbivirus and family Sedoreoviridae, exists as 28 seroloically and genetically different types. The paper discusses about prevalence and distribution of BTV serotypes, antibodies, epidemiology, diagnosis, prevention and control strategies adapted in the state of Karnataka. African Swine Fever, the current status Yashpal Singh Malik College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab-141003 Email: [e-mail] In the current scenario, livestock contributes a large share of global agricultural economy. Incidence of various animal health emergencies in past suggested the vulnerability of this particular industry to emerging infectious diseases. Emerging diseases are the ones, those appear recently in a population or whose incidence or geographic range is increasing at a threatening pace. Emerging diseases with potential to spread across borders, often termed as ‘Transboundary disease’, pose one of the major risk to international trade and impact the dynamism as well as flexibility of agronomic input markets. These diseases may also possess the zoonotic potential and affect human health. Various emerging/re-emerging viral pathogens affecting porcine population include African swine fever virus, Nipah virus, Hepatitis E virus, Japanese encephalitis virus, porcine sapovirus, Seneca virus, Torque teno sus virus, and PRRS virus etc. African swine fever (ASF) is one of the highly contagious transboundary emerging diseases affecting porcine population. Major pig population of India is concentrated in North-eastern region. After it’s first emergence in the North-eastern states during 2020, it localised there for nearly two years. Recently, the disease spread in many northern states and southern state Kerala causing huge mortality among the affected swine herds. Lack of insufficient diagnostic as well as effective therapeutic strategies are major bottle neck of controlling ASF in India. Moreover, there is no suitable vaccine available at present in the country. Biotechnological approaches have helped the scientific community involved in veterinary and human health profession tremendously. In the same line, these approaches are exploited for development of various diagnostic, therapeutic and prophylactic interventions. In the context of ASF, various biotechnological tools have proved much useful for basic research such as understanding the pathogen dynamism and disease severity as well as applied research related to development of suitable diagnostics, antiviral drugs, and prophylactic approaches etc. Keywords: Emerging, Transboundary, Porcine, African swine fever, Biotechnology. Evaluation of neutralizing efficacy of Classical swine fever C-strain specific antibody against genotype 2 Jayashree Sarma1*, Nagendra Nath Barma1, Sophia M. Gogoi1, Durlav Prasad Bora1, Arijit Shome2, Arpita Bharali1, Lukumoni Buragohain3 1Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. 2Department of Biochemistry, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. 3Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India Background: Classical swine fever (CSF) or Hog cholera is a highly contagious viral disease of domestic and wild porcine population. The disease is highly endemic in India including North-Eastern Region (NER) and state of Assam records highest CSF outbreaks. Besides historical sub-genotype, several other sub-genotypes (1.2, 2.1 and 2.2) have been reported from India. Although there are vaccines against CSF, however outbreaks in vaccinated herds and global shift in genotypefrom 1 to 2, have raised the concern over the antigenic variation, protective immune response and neutralizing capacity of C-strain vaccine antibody. Thus, it is essential to determine the cross-protective efficacy of available vaccines against other prevailing genotypes. Objective: The present study was undertaken to explore the cross-neutralization efficacy of C-strain vaccine antibody against different genotypes. Materials and Methods: Archived classical swine fever virus (CSFV) positive samples were selected from the repository of the Department of Veterinary Microbiology, AAU, Guwahati, Assam. The samples were reconfirmed for CSFV antigen/genome by Sandwich ELISA and nested RT-PCR. Then randomly a few samples representing different states of NER were subjected to PCR amplification and sequencing of full-length E2 gene followed by phylogenetic analysis in MEGA X software. The representative samples belonging to different genotypes were isolated in PK-15 cells. Then hyper-immune serum was raised using purified cell culture adapted lapinised C-strain vaccine candidate, and the hyper-immune serum was used to determine the neutralization and cross–neutralization efficacy against different genotypes isolated in PK-15 cells. Results and Conclusion: Out of 49 archived samples, 18 numbers were reconfirmed to be positive in S-ELISA or nested RT-PCR. Samples representing different states of NER were sequenced for full-length E2 gene and phylogenetic analysis revealed that they belong to either 1.1 or 2.2 sub-genotype. Four samples representing two sub-genotypes (1.1 & 2.2) were propagated in PK-15 cells up to 5 passages and titre of the viruses were between 4.49–5.16 log TCID50 per ml. Neutralization and cross –neutralization assay with C-strain specific antibody showed 100% neutralization with sub-genotype 1.1, whereas 84% in sub-genotype 2.2. Thus, the study revealed that sub-genotypes 1.1 and 2.2 are widely circulating in NER and neutralization efficacy of antibodies generated by vaccine strain is lower to heterologous genotypes, though immunogenic epitope is conserved. Keywords: CSF, C-strain vaccine, Genotypes, NER, Neutralization efficacy. Repurposing Goatpox Virus Vaccine for prevention of Lumpy Skin Disease: Lessons learnt from current LSD outbreak Kajal Patel, Rajesh Kumar Singh, Manoj Kumar Chhikara* Hester Biosciences Limited, Mehsana, Gujarat-382721 Email: [e-mail] Background and objectives: Recent Lumpy Skin Disease (LSD) outbreak in India has caused a major loss to the farmers due to high morbidity and mortality in cattle. The Goatpox virus (GPV) vaccine was recommended by the Government of India for prevention of LSD in cattle. Various challenges were observed in implementation of the recommendations and supply of the vaccines. The presentation will focus on the lessons learnt from the manufacturer’s perspective. Materials and Methods: A vero cell based GPV vaccine is being manufactured by Hester Biosciences Limited. The vaccine with virus titre of not less than 10^3.0 TCID50/ml is being supplied to different entities/agencies for vaccination of cattle in affected areas. Efforts have been made to ensure the vaccine supply to the best possible extent to different stake holders. Continuous dialogue with the customers and government agencies are held to fulfil the demand amidst the guideline revisions. Continuous education was imparted to the farmers through training programs, exhibitions and seminars and visits by the technical services team whenever required to clarify the ongoing doubts related to the guidelines. Detailed studies to evaluate GPV vaccine efficacy in prevention of LSD have been initiated. Results and conclusions: The repurposed Hester’s GPV vaccine has proven to be highly effective in preventing LSD in areas of implementation of the vaccine. However, a confusion was prevailing in the field for lack of clarity of virus titre and volume of the vaccine to be given. Significant support is received from the Government and regulatory agencies in coping up with the supply chain and bringing in clarity on the prevailing confusion. Further data on GPV efficacy against LSD will help drive the clear guidelines for implementation of repurposed vaccine while the efforts on homologous LSD vaccine development are ongoing. Evaluation of different stabilizer formulations for lyophilization of Live attenuated Duck plague vaccine Jonmoni Barua1, Sutopa Das1*, Sophia M. Gogoi1, Durlav Prasad Bora1, Rupam Dutta2, Rita Nath3 and N. N. Barman1 1Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. 2Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. 3Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India Presenting Author: Dr Sutopa Das 1* , Mail ID: [e-mail] Background: Duck plague (DP) also known as Duck Viral Enteritis (DVE), Anatid Herpesvirus is an acute contagious herpesvirus infection of ducks and waterfowl and members of the family Anatidae. The only effective method for preventing and controlling DVE is vaccination. India experiences a wide range of temperatures due to its subtropical climate. As a result, maintaining the vaccine's shelf life is very difficult and requires a cold chain. The live attenuated DPV vaccine is thermally inactivated like many other modified live attenuated lyophilized vaccines. The vaccine needs to be transported and stored in a cold chain. Because of this, users must pay high production and transportation costs, which is especially true in developing tropical and semitropical nations like India. Evaluation of the vaccines thus represents the protection provided by immunization. In order to maintain or increase immunization effectiveness through reduction of potency loss due to heat damage; reduce cold chain costs and logistical requirements, their thermo-stability with various stabilizers is of very high demand. Objective: The present study was undertaken to evaluate different stabilizer formulations for lyophilization of Live attenuated Duck plague vaccine. Materials & Methods: The Chicken Embryo Fibroblast adapted duck plague virus vaccine strain available in the DBT-ADMaC, Department of Microbiology, College of Veterinary Science was selected for Evaluation of different stabilizer formulations for lyophilization of Live attenuated Duck plague vaccine. The vaccine virus was revived in CEF and identity was checked with appearance of CPE, PCR and Molecular characterization. After positive identity the vaccine virus was produced at bulk for the study. A total of three stabilizers combinations namely-(1) Lactalbumin hydrolysate (LAH) + Sucrose (S); (2) Pullulan (P) + Trehalose (T)  + Inulin (I); (3) Lactalbumin hydrolysate (LAH) + Trehalose (T) was used for lyophilization of the vaccine virus. The lyophilized vaccine vials were kept at 4 °C, 25 °C, 37 °C and 45 °C. The time of exposure selected for various temperatures were 3 months at 4 °C and 25 °C, while 14 days at 37 °C and 48 h at 45 °C. The vaccines were reconstituted with PBS and NSS as diluents to check thermo-stability at 4 °C, 25 °C, and 37 °C for 48 h. The stability was mainly determined by titrating the treated vaccine virus to find out the infectivity titre to evaluate different stabilizer formulations for lyophilization of Live attenuated Duck plague vaccine. Results and Conclusion: The mean infectivity titre (TCID50/ml) of the DPV vaccine virus propagated in CEF for bulk production was calculated and was recorded to be 6.9 ± 0.17 TCID50/ml. Results revealed that, at 4 °C, all three stabilizers were comparable in maintaining the infectivity titre with minimal or no loss, however, stabilizer LHT showed better stability compared to LS and PTI. At 25 °C, PTI showed better stability than LS and LHT though there was not much significant difference. Being close to the room temperature, all the vaccines can retain the infectivity titre at 25 °C also. At a temperature of 37 °C, the vaccine prepared in the present study using an LHT stabilizer comparatively showed better stability in comparison to the other two stabilizers. At a temperature of 45 °C, all three stabilizers retained their infectivity titre to remain a vaccine candidate even after exposure for up to 48 h. Although LHT showed much better stability than the other two. In case of reconstituted vaccine, at 4 °C LS, PTI and LHT showed a very good titre even after 48 h. The titre (log10 TCID50/ml) did not fall more than 1, it was less than 1 for all the stabilizers with both the diluents used. At higher temperatures (25 °C, and 37 °C) there was a fall in titre by 1 in case of LS diluted with PBS after 36 h, but in case of PTI and LHT even at higher temperatures, the titre did not fall by log 1 with both the diluents. Thus, the study revealed that the vial with LHT stabiliser displayed better titre during the study period when lyophilized vaccines were kept for thermostability evaluation at different temperatures. And NSS was found to be a better diluent than PBS, for reconstitution of the lyophilized vaccine vials and thermostability evaluation. Although PBS can be used as a substitute for NSS. Keywords: DPV vaccine, CEF, LS, PTI, LHT, Lyophilization, NSS, PBS. Marek Disease virus: An important transboundary viral disease of poultry and in-vitro restriction of its oncogenic marker by stimulator of interferon genes (STING). Hosterson Kylla*, Muhammad Munir, Mustafa Ozan Atasoy, Muhammad A. Rohaim, Shilong Chen *Disease Investigation & Surveillance. A.H & Veterinary Department. Meghalaya, India Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YG, United Kingdom Marek’s disease is an economically important neoplastic disease caused by Marek’s disease virus (MDV), an avian alphaherpesvirus that causes a fatal disease in poultry worldwide. The disease is frequently associated with immunosuppression that leads to high morbidity and mortality. OIE data estimates that about half of the world countries have reported cases of MD and costs the poultry industry more than 1 billion US dollar annually. The infection is very much prevalent in the poultry population of Meghalaya State (India) where many outbreak were recorded by molecular detection of important viral genes and in many of cases these outbreak are associated with transboundary of poultry from other states. The innate immune system acts as a first-line of defense against MDV in which Stimulator of interferon gene (STING), a signal junction molecule that hosts the perception of viral nucleic acids and produces type I interferon response plays a very crucial role. Meq oncogene (1020 bp) of MD virus was amplify and clone into a plasmid vector pCAGGs-HA-NH2 for expression study in chicken fibroblast cells (DF-1). STING-Flag in pCAGGs plasmid was used for in-vitro study to demonstrate an interaction of Meq oncogene with STING protein. It is observed that in DF-1 cells, when STING was not expressed, an overexpression of Meq oncogene was observed. However, cells in which STING was transfected and overexpressed together with Meq, it was observed that there was inhibition of expression of Meq oncogene as visualized by confocal microscopy and western blotting. In-vitro experiment indicated that overexpression of STING in cells inhibit the Meq oncogene of MDV, thus suggesting that STING plays an important role in antiviral signaling pathways in chickens, and this can be further explore in-vivo studies in regards to viral oncogenicity. In situ examination and histopathology based investigation of Lumpy skin disease Outbreak in Uttar Pradesh, India Amit Kumar1*, Vikas Jaiswal2, Surandra Upadhyay1, Saumya Jaiswal1, N.N. Mohanty3, Vikas Gupta3 and R.S. Chauhan4 1Division of Animal Biotechnology, College of Biotechnology, SVPUAT, Meerut, India. 2Department of Veterinary Pathology, College of Veterinary Sciences, SVPUAT, Meerut, India. 3National Institute of Animal Health, Baghpat, India. 4Department of Pathology, College of Veterinary Sciences, GBPUAT, Pantnagar, India *Corresponding author Associate Professor & Officer Incharge, Division of Animal Biotechnology, College of Biotechnology, Sardar Vallabhbhai Patel University of Agriculture & Technology (SVPUAT), Modipuram, Meerut-250110, Uttar Pradesh, India Email: [e-mail], M: 9412120813 The Lumpy skin disease has caused more than 67,000 causalities of cattle during July to September 2022 and spread in 8 states of North West India with the mortality rate of more than 5%. Uttar Pradesh is the state with largest cattle population in India, has reported more than 25,000 cases in 2,600 villages of 25 districts in last one month and more than 1.5 million cattle are in the infection zone. Initially Zebu cattle were supposed to be the resistant. However, the deaths of cattle in affected states showed zebu cattle with higher rate of mortality and delayed recovery time. Therefore, to understand the disease and its impact on animals, the present investigation was conducted. The affected animals were clinically examined and postmortem were conducted. The tissues were also used for the molecular confirmation of LSD virus. In situ examination revealed generalized nodular and crust like cutaneous lesions all over the body starting from muzzle to vulva/rectum and swelling of lymph nodes and fetlock joints. Necropsy revealed nodular or circular lesions on organs like tongue, epiglottis, liver, lung, spleen, abomasum, uterus and heart. The mucosal surface of eye, buccal cavity, peritoneum and mesentery were severely affected with localized hemorrhages. The histopathology revealed epidermal hyperplasia, ballooning degeneration, intraplasmic eosinophilic viral inclusions, necrosis, and bacterial infiltration into the skin, hemosiderosis in spleen, interstitial pneumonia, edema and infiltration of inflammatory exudates in lungs, depletion of lymphocytes and atrophy of hepatocytes and sinusoidal dilation in liver. The generalized lesions in and outside of body with pathological changes in almost all the organs suggest the involvement of a highly pathogenic strain in the current outbreak. It could be the cause of the high mortality in cattle during the current epidemic. Keywords: Lumpy Skin Disease, In-situ examination, Histopathology, Outbreak, India. Complete genome analysis of bacterial virus “Escherichia phage PJDM” infecting multidrug resistant APEC Punit Jhandai*1, Dinesh Mittal1 Department of Veterinary Public Health and Epidemiology Lala Lajpat Rai university of Veterinary and Animal Sciences, Hisar-125004 Background: Accumulating evidences of MDR pathogens suggest that it is imperative to explore novel and more efficacious alternatives for combating bacterial infections in the context of public health. Bacteriophages are ubiquitous bacterial viruses (natural bacteria killers) that occur in diverse environments amounting to approximately 1031 phage virions on earth. Objectives: Whole genome sequencing of bacteriophage. Material and methods: The DNA of phage PJDM was extracted according to the experimental protocol described by Gill, 2015. The whole genome sequencing of extracted DNA phage PJDM was carried out using NextSeq 500 with 2 × 150 bp chemistry. Genes in phage sample were predicted using Prokka and searched against NCBI non redundant protein database (Nr) and nucleotide database using Diamond (BlastX and BlastN mode). Results: The whole genome analysis of bacteriophage results revealed that the phage PJDM genome has a double-stranded linear DNA molecule and its genomic DNA contains 57,756 base pairs with a GC content of 43.58%. It was also indicated that the molecular weight of phage DNA was around 21–134 kbp which belong to the family Siphoviridae of order Caudovirales. The open reading frames (ORFs) of Escherichia phage PJDM were identified by blastX search and a total of 98 ORFs were predicted and there were no putative tRNA genes in genome implying its dependence on host tRNA for protein synthesis. Among these genes, 34 genes were predicted to have known functions and are grouped into three functional modules. Functional modules are phage packaging and lysis (7 ORFs), phage structure and assembly (10 ORFs) and DNA metabolism and replication (11 ORFs). Additionally, six auxiliary metabolic genes (AMGs) were predicted and 64 ORFs were predicted to encode hypothetical proteins. Holin, a tiny hydrophobic protein that is encoded by ORF 63, produces a hole in the host by oligomerizing in the cytoplasmic membrane. Endolysin, which cleaves the peptidoglycan in the cell wall, is encoded by ORF 64. They together form the traditional holin-endolysin lysis system. Endolysin enters the cell wall through the pore that holin has created and then completes the host cell wall. Conclusion: Functional annotation of genes of phage PJDM revealed it is lytic in nature against multidrug resistant APEC. Complete genome sequence analysis of the first Indian isolate of bovine ephemeral fever virus (BEFV) Shruti Pyasi1, and Debasis Nayak2* 1Department of Biosciences and Biomedical Engineering, Indian Institute of Technology, Indore 453552, India. 2Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal, MP, 462066, India *Corresponding author: Dr. Debasis Nayak, Cell Room No: 327 Indian Institute of Science Education and Research Bhopal. Email: [e-mail] In the present study, we elucidate the epidemiological characteristics of an epizootic virus, bovine ephemeral fever virus BEFV, the causative agent of BEF disease. We recorded the complete genome sequence of BEFV for the first time from an Indian outbreak (2018–2019). The majority of BEFV cases are acute infections with only mild fever and leukopenia. Despite the fact that these subclinical diseases generate economic losses by reducing animal productivity, they frequently go unreported by farmers. We used a combination of diagnostic assays to confirm BEFV infection, including BEFV-specific PCR and NGS, followed by GenBank accession numbers MN905763. Also, it confirmed the association of BEF disease with cattle, water buffaloes, and bulls, as similarly reported in a previous study. Using genomics approach, the sequence identity of the newly obtained Indian BEFV sequence was compared to all global sequences. The results revealed a considerable genetic diversity of Indian sequence to other BEFV isolates, with a maximum identity of 96.5% with the Middle Eastern, Israel isolate. The genome characterization typically revealed the canonical genome annotations as have been previously published. Moreover, the phylogenetic tree of Indian BEFV isolates based on P and G genes and a complete genome sequence was constructed. The study clustered the Indian BEFV isolate with closely related isolates of Middle Eastern countries, Turkey and Israel. Such findings on BEFV could provide better epidemiological and pathogenic potential, aiding in developing better therapeutic and preventive interventions in India and the world. Increased metabolization of NAD+ by Sirtuin 7 protein supports Newcastle disease virus infection Kamal Shokeen, Sachin Kumar Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India Reactive oxygen species (ROS) accumulation inside the cells instigates oxidative stress leading to the activation of stress-responsive genes. The viral strategies for promoting stressful conditions and utilizing the induced host proteins to enhance their replication remain elusive. Newcastle disease virus (NDV), which causes a highly infectious disease in poultry birds, modulates oxidative stress inside the cells. Although the instigation of oxidative stress during NDV infection has been reported several times, the cellular stress-responsive protein's direct function in virus replication is yet to be well understood. Objectives: The present work shall investigate the impact of oxidative stress on NDV pathogenesis. This study also aims to highlight the plausible stress-responsive proteins involved in viral pathogenesis while exploring the detailed molecular mechanisms of crosstalk between the activated cellular protein and the progress of the NDV replication cycle. Materials and Methods: Here, we did the in vitro quantification studies using Immunoblotting, Real-Time Quantitative PCR, TCID50 assay, and plaque assay. Furthermore, 9-day-old embryonated eggs were used for in ovo study. Results and Conclusions: Our results demonstrate the elevation of SIRT7 levels at transcription and translational levels post-NDV infection, which in turn is associated with the positive regulation of cellular protein deacetylation. The detailed mechanistic study in vitro and in ovo was also carried out utilizing SIRT7 activity modulators to establish its constructive role in infection. Lastly, we concluded that the elevated expression of NDV-mediated SIRT7 protein with an enhanced activity metabolizes the NAD+ to deacetylase the host proteins, thus contributing to high virus replication. Development of a monoclonal antibody-based improved antigen capture ELISA for detection of bluetongue virus. Sushmita Nautiyal1*, Sanchay Kumar Biswas1, Karam Chand2, Madhusudan Hosamani3, Muzamil Bashir2, Smriti Chand2, Kurat Ul Ain2 and M. A. Ramakrishnan2 1Centre for Animal Disease Research and Diagnosis (CADRAD), ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar, Uttar Pradesh - 243 122, India. 2Division of Virology, ICAR-Indian Veterinary Research Institute, Mukteswar Campus, Nainital 263 138, Uttarakhand, India. 3ICAR- Indian Veterinary Research Institute, Hebbal, Bengaluru, India Background: Bluetongue (BT) is an economically important globally re-emerging vector-borne disease of domestic and wild ruminants caused by Bluetongue virus (BTV). Laboratory diagnosis includes detecting virus specific antibodies and demonstration of the viral nucleic acids. An antigen-capture ELISA is a useful tool for detection of BTV antigen as it is rapid, cost effective and convenient to perform while screening of a large number of samples. A polyclonal antibody based BTV sandwich ELISA was developed earlier for detection of viral antigen. The assay could detect viral antigen in clinical samples with limited success. BT is a hemorrhagic disease and being a cell-associated virus detection of BTV antigen in blood is difficult. Presently no antigen capture ELISA (AgELISA) is commercially available for detection of BTV antigen worldwide. The objective of our present assay was to develop an improved assay for detection of BTV antigen in clinical sample and cell culture as mentioned below. Objectives: Evaluation of the effects of detergents and downstream processing methods for development of a monoclonal antibody-based improved antigen capture ELISA for detection of BTV antigen. Materials and Methods: Polyclonal antibody produced against purified virion core antigen and a monoclonal antibody to the BTV group-specific antigen was used as the capture and detection antibody, respectively. Seven different detergents (Triton X-100, Detergent-X, Tween-20, DOC, NaNLS, OBG, and Saponin) and their combinations were tested at different concentrations for the treatment of infected cell culture lysate and spiked blood samples and tested in the AgELISA. Two physical processing methods namely ultrasonic lysis and freeze-thawing were used for downstream processing of the samples. Results and Conclusion: Prior treatment of the culture lysate and clinical samples (blood samples) with 1% Detergent-X (not disclosed due to IPR needs) followed by freeze-thawing was found to be the most suitable processing method to be used in the AgELISA. The AgELISA can detect up to 3.9 × 102 TCID50 of virus present in 50 µl of sample. The assay was found to be specific for BTV and did not show any cross-reactivity with the other common viruses infecting the natural hosts of BTV. The assay is being validated and used routinely for screening of BTV suspected clinical samples. Emergence of Psittaciform 1 orthobornavirus in captive psittacines reveals a serious threat of species conservation in India Pankaj Dekaa, Parikshit Kakatib, Sangeeta Dasa,c*, Ritam Hazarikad, Anil Gargb and Nagendra Nath Barmana aDepartment of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. bLife Science Education Trust, Bengaluru, India. cDepartment of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India. dDepartment of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India *Presenting author & Corresponding author e-mail address: [e-mail] Background: Avian bornaviruses are a recently described genetically diverse group of RNA viruses. Since the discovery of Psittaciform 1 orthobornavirus as the aetiologic agent of proventricular dilatation disease (PDD) in 2008, numerous parrot bornavirus (PaBV) infections have been reported worldwide. Treatment of PDD is usually palliative with no licensed vaccines or antiviral therapies currently available. Objectives: The objectives of our study were to investigate PaBV in captive psittacines in India and their isolation and molecular characterization. Materials and Methods: Eighty three psittacines of 13 different species including birds with suspected PDD based on clinical examination/necropsy (n = 57) and healthy cage mates (n = 26) were screened for PaBV using RT-PCR. Phylogenetic studies were done based on the matrix (M) gene. The virus was isolated in duck embryo fibroblasts and embryonated duck eggs. Results and Conclusions: PaBV infection was detected in 75.4% PDD suspected and 42.3% clinically healthy psittacines of 9 psittaciform species suggesting either horizontal virus transmission or healthy psittacines as PaBV carriers. This indicates the obscure pathogenecity of PaBV infection which needs to be solved. Phylogenetic analysis revealed infection by PaBV genotype 4 (PaBV-4), belonging to the species Psittaciform 1 orthobornavirus. There is, to the best of our knowledge, no publication describing the circulation of PaBv-4 in captive psittacines in India as well as its isolation in embryonated duck eggs. This study highlights the major impact on conservation projects as these birds rely on captive breeding for their survival. Therefore, there is an urgent need to understand the factors that might play a critical role in their emergence supported by future research in development of vaccines or antiviral therapies. Ribavirin effectively inhibits the replication of avian birnavirus through multiple mechanisms Towseef Akram1, Irfan Gul1,2, Mehak Khan1, Jan Muneeb1, Mahrukh Parveez1, Riaz Ahmad Shah2, Syed Mudasir Ahmad2, Nazir Ahmad Ganai3, Naveed Anjum Chikan4, Nadeem Shabir2* 1Division of Animal Biotechnology, Faculty of Veterinary Sciences and Animal Husbandry, Shuhama, Sher-e- Kashmir University of Agricultural Sciences and Technology, Kashmir, 190006, India. 2Department of Biotechnology, University of Kashmir, 190006, India The effect of different antiviral agents on replication of single stranded RNA viruses is well documented, however, little is known about their effect on replication of double-stranded (ds) RNA viruses. In this study we evaluated the effect of four antiviral agents viz. ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride on replication of an avian birnavirus, infectious bursal disease virus (IBDV). Cytotoxic effect of the antiviral agents was evaluated in chicken embryo fibroblasts (CEFs). Further effect of mutagens on the replication of Infectious Bursal Disease Virus (IBDV) was evaluated following which the IBDV was serially passaged for 11 times in the cells at different sub-cytotoxic concentrations of ribavirin, 5-fluorouracil and amiloride. Ribavirin was found to be least cytotoxic on chicken embryo fibroblasts followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin was also found to significantly inhibit the replication of IBDV in CEFs at concentrations as low as 0.05 mM. Further, extinction of IBDV in CEF cultures was achieved during serial passage of the virus at different concentrations of ribavirin, however, emergence of mutagen-resistant virus was not observed. Ribavarin was found to inhibit IBDV replication profoundly through IMPDH-mediated depletion of Guanosine Triphosphate pools. Although other mechanisms like activation of antiviral cytokines and possible inhibition of active pockets of viral RNA-dependent RNA polymerase (RdRP) also seem to play a role. Determination of physicochemical properties of Live attenuated Duck plague vaccine strain propagated in chicken embryo fibroblast Jonmoni Barua1, Sutopa Das1*, Durlav Prasad Bora1, Sophia M. Gogoi1, Rupam Dutta2, Rita Nath3 and N. N. Barman1 1Department of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India.2Department of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India.3Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India Presenting Author: Dr Sutopa Das 1* , Mail ID: [e-mail] Background: Duck plague (DP) also known as Duck Viral Enteritis (DVE), Anatid Herpesvirus is an acute contagious herpesvirus infection of ducks and waterfowl and members of the family Anatidae. The physicochemical properties of a virus are the intrinsic physical and chemical characteristics. There is little clarification on physicochemical characteristics of the Live attenuated Duck plague vaccine virus so far. Clarification on the physicochemical characteristics of the Live attenuated Duck plague vaccine virus which would aid in deactivation of the virus which in turn would be useful when creating field decontamination protocols and effective elimination procedure in contaminated lab materials. Objective: The present study was undertaken to determine the physicochemical properties of duck plague vaccine strain propagated in chicken embryo fibroblast monolayer. Materials and Methods: The Chicken Embryo Fibroblast adapted duck plague virus vaccine strain available in the DBT-ADMaC, Department of Microbiology, College of Veterinary Science was selected for evaluation of the physicochemical properties. The vaccine virus was revived in CEF and identity was checked with appearance of CPE, PCR and Molecular characterization. After positive identity the vaccine virus was produced at bulk for the study. The physical properties evaluation was carried out by exposing the vaccine virus to different temperatures and pH levels, whereas chemical properties were evaluated by exposing the virus to ether, chloroform and enzyme-trypsin. The sensitivity was mainly determined by titrating the treated vaccine virus to find out the infectivity titre to evaluate the physicochemical properties. Results and Conclusion: The mean infectivity titre (TCID50/ml) of the DPV vaccine virus propagated in CEF for bulk production was calculated and was recorded to be 6.9 ± 0.17 TCID50/ml. The vaccine virus was sensitive to the temperature of 56 °C and above for 15 min and there was gradual fall in the infectivity titre. The vaccine virus was also found to be moderately sensitive to temperatures 28 °C and 50 °C without completely falling in the infectivity titre. On exposure to a different level of pH showed that the CEF-adapted DP vaccine virus was stable at pH levels 5,7 and 9 when incubated for 1 h at 4 °C and the vaccine virus was found to be sensitive at pH below 3 and above pH11 when incubated for same afore mentioned hours and temperatures. On chemicals and enzyme treatment of CEF-adapted DP vaccine virus, it was revealed that the vaccine virus was sensitive to ether and trypsin. Also, there was a gradual fall in the titre when treated with chloroform, but it was able to retain its infectivity titre upto 50%. Thus, the study revealed that the vaccine virus are sensitive to temperature of 56 °C and above, pH below 3 and above pH11, chemical-ether and enzyme-trypsin. Keywords: DPV, CEF, Physicochemical properties. An attenuated deletion mutant of EHV1 virus elicits strong immune response in murine model Nitin Virmani, B. C. Bera, Venkataramireddy Balena, Stephanie S. Pradhan, Taruna Anand, and B. N. Tripathi ICAR-National Research Centre on Equines, Sirsa Road, Hisar, Haryana 125 001, India Email: [e-mail] Equine Herpes Virus 1 (EHV1) along with EHV4 is grouped as causal agents of equine rhinopneumonitis and is an OIE notifiable infection. It is one of the most ubiquitous viral pathogens of equines and causes respiratory symptoms, late-term abortion, neonatal mortality, and neurological disorders. The infection continues to be a challenge as it is capable of evading the immune responses of the host in many ways. Traditionally inactivated vaccines were in use but they are known to produce a mild cell-mediated immune response. Despite widespread vaccinations, outbreaks still continue and remain a severe problem for the horse industry as the inactivated vaccines don’t elicit sufficient cellular immune responses. We have developed mutants of EHV1 through the deletion of specific genes (gE, IR6, pUL43, pUL56) employing BAC technology. The mutants have been tested for their attenuation by employing in vitro and in vivo studies. Mice immunized with the mutant viruses showed less pathology in terms of clinical signs, body weight loss, and gross and histopathological lesions accompanied by early shedding of the virus from the respiratory tract. The recombinant virus generated strong humoral and cell-mediated immune responses as adjudged through virus neutralization assay and immunophenotyping of cells. As such the mutant qualifies to be a good MLV vaccine candidate for vaccination against EHV1. Development of an exogenous DNA (eDNA) and validation of the duplex real-time PCR (qPCR) assay with eDNA as internal control (IC) for screening bovine herpesvirus-1 (BHV-1) in frozen semen doses (FSDs) RV Chandrasekhar Reddy1, GU Sai Poojitha1, LN Sarangi1, KSNL Surendra1, P Baji Babu1, Ponnanna NM1, SK Rana2, AV Hari Kumar2 1NDDB R&D Laboratory, Gachibowli, Hyderabad 500032, Telangana, India. 2National Dairy Development Board, Anand 388001, Gujarat, India Background: Bovine herpesvirus-1 (BHV-1) causes infectious bovine rhinotracheitis (IBR) in bovine hosts. Infected bulls shed BHV-1 in semen and other body fluids on reactivation due to immunosuppression. BHV-1 contaminated semen may infect recipient cows. Thus, frozen semen doses (FSDs) produced from the IBR seropositive bulls are screened for BHV-1 prior to use in breeding. Real-time PCR (qPCR) is the assay of choice for screening FSDs owing to its high sensitivity. However, PCR inhibitors in FSDs are known to result in false negatives and compromise accuracy. The use of exogenous DNA (eDNA) as internal control (IC) in the qPCR assay aids the detection of PCR inhibitors in the samples and improves assay accuracy. Objective: Design and synthesis of eDNA, incorporation of eDNA as IC in the qPCR assay, and standardization and validation of duplex-qPCR assay (IC-qPCR) for the detection of BHV-1 in FSDs. Materials and Methods: eDNA of 126 bp unique sequence was designed, synthesized and cloned in a TOPO TA plasmid vector. The optimal concentration of plasmid with eDNA was determined for use as IC in the qPCR assay. The IC-qPCR assay was standardized and validated for the detection of BHV-1 in FSDs with a commercial IC-qPCR kit as standard. Results and Conclusions: eDNA did not interfere in the detection of BHV-1 in semen in the qPCR. The optimal concentration of eDNA as IC was determined was 10,000 copies/µL. IC-qPCR was highly repeatable as determined by the intra and inter-assay precision study (CV  < 2%). A threshold cycle above 38 (Ct ≥ 38) was considered indicative of PCR inhibitor and/or inadequate processing of samples for downstream qPCR assay. The IC-qPCR developed in-house showed 100% agreement with the commercial kit. The results suggest improved accuracy of IC-qPCR than the qPCR alone in screening FSDs for the detection of BHV-1. Sheep Pox and Contagious Ecthyma Outbreak Investigation, and a Non-antibiotic Approach for Clinical Management of Contagious Ecthyma M. A. Kawa, S. Bashir, Q. A. Nazir, G. N. Sheikh, M. Shabir1, G. B. M. Reddy2 and A. A. Dar* Division of Veterinary Epidemiology & Preventive Medicine, Faculty of Veterinary Sciences & Animal Husbandry (SKUAST – Kashmir), Jammu & Kashmir – India. 1Division of AGB, FVSc & AH, Shuhama Alusteng. 2Scientist, ICAR-NIVEDI, Bangaluru Karnataka – India *Corresponding Author: [e-mail] Sheep pox (SP) and contagious ecthyma (CE) are economically important viral diseases of sheep & goat, characterized by papular/nodular and proliferative lesions respectively on multiple body sites. Outbreaks (SP, N = 3; CE, N = 3) reported during January–November 2018 from both organized and unorganized farms were investigated. Diagnostic confirmation was done by PCR targeting P32 gene of Capri pox virus (for SP) and B2L gene of ORF virus (for CE). PCR products were then sequenced for comparative analysis. Also, a non-antibiotic approach using topical Zinc Oxide (ZnO) based preparations was evaluated in CE affected lambs (aged < 3 months) for alleviating the severity of lesions and hastening of clinical recovery. All the outbreaks reported and investigated occurred in January, February, October and November months indicating a seasonal/time-related occurrence. The lesions were mostly restricted to oral cavity in CE cases, however, in SP cases the lesions were observed on wool less/sparsely woolled parts of body (axilla, perineal, peri-orbital, base of tail, scrotum, ventral abdomen) in addition to oral cavity. All the outbreaks investigated were confirmed by PCR and sequences have been submitted to GenBank. In SP outbreaks, the overall morbidity rate observed was 21.76% (16.0% in > 6 months age group, 41.6% in 0–3 months age group and 45.1% in 3–6 months age group). The overall mortality rate noticed was 4.4% (2.0% in > 6 months age group, 12.9% in 3–6 months age group and 13.8% in 0–3 months age group). Abortion occurred in three SP affected ewes in one outbreak. In CE outbreaks, the overall morbidity rate observed was 10.68% (1.65% in > 6 months age group, 30.14% in 0–3 months age group and 50.0% in 3–6 months age group). However, none of the animals died due to CE in the investigated outbreaks (mortality rate zero percent). The clinical trial yielded significantly appreciable results vis-à-vis hastening of recovery in CE cases. However, the study needs to be validated on more number of cases using double/triple blinding approach. Use of ZnO-based preparations could offer a novel and a non-antibiotic approach for management of CE. Further, the results shall be discussed in detail during the presentation. A novel computational approach for rapid screening of antibody therapeutics for efficacy against SARS-CoV-2 variants of concern including Omicron variant Naveen Kumar1*, Rahul Kaushik2, Kam Y. J. Zhang2, Vladimir N. Uversky3,4, Upasana Sahu1, Richa Sood1, Sandeep Bhatia1 1Zoonotic Diseases Group, ICAR- National Institute of High Security Animal Diseases, Bhopal 462022, India. 2Laboratory for Structural Bioinformatics, Center for Biosystems Dynamics Research, RIKEN, 1-7-22 Suehiro, Yokohama, Kanagawa 230-0045, Japan. 3Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA. 4Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center 'Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences', Moscow region, 142290 Pushchino, Russia * Correspondence: E-mail address: [e-mail] (Naveen Kumar) Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to evolve carrying flexible amino acid substitutions in the spike protein's receptor binding domain (RBD). These substitutions modify the binding of the SARS-CoV-2 to human angiotensin-converting enzyme 2 (hACE2) receptor and have been implicated in altered host fitness, transmissibility and efficacy against antibody therapeutics and vaccines. Reliably predicting the binding strength of SARS-CoV-2 variants RBD to hACE2 receptor and neutralizing antibodies (NAbs) can help assessing their fitness, and rapid deployment of effective antibody therapeutics, respectively. Here, we introduced a two-step computational framework with three-fold validation that first identified dissociation constant as a reliable predictor of binding affinity in hetero-dimeric and –trimeric protein complexes. The second step implements dissociation constant as descriptor of the binding strengths of SARS-CoV-2 variants RBD to hACE2 and NAbs. Then, we examined several variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and Omicron and demonstrated that these VOCs RBD bind to the hACE2 with enhanced affinity. Furthermore, the binding affinity of Omicron variant’s RBD was reduced with majority of the RBD-directed NAbs, which is highly consistent with the experimental neutralization data. By studying the atomic contacts between RBD and NAbs, we revealed the molecular footprints of four NAbs (GH-12, P2B-1A1, Asarnow_3D11, and C118)—that may likely neutralize the recently emerged omicron variant—facilitating enhanced binding affinity. Finally, our findings suggest a computational pathway that could aid researchers identify a range of current NAbs that may be effective against emerging SARS-CoV-2 variants. Development, characterization and diagnostic application of monoclonal antibodies to Canine distemper virus Arfa Fayaz#, Kaushal Kishor Rajak*, Ashok Kumar, Mukesh Bhatt, Monu Karki, Vishal Rai, Kiran, Ajay Kumar Yadav, Rabindra Prasad Singh* Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar # Presenting Author *Correspondence e-mail: [e-mail], [e-mail] Background: Canine distemper virus (CDV) is a multi-host pathogen, highly contagious with high morbidity and mortality rates in the susceptible hosts’ viz. domestic dogs, wild canines and felines. Since the cases of canine distemper are increasing widely among different animal populations, it becomes important to develop simple, specific and high throughput diagnostics for large scale sample screening and accurate diagnosis. Objectives: The present study was initiated with the objective of development, characterization of monoclonal antibodies (mAbs) using an indigenous strain of CDV (CDV/Dog/Bly/Ind/2018) for diagnostic applications. Materials and Methods: A total of six mouse mAbs were developed. All the mAbs generated belonged to IgG class and were found to be CDV-specific as determined by immunofluorescence antibody test. Two of the mAbs, CD-2F8 and CD-3D8 were directed against Nucleocapsid protein of CDV as determined in western blot. A close antigenic relationship was found among CDV and PPR virus as compared to measles virus in cell-ELISA. A competitive-ELISA (c-ELISA) was developed using mAb CD-3D8 for the detection of CDV antibody. Results: A total of 322 serum samples were screened and results were compared to a gold standard test i.e. virus neutralization test. The test revealed a high relative diagnostic sensitivity (95.08%) and specificity (96.50%). The c-ELISA test was found to be suitable both for sero-surveillance and sero-monitoring (CDV-Ondersteeport and CDV/Dog/Bly/Ind/2018 vaccine candidate). The test also demonstrated its suitability for antibody titration. Conclusion: The present investigation therefore resulted in development of mAb based c-ELISA for CDV sero-surveillance, sero-monitoring and mAbs for R&D. These reagents are likely to help in controlling CDV effectively within the country. Veterinary Virology (Poster Presentation) Development of a probe-based Real-Time PCR assay for detection of Porcine Circovirus 2 Arpita Bharali1, Lukumoni Buragohain1, Nagendra Nath Barman1, Pankaj Deka1, Jayashree Sarma1* 1College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India *Presenting Author: Jayashree Sarma, Email ID:[e-mail] Background: Porcine circovirus 2 (PCV2) is the causative agent of post-weaning multisystemic wasting syndromeand it is one of the economically important pig diseases. PCV2 can infect both domestic and wild pig population. PCV2 is a ssDNA virus of the family Circoviridae and genus Circovirus. PCV2 infection is reported worldwide. PCV2 is circulating in India since 2006 and it is highly prevalent in North-Eastern Region (NER) of India. Early and accurate diagnosis is essential to effectively control and prevent an infectious disease. Moreover, many pigs may remain as carrier for PCV2 without showing any clinical symptoms, thus it is necessary to identify such carrier with confirmatory test so that further spread of the disease could be restricted. Objectives: The present study aims to develop a probe-based Real-Time PCR (qPCR) assay for accurate, rapidand confirmatory diagnosis of PCV2 infection. Materials and Methods: The Rep (ORF1) gene of PCV2 is less diverse at nucleotide level compared to the Cap (ORF2) gene; therefore, a pair of primers and a probe was designed with conserved regions by aligning several numbers of Rep gene sequences. The optimum qPCR condition was determined with appropriate concentration of probe and primers. The sensitivity of the assay was determined by serial dilution (tenfold) of a standard plasmid that contains the target sequence. The specificity was determined by using DNA/cDNA templates of other pig pathogens. The assay was tested with clinical samples and the results were compared with conventional PCR. Results and Conclusion: The probe-based Real-Time assay was optimized for detection of PCV2 DNA. Thesensitivity of the qPCR assay was superior as compared to conventional PCR. The assay did not produce any amplification against other pig pathogens. In detection of clinical sample also it was found be more sensitive than conventional PCR. The developed qPCR will be beneficial to rapidly confirm the PCV2 infection in pig population. Keywords: Diagnostics, PCV2, qPCR assay. Molecular detection of Lumpy Skin Disease virus in Jammu division, UT of J&K Sabahat Gazal, G. A. Badroo, Deep Shikha*, Salena Janjua, Shaista Akhter, M. Rashid, A. K. Taku Division of Veterinary Microbiology and Immunology, F.V.Sc & A.H, SKUAST-Jammu, R.S. Pura-181102 Abstract: Lumpy skin disease (LSD) is a transboundary viral disease of cattle and buffaloes caused by Lumpy skin disease virus (LSDV), a DNA virus belonging to genus Capripoxvirus within the family Poxviridae. LSDV has double-stranded DNA genome approximately 151 kb in length. LSDV genome contains 156 open reading frames and encodes 30 homologues of poxviral proteins known to be structural or involved in virion morphogenesis and assembly. LSDV is antigenically and genetically closely related to sheeppox virus (SPPV) and goatpox virus (GTPV) with nucleotide sequence identities of 96% between species. The 2022 outbreak of Lumpy skin disease in India began in Gujarat and rapidly spread to other states and UTs including J&K where the disease was reported around the month of July/August. The study was aimed at determining the prevalence of LSD in the Jammu division, UT of J&K. A total of 1376 samples from animals exhibiting clinical signs of LSD as well as in contact animals from different districts of Jammu were screened for presence of LSD virus by Real Time PCR. Briefly, viral DNA was extracted from the nasal swab samples using Viral nucleic acid extraction kit as per manufacturer’s recommendation. The viral DNA was then used for amplification of LSDV P32 gene by Real Time PCR using specific primers as recommended by OIE. Out of a total of 1376 nasal samples tested, 554 (40.2%) were found positive for LSDV in Jammu division. In Jammu district, the prevalence of LSD was found to be 41% (85 of 206 samples found positive). Sero-surveillance for anti-3AB3 non-structural protein antibodies against foot-and-mouth disease virus in bovine of district Hisar, Haryana during 2022 Ankit Pannu1*, Swati Dahiya1, Anshul Lather1, Amandeep Kaur1, Punesh Sangwan1, Neelam Rani2 and J. K. Mohapatra3 1Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004, Haryana, India. 2Department of Animal Husbandry and Dairying, Haryana, India. 3ICAR-Directorate of Foot and Mouth Disease, International Centre for Foot and Mouth Disease, Bhubaneswar-752 050, Odisha, India Foot-and-mouth disease (FMD) is a World Organization for Animal Health (OIE)-listedhighly contagious and economically devastating disease of cloven-hoofed animals like cattle, buffalo, sheep, goat, pigand other wildlife species. The etiological agent, FMD virus (FMDV), belongs to genus Aphthovirus, family Picornaviridae andorder Picornavirales. FMDV non-structural protein (NSP) based indirect ELISA testusing recombinant r3AB3 expressed in E. colideveloped indigenously by ICAR-Directorate of FMD, Bhubaneswar, Odisha is used in population serosurveys to detect anti-NSP antibodies as an indicator of exposureof animal tothe virus so as to differentiate infected from vaccinated animals (DIVA). Demonstration of consistent NSP-antibody free status in the population is one of the prerequisite indicators for effective vaccination campaign, declaration of disease-free zones and also for regaining FMD free status consequent to an occasional virus incursion. Routine sero-surveillance is being carried out countrywide under FMD Control Program/National Animal Disease Control Programmelaunched by Govt. of India. Under this programme, a total of 377 serum samples of cattle and buffaloes of 6–18 months age fromdistrict Hisar of Haryana were tested by 3AB3-NSP ELISA in 2022. The circulation of virus was found to be less in buffaloes as significantly higher proportion of cattle (11.4%; 12/105) had 3AB3 specific antibodies as compared to buffaloes (2.9%; 8/272). A decreasing trend was observed inoverall DIVA positivity in bovine in 2022 i.e. 5.3% (20/377) as compared to 6.3% (29/458) in 2021in district Hisar indicating a decrease in virus circulation in this area. Ideally follow up investigation of the NSP seropositive animals through probang sampling is recommended in a region reporting no/low incidence of FMD over a period of time. Hence, ICAR-DFMD has endorsed probang sampling from Haryana. Further studies are under progress to detect FMDV in subclinical/persistently infected animals and their possible role in transmission and spread of disease. In silico analysis of promoter region of Bovine TLR4 gene in relation with the susceptibility of bovine subclinical mastitis among Holstein Frisian cross bred dairy cattle. Rahil Razak Bhat1, Ambreen Shabir2, Nadiem Nazir Bhat1, Ishraq Hussain1, Shiekh Bilal Ahmad1, Shazada Mudasir Rashid1, Javeed I. A. Bhat1 1Division of Veterinary Biochemistry SKUAST-Kashmir. 2Division of Fish Genetics and Breeding, SKUAST-Kashmir Bovine mastitis is polygenetic trait controlled by many nonimmune and immune system associated genes (Khan et al., 2022). To our best knowledge Genetic mapping has identified about 2599 quantitative trait loci (QTL) which are associated with bovine mastitis resistance or susceptibility on bovine genome https://www.animalgenome.org/cgibin/QTLdb/BT/summary?summ=type&qtl=192,954&pub=1,103&trait=683&gene=202 (accessed on 10 August 2022). TLR4 gene has been identified as one of the candidate gene for bovine subclinical mastitis in dairy cattle via functional genomic approach (Ogorevc et al., 2009). In this study, we sought to investigate the sequence variants of bovine TLR4 gene within promoter regions using in silico tools. In the present study, 180 Holstein Frisian cross bred dairy cattle were selected including 10 normal animals. The blood was collected from animals and subjected to DNA extraction followed by PCR of two promoter regions (PPR1 & PPR2). The prominent PCR products of PPR1 (546 bp) and PPR2 (331 bp) of every animals were sequenced at Xcelris Medical Genetics labs Ltd. (Ahmadabad-India, Ltd.). The DNA sequence data of all 180 (including 10 normal/control and 180 SCM) PCR products was analysed by using the bioinformatics softwares viz: Chromas v 2.6.5 and aligned by Bio-edit v 7.2.6 tools. The post sequenced output DNA sequence files in ab1 format were displayed via Chromas bioinformatical tool. The in silico analysis of PPR1 and PPR2 revealed 8 genetic variants which may effect on transcription pace of TLR4 gene or effect directly on the immune status of dairy cattle. The current study revealed that promoter region of bovine TLR4 gene of Holstein Frisian cross bred cattle is highly polymorphic and the study needs to expand with large number of animals to validate their biomarker role in bovine subclinical mastitis. Pulsed-field gel electrophoresis typing of Clostridium chauvoei Q. Beigh, I. Hussain, S. A. Wani Correspondence: [e-mail] Clostridium is the main aetiologic agent of nosocomial diarrhoea and is responsible for an important increase in hospital stays, with high healthcare and economic repercussions. Typing of the micro-organism can be useful in the detection and control of epidemic outbreaks and endemic situations, and in the characterization of recurrence. Different approaches have been used for typing bacteria, although Pulsed-field gel electrophoresis is the ‘gold standard’ technique for bacterial typing and has proved to be discriminatory and reproducible for typing Clostridium species. Nevertheless, a high proportion of strains are non-typable by this technique due to the degradation of the DNA during the process. The introduction of several modifications in the PFGE standard procedure increased typability from 40% (90 isolates) to 100% (220 isolates) while maintaining the high degree of discrimination and reproducibility of the technique. Genotyping of the representative isolates of C. chauvoeiisolatesfrom faecal samples of sheep, cattle and goats collected from different organised and unorganized farms of Kashmir valley was performed by pulsed field gel electrophoresis. In the present study, thirty six (36) C. chauvoei isolateswere identified and were subsequently examined by PFGE. Among these (36) isolates, (25) isolates gave reproducible patterns while (11) isolates gave no pattern. PFGE analysis of 25 isolates resulted in different PFGE patterns of which some were closely related. Many isolates identified from the animals of the same farm revealed similar PFGE patterns and vice versa. But in other cases, different pulsotypes were identified from the animals of the same farm also, although all the isolates produced an ~ 1100-kb fragment in PFGE. Thus the difference between the banding patterns of these pulsotypes can be used as a tool in epdemiological studies. It has been used to examine the genomic variation in the isolates for the investigation of epidemiologic relationships between isolates from different sources and outbreaks. In the present study, genetic diversity among different isolates of C. chauvoei was found. PFGE clearly discriminated among unrelated isolates of C. chauvoei and established a clonal relationship between related strains. The general consensus regarding strains was previously investigated by PFGE is that strains originating from the same outbreak have similar PFGE patterns and that non-outbreak strains have lower genetic relatedness. Molecular detection of Porcine Rotavirus A from different agro-climatic zones of Haryan Deepika Sheoran*, Sanjeevna K. Minhas, Vandna Bhanot, Parveen Kumar, Ritu Panghal, Raman, Akhil Kumar Gupta and Rajesh Chhabra Department of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, India Rotavirus (RV) A are leading viral diarrheal agent associated with acute diarrhoea in nursing and weaned piglets leading to severe economic losses to piggery industry worldwide. RVs destroy villous enterocytes and cause villous atrophy in small intestines, resulting in profuse diarrhoea, vomition and dehydration. In India, Porcine RV group A have been reported regularly from North-Eastern states of India with limited reports from other parts of country. The present study was carried out to detect the presence of porcine RVA circulating in piggery farms of Haryana. A total of 96 rectal swab samples (39 diarrheic and 57 non-diarrheic) were subjected to RT-PCR based on VP6 gene. A total of 38.54% fecal swab samples (37/96) were found positive including 51.28% (19/39) diarrheic and 29.82% (18/57) non-diarrheic piglets revealing significant relation between presence of rotavirus and diarrhoea. In semi-arid zone of Haryana 38.1%, whereas in arid zone of Haryana 34% fecal swab samples were found positive for porcine rotavirus. Within different age group, suckling piglets were found to be most susceptible age group. The study will aid in development of an efficient control program thereby, raising household economy of the rural farmers. Emergence of a genotype I lineage 1 variant avian infectious bronchitis virus in Assam, India Sangeeta dasa,b*, Rajesh Chhabrab, Anidrita Baruahc, Ritam Hazarikad, Rofique Ahmedc, Mihir Sarmae Ilakshy Dekaf and Pankaj Dekaa aDepartment of Veterinary Microbiology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. bDepartment of Veterinary Microbiology, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India. cDepartment of Preventive Medicine and Epidemiology, College of Veterinary Science, Asam Agricultural University, Guwahati, Assam, India. dDepartment of Animal Biotechnology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. eDepartment of Poultry Science, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam, India. fSubject Matter Specialist (Animal Science), Krishi Vigyan Kendra, Kamrup *Presenting author e-mail address: [e-mail] Background : Infectious bronchitis (IB) is an acute and highly contagious viral disease of poultry affecting chicken of all ages. The causative agent IB virus (IBV) is a Gammacoronavirus within the family Coronaviridae. Viral genetic mutations and recombination events particularly in the spike protein (S1) of IBV constantly give rise to emerging IBV variants. Vaccination is considered as the most reliable approach for IBV control, but current vaccines have been found to be ineffective due to constant emergence of new variant viruses. Objectives: The objective of our study was to detect IBV genotypes prevalent in Assam, India. Materials and Methods: Oro-pharyngeal swabs and tissue samples from unvaccinated broiler chickens showing respiratory symptoms were tested using RT-PCR targeting the N gene of IBV. The virus was isolated from infected swab/tissue samples in 9 days old specific pathogen free embryonated chicken eggs through allantoic cavity route. Phylogenetic studies were done based on the S1 gene of IBV. Results and Conclusions: Clinically, the birds showed gasping and tracheal rales. Necropsy revealed distended ureters. Virus was isolated and identified by curling and dwarfing of the dead embryos and further confirmed by RT-PCR. Positive PCR amplicons were sequenced and phylogenetic analysis clustered the IBV isolate from Assam with genotype I lineage 1 IBV prototype sequence belonging to Beaudette and Mass 41 strains but the isolate exhibited a relatively high degree of sequence divergence with reference strains. Our findings suggest that the IBV isolate might have emerged from recombination with the local circulating virus or vaccine strains. This will have important implications for IB prevention strategies. From cell to well: Recombinant protein-based ELISA for detection of ASFV Deepa Mehta, JRF, IIT Guwahati Background: African swine fever virus (ASFV) is the causative agent of a highly contagious, non-zoonotic transboundary disease of all members of the family Suidae. The disease is endemic in different parts of the world and in the absence of any commercial vaccine or treatment option, rapid and reliable diagnostic tools are of paramount importance for the confirmation of clinical cases. Several studies showed that p30, p54 and p72 are major immunogenic proteins eliciting protective immunity in host; thus, can act as promising diagnostic markers for timely ASFV detection. Antigen expression in the bacterial system presents a rapid and low-cost method that obviates the need for expensive tissue culture scale-ups or special equipment. Objective: Aim of the work is to establish an optimised protocol for the overexpression and purification of ASFV immunogenic proteins in bacterial expression system. Further, their utilisation for the development of rapid onsite immunodiagnostic assay. Materials and method: In the present study, an Escherichia coli-based expression system has been optimised to individually express recombinant p30, p72 and a truncated form of p54 ASFV protein. The optimized expression conditions were then used for their bulk production and subsequent purification using 6xhis/Nickel-NTA affinity chromatography. Results and conclusion: Here, we have successfully established expression and purification methodology for ASFV immunogenic proteins. Afterwards, the purified proteins will be utilised in developing ELSA based diagnostic assays with enhanced sensitivity, specificity and accuracy. Additionally, each of the developed ELISA will be accessed for their ability for informing on the presence of ASFV infection and viral load. Further studies will be conducted to validate this recombinant protein based ELISAs under field conditions. Molecular detection and identification of serotype/s of foot-and mouth-disease (FMD) virus in Kashmir, India Aamina Dilawar1, Nuzhat Hassan1*, Shaheen Farooq2, Amatul Muhee1, Abdul Q Mir3, SA Hussian1, Arif Rasool2, SK Alamooru2 1Division of Veterinary Epidemiology and Preventive Medicine, 2Division of Veterinary Microbiology & Immunology & 3Division of MRCSG, Faculty of Veterinary Sciences and Animal Husbandry, Shuhama, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir-190006 Abstract Background: Foot and mouth disease (FMD) is one of the highly contagious viral diseases of cloven-hoofed animals and is globally responsible for huge economic losses, especially in domestic animals. The disease is prevalent throughout the country, endemic in Kashmir and occurs in the form of frequent outbreaks. Objective: The present study was aimed to detect the foot and mouth disease virus in natural outbreaks and to identify serotype/s circulating among the affected cattle in Kashmir valley. Material and Methods: A total of 89 samples consisting of tongue epithelium were collected from four districts of Kashmir valley viz Baramulla, Bandipora, Ganderbal and Srinagar. Results: Sixty-five (73.0%) samples were detected positive for FMDV by polymerase chain reaction targeting 5-untranslated region (5-UTR) gene fragment. Serotyping of samples revealed serotype O in all 65 (73.0%) FMDV-positive samples using primer sets ARS4/NK61 and A-1C562/NK61. The representative positive samples were outsourced for sequencing which revealed that serotype O of the present study is genetically close to FMDV serotype O PanAsia strain of Pakistan. Conclusion: It was concluded that serotype O was the only FMDV serotype responsible for natural outbreaks in Kashmir valley. The close-border proximity with adjoining countries and thereby the movement of animals may be the source of analogous FMDV serotypes in Kashmir. Current Update on the Epidemiology of Foot rot in Kashmir M. U. Qurra, I. Hussain*, S. Farqooq, Z. A. Kashoo, M. A. Bhat, S. Qureshi and A. H. Wani Division of Veterinary Microbiology & Immunology, Faculty of Veterinary Sciences & Animal Husbandry Shuham (Alusteng) Srinagar-190006 *Corresponding Author: isfaqhussain @ yahoo.com A total of 5475 sheep from different organised farms (Gowbal, Daksum, Kral Pathri, Fakir Gujri, MRCSG, Shuhama), 61 flocks comprising 2807 sheep in unorgansied sector in four different districts namely Ganderbal, Budgam, Srinagar and Pulwama and 14 Bakerwal sheep flocks consisting of 980 sheep were inspected from December, 2018 to May 2019 and foot rot was recorded in 0.67%, 6.7% and 3.36%, animals, respectively. A total of 260 swab samples (organized farms 37, Unorganized sector 190 and Bakerwal sheep 33) with lesion score of 2–4 were collected as per availability of foot rot affected sheep and D. nodosus was detected in 56.3 to 60.6% samples by direct PCR using 16 s rRNA gene specific primers. The samples that were positive for D. nodosus were further subjected to serogroup specific multiplex PCR to determine the serogroup of D. nodosus. Serogroup B was most predominant (83.17%) followed by serogroup E (13.08%) and mixed infection with B and E (3.7%) in unorganized sector. Similarly, serogroup B (95.45%) was most common followed by mixed serogroup B and E (4.5%) in organized farms. Likewise, serogroup B and E were detected in 90% and 10% positive samples of Bakerwal sheep. Serogroup B was exclusively detected in the samples collected from Budgam, Pulwama and Srinagar, while both serogroups B and E were detected from Ganderbal district. Concurrent infection of porcine parvovirus and porcine circovirus, Punjab, India Adarsh Mishra1, Deepali Kalambhe2, Yashpal Singh Malik1# 1College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab. 2Centre of One Health, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab # Presenting and corresponding author: [e-mail] Background: Porcine parvoviruses (PPVs) and porcine circoviruses, are among the most common pathogens linked with serious health implications in pigs. PPVs are responsible for stillbirth, mummification, embryonic death and infertility (SMEDI) among the affected population while porcine circovirus 2 (PCV2) is responsible for multi-systemic syndromes and reproductive disorders, collectively known as porcine circovirus‐associated disease (PCVAD). Objective: Surveillance of porcine parvovirus 1 (PPV1), porcine parvovirus 2 (PPV2) and/or PCV2 infections from suspected cases of the diseases. Materials and methods: For the detection of PPV1, PPV2 and/or PCV2 infections, polymerase chain reactions (PCRs) optimized in the laboratory were used. A total of 20 samples comprising of blood samples (n = 10), tissue samples (n = 10) of various organs (spleen, lung and liver) collected during 2022 from suspected cases of the diseases in and around Punjab were screened. Result: The tissue samples (spleen and lung) (n = 2) collected from the pig farm from Rupnagar district of Punjab were found positive for PPV1, PPV2 and PCV2 infections. Out of a total of 220 animals, mortality of 101 adult animals was reported in 3 days. The second incidence was reported from Patiala district of Punjab. The affected animals were of 3–4 months of age and died with lethargy and body temperature around 41–42˚ Celsius. PPV1 with PCV2 were detected in the spleen, lung and liver tissue samples (n = 6). Conclusion: The findings highlight circulation of PPV1, PPV2 and PCV2 infections in porcine herd. Keyword: Emerging viral diseases, porcine parvovirus, porcine circovirus, epidemiology, Punjab. Nested PCR and qPCR-based assays for early and rapid detection canine distemper virus Kaur, I.1, Choudhary, S.1, Choudhary, R. K.1, Randhawa, S. S.2, Kansla, A. S.2 and Malik, Y. S1# 1College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University Firozpur Road, Ludhiana, 141004, Punjab. 2Department of Veterinary Medicine, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University Firozpur Road, Ludhiana, 141004, Punjab # Correspondence: [e-mail] Background: Canine distemper virus (CDV) is the infectious microparasite responsible for “Hard pad disease” in domestic and wild canines worldwide. Early diagnosis of the disease helps cope with the control and differential diagnosis from rabies (which produces similar neurological signs). In this study, we developed nested PCR and qPCR-based diagnostics tests to amplify the L-gene of CDV. Objective: In this study, we developed a nested PCR and qPCR-based assays for diagnosing CDV. Materials and Methods: A total of 32 canine samples (blood/naso-ocular discharge) were collected from veterinary clinic and hospitals. After isolation of viral RNA, in house designed nested PCR and qPCR primers were performed. In nested PCR, the outer primers amplified 267 bp product and inner primers amplified 127 bp of amplicoin. The same inner primers were used to optimize qPCR assay targeting L-gene of CDV. Results: The results showed 40.6% (13/32) CDV-positive samples in the nested PCR. Furthermore, in the qPCR results all the PCR-positive cases remained positive. To note, three PCR-negative samples were found qPCR-positive, indicating that qPCR was more sensitive in detecting CDV infection. A ten-fold serial dilution of viral DNA showed a sensitivity of qPCR assay 1.7X10−10 ng, equivalent to 1.24 copies of viral DNA. Amplifcation efficiency of qPCR assay was 109% with high coefficient of determination (R2 = 0.99). The study points only towards early diagnosis of CDV in domestic dogs. Conclusion: Our diagnostic assays of nested PCR and qPCR could empower veterinary diagnosticians to diagnose early and mild infections of CDV in dogs. Keywords: Canine distemper virus, nested PCR, qPCR, early diagnosis. A novel HRM assay for the simultaneous detection of two non-structural genes of the African swine fever virus Choudhary RK1, Choudhary S1, Kumar S2, Barman NN3, Lukumoni B3, and Malik YS1# 1College of Animal Biotechnology Guru Angad Dev Veterinary and Animal Sciences University Firozpur Road, Ludhiana, 141,004, Punjab. 2Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati- 781,039, Assam, India. 3College of Veterinary Science, Assam Agricultural University, Khanapara Campus, Guwahati-781022, Assam, India # Correspondence: [e-mail] Background: Rapid and reliable method of detecting the African swine fever virus (ASFV) is essential in emergency control and preventive measures for the disease. Several approaches were used to diagnose ASFV based on classical and real-time PCR. In real-time PCR, probe-based assays are expansive and can be avoided using high-resolution melting (HRM) curve analysis. HRM offers genotyping and mutation screening and multiplex assay design. Materials and Methods: Two nonstructural genes of ASFV, namely, RNA polymerase subunit 1 (RNAP1 or NP1450L) and RNA polymerase subunit 6 (RNAP6 or C147L), were chosen to amplify 141 bp and 197 bp products, respectively. Our assay was based on HRM curve analysis of PCR amplicons using Evergreen-a double-stranded DNA binding dye. Differences in fragment size and GC content were used to differentiate between the two amplicons. Results: Initial evaluation and optimization of RNAP1 and RNAP6 were performed using plasmid harboring target fragments in a single plex reaction. The Disclination power of the assay was validated on DNA extracted from clinically affected ASFV pigs (n = 5). Results showed that qPCR runs generated two well-separated melting regions for each gene and could distinguish two product amplification. Negative samples, other pig-specific viruses, and no template controls showed no gene-specific amplification of the product. Conclusions: Our assay provides a rapid, cost-effective, and specific detection of ASFV in clinical samples of pigs. Keywords: ASFV, HRM, Duplex assay, qPCR. A review of the Socio-economic impact of Lumpy skin disease outbreak in India Irtiqa Manzoor1, Danish Rashid21 1Department of Veterinary Medicine, College of Veterinary and Animal Sciences G.B. Pant University of Agriculture and Technology, Pantnagar, U.S. Nagar-263145 Uttarakhand, India. 2Veterinary Assistant Surgeon, Department of Animal Husbandry, Government of Jammu & Kashmir, India Corresponding Author: [e-mail] Currently, Lumpy Skin Disease (LSD), an emerging bovine viral disease has been portrayed as a devastating threat to cattle in the Indian subcontinent. The economic significance of this disease is of great concern as far as international trade, and agricultural bioterrorism is concerned. An arthropod-borne contagious illness presents lump-like nodules in the external skin and mucous membrane with fever and swollen lymph nodes. Albeit the reported mortality of the current outbreak in India is only up to 15%, the rapid spread of the disease has led to substantial and severe socio-economic losses. It causes a reduction in feed intake, a considerable decrease in milk yield, and the occurrence of secondary mastitis associated with lesions on the teat. The losses also accrue from reduced body condition, damaged hides, loss of draught power, abortion in cows, infertility in bulls, lack of semen for artificial insemination, a decline in the growth rate of beef cattle, export restrictions due to higher morbidity, treatment, vaccination costs and death of the infected animals. The sharp drop in milk production is of paramount importance in the current outbreak as the dairy sector of India contributes to approximately 23% of global milk production. A risk assessment study conducted by FAO based on the information available from 2019 to October 2020 revealed that the economic impact of Lumpy Skin Disease for Southeast Asian countries was up to $1.45 billion in direct losses of livestock and production. For effective disease control, strict quarantine measures, control of animal movement, vector control, vaccination, bio-security measures, and awareness programs can be implemented at the earliest to prevent further disease incidence. Successful treatment of Feline Panleukopenia Virus (FPV) in Persian cats Iqra Shafi Khan, Iqra Hussain, Amatul Muhee, Nuzhat Hassan, Shahnaz Bashir, and Showkat Nabi Division: Department of Veterinary Epidemiology and Preventive Medicine FVSc and AH SKUAST-K Background: Feline panleukopenia is a cat disease that is extremely contagious and frequently fatal caused by the feline parvovirus. FPV's single-stranded DNA makes it necessary for cellular DNA polymerase to perform replication, which results in rapidly dividing cells during the S-phase of cell division. Cells particularly affected by this disease include intestinal epithelium, lymphatic tissue, and bone marrow. The most typical clinical symptoms include fever, nausea, diarrhea, anorexia, and/or dehydration. The disease often progresses acutely, especially in kittens, which results in death. Objective: Eight cases presented with a history of anorexia, vomiting diarrhea, and fever were selected for the study. The cases were diagnosed with feline parvovirus based on clinical signs, direct virus detection (using point-of-care (POC) antigen test and hematology. Results and conclusion: Eight cats in the age group of 3–4 months unvaccinated with a history of anorexia, vomiting diarrhea, and fever were selected for the study. The cases were diagnosed with feline parvovirus based on clinical signs, and hematology. On physical examination cats were dull, severely dehydrated (> 5%), CRT > 2 s, and had abnormal vital parameters with an average rectal temperature of 104.28° F ± 0.19. All the cats were positive on direct virus detection (using point-of-care (POC) antigen test. The feline parvovirus infects rapidly dividing cells of bone marrow causing pan leukopenia. In support to confirm leukopenia, the cats were subjected to hematological analysis. All the cats showed leukopenia with an average total leucocyte count of 0.83 ± 1.7 × 103. Furthermore, the cats showed severe neutropenia with an average neutrophil count of 0.180.3 ±  × 103. However, the hemoglobin 8.46 ± 0.18 g/dL and PCV 25.08 ± 0.39% revealed moderate anemia. The cats were treated with standard treatment of aggressive fluid therapy, broad-spectrum antibiotics, antacids, and antiemetics. Every case was treated with a non-pegylated human granulocyte colony-stimulating factor (G-CSF) analog with a dose rate of 6 mcg/kg BW sc for three consecutive days. All the cats responded and recovered well after five days of treatment. Leucocyte counts were repeated after 7 days and counts increased remarkably in all cats with an average count of 9.02 × 103. FPV is a remerging and most common infectious diseases of cats with proper treatment protocol and diagnosis the cats recover completely. Detection and Characterization of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli from Poultry in Central Kashmir Gull, Bisma., Altaf, Mohd., Qureshi, Sabia., Kashoo, Zahid., Farooq, Shaheen., Gulzar, Maliha., Hafiz, Mahruk., Qureshi, Arham. and Faheem U din Division of Veterinary Microbiology and Immunology, Faculty of Veterinary Sciences and AH, Shuhama, Alusteng, Ganderbal The emergence and rapid spread of antimicrobial resistance is now a global concern. Global studies has reported a wide range of prevalence of ESBL producing Esherichia coli (EPE) among commercially grown poultry flocks. The risks to human health posed by EPE from non-human reservoirs are not fully understood. Surveillance for ESBL-producing bacteria could provide information on the fecal flora, which may contaminate the products entering the food chain. The present study was undertaken to study the prevalence and determine the diversity among β lactamase genes isolated from poultry. The samples were collected from poultry birds of commercial poultry farms and private households of three districts of Kashmir viz., Ganderbal, Srinagar and Budgam. Out of 50 pooled rectal swabs comprising of 25 from commercial poultry farms and 25 from private households, 20% samples were found positive for EPE from commercial poultry farms only. All isolates recovered in present study were found positive phenotypically by double disc synergy test (DDST). These phenotypically positive isolates were then subjected to geotypic test for detection of TEM, SHV AND CTX-M genes. All isolates (100%) were positive for TEM, (20%) SHV, (20%) CTX-M Group 1, (30%) CTX-M Group 9 and none of the isolate came positive for CTX-M Group 2. The study revealed that resistance pattern was shown only by those poultry birds which have taken from commercial poultry farms and not from private households. Keywords: ESBL, Poultry, Pooled rectal swabs, prevalence, commercial poultry farms. Genotyping of Group A Rotaviruses from Calves of Kashmir Valley Iqra Hussain*, S. A. Hussain, Iqra Shafi Khan, Showkat Ul Nabi, Shabu S and Mir Nadeem Hassan Division of Veterinary Public Health & Epidemiology, Faculty of Veterinary Sciences & A.H. (SKUAST of Kashmir), Shuhama (Alusteng), Kashmir Acute-gastroenteritis is one of the most common cause of morbidity and mortality in infants/young children and many animal species. Among viruses, rotavirus is the most common and leading cause of acute watery dehydrating diarrhea often noticed in the valley of Kashmir. The present study was carried out to screen the diarrhoeic samples from calves (upto 06 months of age) (n = 100) for the presence of Group A rotaviruses with its molecular typing of strain based on structural genes (VP7 and VP4). Eleven samples were found positive for the presence of the virus using Latex Agglutination Test (LAT). Genotypic characterization (VP7 gene, G genotype) of the rotaviruses revealed predominance of G10 (44.4%) followed by G6 (22.2%) and G3 (11.15%). Characterization of VP4 gene (P genotype) revealed dominance of P[11] (55.5%) followed by P[5] (22.2%). The genotypic combination from the positive samples of calves (bovine) presented as G10P [11] and G6P [11] to be 22.2% followed by G10P [5] and G3P[11] as 11.1%. The dynamic pattern of predominant genotypes underlines the need for continued surveillance of the circulating types of rotavirus for effective and suitable vaccination strategy. Occurrence of Campylobacter jejuni and Campylobacter coli and its virulence gene profile isolated from sheep rectal swabs from Kashmir Mahrukh Hafiz, Sabia Qureshi, Maliha Gulzar, Faheem u Din, Junaid Mehraj Lone, Arham Qureshi, Zahid A Kashoo, M. Altaf Bhat, M.I hussain, Shaheen Farooq Campylobacter laboratory, Division of Veterinary Microbiology & Immunology, FVSC & A.H Shuhama (Aulesteng) SKUAST-K, J&K, India-190006 Human cases of Campylobacter gastroenteritis have grown, making it the second most important foodborne pathogen globally after Salmonellosis. Researchers have strongly suggested that the animal faeces mainly have been serving as the source of human Campylobacteriosis to their handlers and hence the present study was conducted to assess the occurrence of C jejuni and C coli from the sheep rectal swabs. For this polymerase chain reaction (PCR) based on mapA gene of C. jejuni and ceuE gene of C. coli was performed. A total of 200 rectal swab samples were collected from organized sheep farms of district Srinagar (Sheep breeding farm Khimber, n = 50), Ganderbal (Sheep breeding farm Goabal, n = 50), Mountain Research centre for sheep and goat (MRCSG –Shuhama (n = 50) and Private Sheep breeders (n = 50). From the 150 samples of organized sheep farms 7 samples had a concurrent presence of both C. jejuni and C. coli, 8 were positive for C. jejuni and 5 were positive for C. coli only. Out of the 50 samples collected from private farms 3 were positive for both C. jejuni and C. coli, 2 were positive for C. jejuni and 3 for C. coli only. All the isolates were screened for the virulence genes flaA, cdtB, cadF, wlaN which plays an important role in transmission of infection to humans. None of the isolates was positive for wlaN gene but all harboured flaA, cdtB, cadF being a matter of concern due to zoonotic potential of such strains. Keywords: Campylobacter jejuni, Campylobacter coli, mapA, ceuE, flaA, cdtB,, wlaN, cadF. IVS Young Scientist Award A comparative global transcriptomic study of Nicotiana benthamiana plants inoculated with cloned Sri Lankan cassava mosaic virus DNA-A & DNA-A + DNA-B Neelam Jagram*, Indranil Dasgupta* *Department of Plant Molecular Biology, University of Delhi South Campus New Delhi- 110021 Delhi, India Background: Sri Lankan cassava mosaic virus (SLCMV) is a bipartite begomovirus widespread in the cassava crop in the Indian subcontinent and is the cause of cassava mosaic disease. Interestingly, SLCMV DNA A behaves as a monopartite begomovirus and can induce upward leaf curling and vein swelling symptoms in Nicotiana benthamiana and the presence of DNA B accelerates symptom development. Host plants display a suite of differentially expressed genes (DEGs) upon virus infection, the analysis of which can shed light on the defence systems employed by plants and the mechanisms of pathogenesis. Objective: This study was undertaken to understand how the presence of DNA B influences DEGs in the case of SLCMV. Materials and Methods: In this study, RNA-seq was used to analyse differentially expressed genes (DEGs) in Nicotiana benthamiana plants inoculated with infectious clones of SLCMV DNA-A only and SLCMV DNA-A + SLCMV DNA-B. Results and Conclusions: An estimation of the accumulation of DNA-B showed that its levels vary as the infection progresses. Using the MGI sequencing platform, DEGs were identified at time points showing varying accumulation of DNA-B and compared to that without DNA-B, followed by KEGG-based pathway analysis. Computational prediction of differential expression was validated using quantitative reverse-transcription PCR, confirming the robustness of the analytical methods. Nuclear localization of accessory viral protein W of avian paramyxovirus triggers cell death B. Nagaraj Nayak1,#, Devasmita Dutta1, Revathy Shanmugasundaram1, Kalaimagal Rajagopal1, Madhuri Subbiah1,#a,* 1National Institute of Animal Biotechnology, Hyderabad, India. #Graduate studies, Regional Centre for Biotechnology (RCB), New Delhi, India - Registered to PhD with RCB. #aAdjunct Faculty, Regional Centre for Biotechnology, New Delhi, India *Corresponding author Background: Newcastle disease virus (NDV), an avian paramyxovirus is a promising oncolytic virus known to combat many different types of cancers. Previously published research data suggest involvement of structural proteins of NDV in oncolysis. Here, we report the role of accessory viral protein, W, of NDV strain Komarov in augmenting its oncolytic potential. Objectives: To study the role of the accessory viral protein, W, as a contributor to the oncolytic property of NDV. Materials and Methods: DNA laddering and TUNEL assay for detecting DNA fragmentation in the cells overexpressing W protein. Immunofluorescence for imaging active caspase-3 and fluorescence microscopy to observe the various nuclear morphological changes in the W protein transfected cells. Immunoblotting to examine caspase activation in the W protein transfected cells. Flow cytometry analysis after staining the W protein transfected cells with propidium iodide for determining cell death. Results and Conclusion: W protein, an accessory viral protein of NDV strain Komarov generated by unique RNA editing mechanism, was found to localize in the nucleus. The nuclear localization signal was identified by mutational studies and it was observed that the overexpression of W protein and subsequent localization in the nucleus caused distortion of nucleus and ultimately cell death. Laddering of the cellular DNA (characteristic feature of apoptosis), was observed in W protein expressing cells. This was further confirmed by TUNEL assay by labelling of the 3′-hydroxyl termini in the double-strand DNA breaks generated during apoptosis. Various nuclear morphological changes which are consistent with the apoptosis like nuclear condensation, fragmentation, blebbing and apoptotic bodies formation were observed in the W protein transfected cells. Our results showed that about 30–40% of the cells transfected with W protein showed abnormal nucleus. Immunofluorescence showed the presence of active caspase-3 in the W protein transfected cells. Further, immunoblotting showed that the expression of W protein results in the cleavage caspase-3, caspase-7 and PARP-1 proteins. Flow cytometry analysis showed ~ 25% increase in apoptotic cells when transfected with W protein. Our findings suggest that the W protein triggers cell death via induction of apoptosis and can be a potential oncotherapeutic molecule. Exogenous application of suppressor gene derived dsRNAs in chilli plants against chilli leaf curl virus Oinam Washington Singh, Dipinte Gupta, Anirban Roy, and Bikash Mandal* Advanced Centre for Plant Virology, Division of Plant Pathology, ICAR-IARI, New Delhi, India (Correspondence to Dr. Bikash Mandal: [e-mail]) Begomoviruses (family Geminiviridae) are the most significant plant viruses infecting food, fiber and ornamental crops in India. They generally cause leaf curl, mosaic and yellow mosaic disease and are spread exclusively through whitefly (Bemisia tabaci). Chilli leaf curl virus (ChiLCV), a monopartite begomovirus causes major epidemic and substantial yield losses in chilli in India. Effective management of the chilli leaf curl disease is a major challenge due to non-availability of resistance cultivars. In the present study, an alternative novel approach was attempted to prevent the virus by inducing RNA interference in the host plant through the external application of dsRNAs. Four different dsRNAs (dsC2, dsC4, dsV2, and dsβC1) were prepared from suppressors genes of ChiLCV DNA-A and the associated betasatellite by using the vector, L4440 and the bacterial strain, HT115. The individual dsRNA was applied through rubbing the chilli leaves pre-dusted with Celite powder, followed by the virus inoculation through whitefly feeding at one-day post-treatment. All the dsRNA was effective as evident by the reduction of disease incidence up to 14–85.7% at 30 days post virus inoculation (dpi) depending on the type of dsRNA used. Further, the absolute quantification of the virus through real-time PCR in the dsRNA-treated plants showed a reduction of viral copy number up to 84.94–99.95% at 21 dpi. The study for the first time showed that the exogenous application of dsRNAs derived from the suppressor genes are able to impart protection in chilli plants against ChiLCV. Molecular detection of porcine astrovirus, porcine circovirus and porcine rotavirus in diarrheic pigs in Haryana, India Vaishali, Renu Gupta, Mohit Kumar, Nitish Bansal, Anand Parkash, Ramesh Kumar and Naresh Jindal Department of Veterinary Public Health and Epidemiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar 125 004 Background: Porcine astrovirus (PAstV), porcine circovirus (PCV 2) and porcine rotavirus (PR A) are responsible for causing gastroenteritis in pigs globally. Objectives: The present study was conducted to determine the presence of PAstV, PCV 2, and PR A in diarrheic pigs from different districts of Haryana, India. Materials and Methods: Three hundred and six fecal samples were collected from diarrheic pigs in 54 piggery units in the state during March 2021 to March 2022. The samples were collected from three age groups and from three agroclimatic zones. The samples were screened for PAstV, PCV 2, and PR A using reverse transcription-polymerase chain reaction. The reaction conditions were optimised using positive control for each virus. The PCR products were gel electrophoresed and sequenced to confirm the identity. Results: Of these samples, 209 samples (68.3%) showed the presence of either of these three tested viruses; 154 samples (50.3%) were positive for PAstV by using polymerase gene specific primers, 108 samples (35.29%) for PCV 2 by capsid gene specific primers and 32 samples (10.45%) for PR A using e2 gene specific primers. The amplified product size for PAstV, PCV 2 and PR A was 422 bp, 492 bp and 309 bp, respectively. One hundred and thirty-three samples (43.46%) had one virus while 76 samples (24.83%) had two or three tested viruses. Combination of PAstV and PCV 2 was detected in 50 samples (16.33%) while that of PAstV and PR A, and PCV 2 and PR A in 14 (4.5%) and 5 samples (1.6%), respectively. The PAstV alone was detected in 13 piggery units (24.07%) while PCV 2 alone in 5 units (9.25%). The PR A alone was not detected in any of the piggery units. The combination of PAstV and PCV 2 was detected in 18 units (33.33%) while that of PAstV and PR A in 2 units (3.7%). Three units did not show the presence of tested virus. The PAstV and PR A were detected more in pigs of 3–6 weeks of age while PCV 2 was in 0–3 weeks of age. PAstV was present more in semi-arid zone, PCV 2 in dry sub humid zone and PR A in arid zone in the state. Phylogenetic analysis of 39 PAstVs revealed that PAstV 4 and 2 are circulating in Haryana. Further nucleotide homology in PAstV sequences of this study with previously published sequences was 51–100%. Conclusions: Porcine astrovirus, porcine circovirus and porcine rotavirus are present alone or in combination in piggery units in Haryana. The presence of two or more viruses in a piggery unit is of concern as these viruses may increase the severity of disease because of additive or synergistic actions. Adaptation of Mungbean yellow mosaic India virus in diverse crops: a greater epidemic consequence Mohammad Ansar Department of Plant Pathology, Bihar Agricultural University, Sabour-813 210, Bhagalpur, India *Corresponding author: [e-mail] Currently yellow mosaic disease is a serious problem and restraining factor for leguminous crops in the Indian subcontinent. More than eight various bipartite and monopartite begomovirus species are known to cause mosaic symptom in dicot plants including different legumes. A long to short duration crops including pigeonpea, mungbean, uradbean, French bean, soybean and cowpea were depicted golden mosaic symptom. Apart from legumes few of cucurbits and solanaceous plants produced such symptom. From each host, 15 symptomatic plants samples tested against different viruses. Among them, all tested legumes, cucurbit and solanaceous plant found positive for whitefly transmitted geminivirus in PCR assay. In order to further confirmation full genome amplified by rolling circle amplification (RCA) and characterized by sequencing of 2.7 kb genome. The genomic sequence of DNA-A and DNA-B shared highest nucleotide identity between 93–98% with Mungbean yellow mosaic India virus (MYMIV) isolates. The AV1 gene IR-region specific primer of MYMIV successfully amplified the coat protein and intergenic region of all tested legumes, few of cucumber, tomato, and chilli plants. The nucleotide of both CP and IR religion was aligned and recognize the highly conserved region among isolates of legumes, cucurbit and solanaceous plants. In artificial whitefly-mediated transmission experiments, MYMIV efficiently transmitted to each different group of plants and produced the typical symptom. The present study explored the overlapping of seasonal crops serving a green bridge of MYMIV and involved in developing infection in three distinct families. The generated information on sequence data and whitefly-mediated transmission explored that throughout availability of dicot pants accountable for developing epidemics in diverse crops. Looking to the wide potentiality of MYMIV further study needed to relook the adoptability beyond the legume family. Structure-based identification and evaluation of antiviral activity of potent small molecule inhibitors targeting alphavirus RNA-dependent RNA polymerase Authors: Ravi Kumar, Akshay Pareek, Pravindra Kumar, Shailly Tomar Authors’ affiliations: Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, India – 247667 Email: [e-mail] Abstract Background: Alphavirus nsP4 replication protein possesses RNA dependent RNA polymerase (RdRp) activity and is a potential antiviral target. Alphaviruses are continuously re-emerging and are a global threat to human health. Until date, no antiviral drug is commercially available against alphaviruses. Objectives: Here, in this study for the first time small molecule inhibitors targeting nsP4 replication protein were identified and characterized as an effective antiviral against alphaviruses. Materials and Methods: RdRp Δ97 protein was purified and biophysical techniques were employed for analyzing interaction of various ligands with target nsP4 protein. Further, Binding to purified protein were analysed using surface plasmon resonance (SPR). For identification of potential inhibitors, molecular docking was done using 3D model of chimeric RdRp domain of chikungunya nsP4 generated using Phyre2. Further, evaluation of antiviral activity of these molecules was done against alphaviruses (Sindbis and Chikungunya virus) using in vitro cell-based antiviral assay. Results and Conclusions: Four potential inhibitors, piperine (PIP), 2-thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA) targeting the nucleotide-binding site in RdRp were identified. SPR experiments validated the binding of all four molecules to RdRp and affinity of PIP, 2TU, PZA, and CGA, were 0.08, 0.13, 0.66, and 9.87 µM, respectively. These were effectively inhibiting the Sindbis and Chikungunya virus and the EC50 values of PIP, 2TU, PZA, and CGA, were 6.68, 27.88, 35.56, and 53.62 µM, respectively. In conclusion, these non-nucleoside small molecules have the potential to be used for the development of an effective antiviral therapy for treatment of alphaviral infections. Effectiveness of two dose Covaxin, an Inactivated COVID-19 vaccine in protection against SARS-CoV-2 infection: Six-month prospective cohort study 1Rima R. Sahay*, 1Pragya D. Yadav, 2Ashok Nandapurkar, 1Rutuja Dhawde, 1Annasaheb Suryawanshi, 1Deepak Y. Patil, 1Anita M. Shete, 1Gajanan N. Sapkal, Milind Kulkarni, 1Yogesh K. Gurav, 1Gururaj R. Deshpande, 2Jagadevi S. Ghodke, 1Rajlaxmi Jain, 1Raj Hawale, 1Kaumudi Kalele, 1Jyoti Yemul, 1Pranita Gawande, 1Priya Abraham Maximum Containment Facility, ICMR-National Institute of Virology, Microbial Containment Complex (MCC), 130/1, Sus Road, Pashan Pune- 411021, Maharashtra, India E-mail: [e-mail], Mobile no: + 91-8928955888 Introduction: SARS-CoV-2 affected millions of lives globally and led to devastating impact on public health. India had also witnessed the dreadful effect of SARS-CoV-2 pandemic. Within a short span of time, various SARS-CoV-2 vaccines were developed using different platforms across the world. India has also developed one such indigenous whole-virion inactivated SARSCoV-2 vaccine named as BBV152 (Covaxin). The Covaxin has been found to be immunogenic and second most widely used vaccine in India. Recent studies have also shown significant increase in the humoral and neutralizing antibody response post the administration of booster dose against Omicron variant. Apparently, there is limited data on the long-term persistence of the immune response against the Covaxin in Indian context. Methods: We evaluated an effectiveness of the Covaxin and comparing its specific immune responses in two categories through prospective cohorts recruited at the vaccination centre, Pune during June 2021 to March 2022. We defined the study population in two groups who were COVID-19 naïve individuals (group-1) and COVID-19 recovered individuals (group-2) prior to the immunization with Covaxin. The two cohorts and the study participants were decided considering the baseline antibody titres against SARS-CoV-2, the COVID-19 positivity rate, sample power and loss to follow up. The study population was assessed during three follow-ups at second dose, one and six months post second dose to determine the immune response and effectiveness using S1-RBD IgG ELISA and neutralizing antibody response (NAbs) by plaque reduction neutralization test (PRNT). Results: We enrolled participants between age group of 18–80 year (median 32 years). In group-1 and group-2, we recruited 118 and 128 participants respectively. The cohort retention was found to be > 85%, > 70% and > 40% in 1st, 2nd and 3rd follow up respectively. Loss to the 3rd follow up was coincided with third wave with omicron variant. A rise in geometrical mean titre (GMT) of S1-RBD IgG were observed amongst the participants of both the groups at one-month post immunization (Group 1: S1-RBD: 154.4 to 446.3, Group 2 S1-RBD: 918 to 1127). However, the GMTs at six months post vaccination found to be slightly raised in Group 1 compared to one-month follow-up. Considering the hybrid immunity in group 2 participants, the GMTs of NAbs were higher than group 1 participants at each follow-up against B.1, Delta, Omicron BA.1 and BA.2. Both the groups had shown significant reduction in the levels of NAbs against Delta, Omicron BA.1 and BA.2 compared to B.1. The lowest GMTs of NAbs was observed against BA.1 variant. The IgG and NAbs persisted till six months in 90% participants in both categories except BA.1 variant. Breakthrough cases were reported at one-month (n = 1) and six-months (n = 2) post vaccination respectively from group 1. While reinfection cases (n = 3) were detected at six months post vaccination from group 2 due to Omicron BA.1 variant. Conclusion: A two-dose regimen of the Covaxin vaccine enhanced humoral immune response in adults with/without past COVID-19 infection and protected more than 90% adults against SARSCoV-2 infection. Additionally, IgG and NAb responses persisted for six months postvaccination. Keywords: SARS-CoV-2, Covaxin, adult population, immunogenicity, effectivenes.