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Abstract
West Nile virus (WNV) infections were first detected in Germany in 2018, but information
about WNV seroprevalence in horses is limited. The study’s overall goal was to gather
information that would help veterinarians, horse owners, and veterinary-, and public
health- authorities understand the spread of WNV in Germany and direct protective
measures. For this purpose, WNV seroprevalence was determined in counties with and
without previously registered WNV infections in horses, and risk factors for seropositivity
were estimated. The cohort consisted of privately owned horses from nine counties
in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus
ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA
and confirmed by virus neutralization test (VNT). Information about potential risk
factors was recorded by questionnaire and analyzed by logistic regression. A total
of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six
tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%),
while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized
Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and
was significantly higher in counties with previously registered infections (p = 0.005).
The risk factor analysis showed breed type (pony), housing in counties with previously
registered infections, housing type (24 h turn-out), and presence of outdoor shelter
as the main significant risk factors for seropositivity. In conclusion, we estimated
the extent of WNV infection in the resident horse population in Eastern Germany and
showed that seroprevalence was higher in counties with previously registered equine
WNV infections.
West Nile virus (WNV) was first detected in the Western Hemisphere in 1999 during an outbreak of encephalitis in New York City. Over the next 5 years, the virus spread across the continental United States as well as north into Canada, and southward into the Caribbean Islands and Latin America (1). This article highlights new information about the epidemiology and transmission dynamics of human WNV disease obtained over the past 5 years of intensified research. Epidemiology WNV is transmitted primarily by the bite of infected mosquitoes that acquire the virus by feeding on infected birds. The intensity of transmission to humans is dependent on abundance and feeding patterns of infected mosquitoes and on local ecology and behavior that influence human exposure to mosquitoes. Although up to 55% of affected populations became infected during epidemics in Africa, more recent outbreaks in Europe and North America have yielded much lower attack rates (1,2). In the area of most intense WNV transmission in Queens, New York, in 1999, ≈2.6% of residents were infected (most of these were asymptomatic infections), and similarly low prevalence of infection has been seen in other areas of the United States (3,4). WNV outbreaks in Europe and the Middle East since 1995 appear to have caused infection in 1,000 potentially WNV-viremic blood donations were identified, and the corresponding blood components were sequestered. Nevertheless, 6 WNV cases due to transfusion were documented in 2003, and at least 1 was documented in 2004, indicating that infectious blood components with low concentrations of WNV may escape current screening tests (19). One instance of possible WNV transmission through dialysis has been reported (20). WNV transmission through organ transplantation was also first described during the 2002 epidemic (15). Chronically immunosuppressed organ transplant patients appear to have an increased risk for severe WNV disease, even after mosquito-acquired infection (16). During 2002, the estimated risk of neuroinvasive WNV disease in solid organ transplant patients in Toronto, Canada, was approximately 40 times greater than in the general population (16). Whether other immunosuppressed or immunocompromised patients are at increased risk for severe WNV disease is uncertain, but severe WNV disease has been described among immunocompromised patients. WNV infection has been occupationally acquired by laboratory workers through percutaneous inoculation and possibly through aerosol exposure (21,22). An outbreak of WNV disease among turkey handlers at a turkey farm raised the possibility of aerosol exposure (17). Dynamics of Transmission: Vectors WNV is transmitted primarily by Culex mosquitoes, but other genera may also be vectors (23). In Europe and Africa, the principal vectors are Cx. pipiens, Cx. univittatus, and Cx. antennatus, and in India, species of the Cx. vishnui complex (6,24). In Australia, Kunjin virus is transmitted primarily by Cx. annulirostris (11). In North America, WNV has been found in 59 different mosquito species with diverse ecology and behavior; however, 40%. Field studies during and after WNV outbreaks in several areas of the United States have confirmed that house sparrows were abundant and frequently infected with WNV, characteristics that would allow them to serve as important amplifying hosts (23,25,37). The importance of birds in dispersing WNV remains speculative. Local movements of resident, nonmigratory birds and long-range travel of migratory birds may both contribute to the spread of WNV (38,39). Although WNV was isolated from rodents in Nigeria and a bat in India, most mammals do not appear to generate viremia levels of sufficient titer to contribute to transmission (24,40–42). Three reptilian and 1 amphibian species (red-ear slider, garter snake, green iguana, and North American bullfrog) were found to be incompetent as amplifying hosts of a North American WNV strain, and no signs of illness developed in these animals (43). Viremia levels of sufficient titer to infect mosquitoes were found after experimental infection of young alligators (Alligator mississippiensis) (44). In Russia, the lake frog (Rana ridibunda) appears to be a competent reservoir (45). Nonmosquitoborne WNV transmission has been observed or strongly suspected among farmed alligators, domestic turkeys in Wisconsin, and domestic geese in Canada (17,46,47). Transmission through close contact has been confirmed in both birds and alligators in laboratory conditions but has yet to be documented in wild vertebrate populations (23,36,44). Control of WNV Transmission Avoiding human exposure to WNV-infected mosquitoes remains the cornerstone for preventing WNV disease. Source reduction, application of larvicides, and targeted spraying of pesticides to kill adult mosquitoes can reduce the abundance of mosquitoes, but demonstrating their impact on the incidence of human WNV disease is challenging because of the difficulty in accounting for all determinants of mosquito abundance and human exposure. One study indicated that clustering of human WNV disease in Chicago varied between mosquito abatement districts, suggesting that mosquito control may have some impact on transmission to humans (14). Persons in WNV-endemic areas should wear insect repellent on skin and clothes when exposed to mosquitoes and avoid being outdoors during dusk to dawn when mosquito vectors of WNV are abundant. Of insect repellents recommended for use on skin, those containing N,N-diethyl-m-toluamide (DEET), picaridin (KBR-3023), or oil of lemon eucalyptus (p-menthane-3,8 diol) provide long-lasting protection (48). Both DEET and permethrin provide effective protection against mosquitoes when applied to clothing. Persons' willingness to use DEET as a repellent appears to be influenced primarily by their level of concern about being bitten by mosquitoes and by their concern that DEET may be harmful to health, despite its good safety record (49). To prevent transmission of WNV through blood transfusion, blood donations in WNV-endemic areas should be screened by using nucleic acid amplification tests. Screening of organ donors for WNV infection has not been universally implemented because of concern about rejecting essential organs after false-positive screening results (50). Pregnant women should avoid exposure to mosquito bites to reduce the risk for intrauterine WNV transmission. Future Directions WNV disease will likely continue to be a public health concern for the foreseeable future; the virus has become established in a broad range of ecologic settings and is transmitted by a relatively large number of mosquito species. WNV will also likely continue to spread into Central and South America, but the public health implications of this spread remain uncertain. Observations thus far in North America indicate that circulation of other flaviviruses, such as dengue, viral mutation, and differing ecologic conditions may yield different clinical manifestations and transmission dynamics. Over the next few years, research efforts might well be focused in several areas. Research into new methods to reduce human exposure to mosquitoes is crucial and can help prevent other mosquitoborne illnesses. This should include development of new methods to reduce mosquito abundance, development of new repellents, and behavioral research to enhance the use of existing effective repellents and other personal protective measures against mosquito bites. A better understanding of the dynamics of nonmosquitoborne transmission is essential to prevent disease among infants of infected mothers and recipients of blood transfusions and transplanted organs. Currently available prevention strategies such as the dissemination of knowledge and products for personal protection from mosquito exposure and the application of existing techniques for reducing mosquito abundance in communities at risk of WNV transmission need to be vigorously implemented. National and international surveillance for WNV transmission will be important to monitor spread of the virus and the effect of control strategies. Finally, further research into the ecologic determinants of WNV transmission, including climatic factors and dynamics of reservoir and vector populations, could help in determining geographic areas of higher risk for WNV disease.
Geographically, West Nile virus (WNV) is the most widespread member of the Japanese encephalitis virus (JEV) complex within the genus Flavivirus and the family Flaviviridae. The first strain (B 956) was isolated from a human patient in the West Nile district of Uganda in 1937; later the virus was also detected in several mosquito species, horses, humans, and other hosts in Africa, Europe, Asia, and Australia (where it has been named Kunjin virus) ( 1 – 3 ). WNV was introduced into the United States in 1999, and it spread quickly over large parts of North America and reached Mexico ( 4 – 7 ). The clinical impact of WNV varies in different regions. In the Old World, WNV causes relatively mild infections with influenzalike symptoms or no apparent disease ( 2 ); encephalitis and fatalities in the human population, horses, or poultry are spasmodic ( 3 , 8 , 9 ). In the New World, WNV exhibits increased virulence among the local wild bird populations and causes more frequent severe central nervous system symptoms and deaths in humans and horses ( 6 , 10 ). Although exactly how WNV was introduced into New York is unclear, phylogenetic comparison of the viral nucleic acid sequences has shown a close relationship between the American WNV isolates and strains isolated from encephalitic geese and storks in Israel in 1998 ( 11 – 13 ). Experimental infections of rodents indicated that the neurovirulence of WNV correlates with its genotype, and the North American strains are highly neurovirulent for mice ( 14 ). WNV shows relatively high levels of sequence diversity. Comprehensive studies on the phylogenetic relatedness of WNV strains show that they form at least 2 main lineages ( 15 – 17 ). Lineage 1 is composed of WNV strains from different geographic regions, and it is subdivided into at least 3 clades. Clade A contains strains from Europe, Africa, the Middle East, and America; clade B represents the Australian (Kunjin) strains; and clade C contains Indian WNV isolates. Lineage 2 contains the B 956 prototype strain and other strains isolated so far exclusively in sub-Saharan Africa and Madagascar. In addition to the 2 major WNV lineages, we recently proposed 2 lineages for viruses that exhibited considerable genetic differences to the known WNV lineages: lineage 3 consists of a virus strain isolated from Culex pipiens mosquitoes at the Czech Republic/Austria border (named Rabensburg virus), and lineage 4 consists of a unique virus isolated in the Caucasus. These 2 viruses, however, may also be considered independent flaviviruses within the JEV complex ( 18 ). WNV has been known to be present in central Europe for a long time. Seroprevalence in humans was reported in several countries, including Hungary, and WNV strains were isolated from mosquitoes, humans, migrating birds, and rodents during the last 30 years ( 3 ). Until 2003, however, WNV infections in Hungary have never been associated with clinical symptoms, although a severe outbreak of West Nile encephalitis in humans was reported in 1996 and 1997 in neighboring Romania. In late summer 2003, an outbreak of encephalitis emerged in a Hungarian goose flock, resulting in a 14% death rate among 6-week-old geese (Anser anser domesticus). Based on histopathologic alterations, serologic investigations, and nucleic acid detection by reverse transcription–polymerase chain reaction (RT-PCR), WNV was diagnosed as the cause of the disease ( 19 ). Chronologically and geographically related to the outbreak in geese, a serologically confirmed WNV outbreak was also observed in humans, which involved 14 cases of mild encephalitis and meningitis ( 20 ). One year later, in August 2004, a goshawk (Accipiter gentilis) fledgling showed central nervous system symptoms and died in a national park in southeastern Hungary. When histopathologic methods and RT-PCR were used, WNV antigen and nucleic acid were detected in the organs of the bird. Furthermore, the virus was isolated after injection of suckling mice. Here we report the sequencing and phylogenetic results of these 2 encephalitic WNV strains that emerged recently in central Europe. Materials and Methods Brain specimens from one 6-week-old goose, which died during the encephalitis outbreak in a Hungarian goose flock, and brain samples from a goshawk, which also died from encephalitis, were used for WNV nucleic acid determination. The brain samples were homogenized in ceramic mortars by using sterile quartz sand, and the homogenates were suspended in RNase-free distilled water. Samples were stored at –80°C until nucleic acid extraction was performed. Viral RNA was extracted from 140 μL of brain homogenates by using the QIAamp viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. First, a universal JEV-group specific oligonucleotide primer pair designed on the nonstructural protein 5 (NS5) and 3´-untranslated regions (UTR) of WNV (forward primer: 5´-GARTGGATGACVACRGAAGACATGCT-3´ and reverse primer: 5´-GGGGTCTCCTCTAACCTCTAGTCCTT-3´ [21]; ) was applied on the RNA extracts in a continuous RT-PCR system employing the QIAGEN OneStep RT-PCR Kit (Qiagen). Each 25-μL reaction mixture contained 5 μL of 5× buffer (final MgCl2 concentration 2.5 mmol/L), 0.4 mmol/L of each deoxynucleoside triphosphate, 10 U RNasin RNase Inhibitor (Promega, Madison, WI, USA), 20 pmol of the genomic and reverse primers, 1 μL enzyme mix (containing Omniscript and Sensiscript Reverse Transcriptases and HotStarTaq DNA polymerase) and 2.5 μL template RNA. Reverse transcription was carried out at 50°C for 30 min, followed by a denaturation step at 95°C for 15 min. Thereafter, the cDNA was amplified in 40 cycles of heat denaturation at 94°C for 40 s, primer annealing at 57°C for 50 s, and DNA extension at 72°C for 1 min, and the reaction was completed by a final extension for 7 min at 72°C. Reactions were performed in a Perkin-Elmer GeneAmp PCR System 2400 thermocycler (Wellesley, MA, USA) and in a Hybaid PCR Sprint thermocycler (Thermo Electron Corporation, Waltham, MA, USA). After RT-PCR, 10 μL of the amplicons was subjected to electrophoresis in a 1.2% Tris acetate-EDTA-agarose gel at 5 V/cm for 80 min. The gel was stained with ethidium bromide; bands were visualized under UV light and photographed with a Kodak DS Electrophoresis Documentation and Analysis System using the Kodak Digital Science 1D software program (Eastman Kodak Company, Rochester, NY, USA). Product sizes were determined with reference to a 100-bp DNA ladder (Promega). Where clear PCR products of the previously calculated sizes were observed, the fragments were excised from the gel, and DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Fluorescence-based direct sequencing was performed in both directions on PCR products. Sequencing of PCR products was carried out with the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer), according to the manufacturer's instructions, and an ABI Prism 310 genetic analyzer (Perkin-Elmer) automated sequencing system. Nucleotide sequences were identified by Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/blast) search against gene bank databases. Based on the sequence information obtained from the amplification products, complete WNV sequences that exhibited the highest nucleotide identities with the Hungarian genotypes were selected from the GenBank database to design primers that amplify overlapping RT-PCR products covering the entire genome of the strains. Oligonucleotide primers were designed with the help of the Primer Designer 4 for Windows 95 (Scientific and Educational Software, Version 4.10; Microsoft, Redmond, WA, USA) and were synthesized by GibcoBRL Life Technologies, Ltd. (Paisley, Scotland, UK). Detailed information on all primers is in the Tables A1 and A2. PCR amplification products were directly sequenced in both directions; the sequences were compiled and aligned to complete genome sequences of selected representatives of WNV lineages 1a, 1b, 2, and putative lineages 3 and 4 (listed in Table). Phylogenetic analysis was performed by using the modified neighbor-joining method (ClustalX [22]; ), and trees were constructed to demonstrate the relationship between the Hungarian WNVs and other WNV strains (Figure). Figure Phylogenetic tree based on the complete nucleotide sequences of selected West Nile virus strains demonstrating the genetic relatedness of these strains (abbreviations are listed in Table). Boxes indicate different lineages and clades. The Hungarian strains reported in this article are highlighted with gray background). RabV, Rabensburg virus; JEV, Japanese encephalitis virus. Scale bar depicts degree of relatedness. Table West Nile virus strains included in the phylogenetic analysis Name Code Accession no. Isolation Year Host Origin Lineage, clade WNV HNY1999 NY99a AF202541 1999 Human New York, USA 1a WNV NY99flamingo38299 NY99b AF196835 1999 Flamingo New York, USA 1a WNV IS98STD Is98 AF481864 1998 Stork Israel 1a WNV goose-Hungary/03 Hu03 DQ118127 2003 Goose Hungary 1a WNV Italy1998Equine It98 AF404757 1998 Horse Italy 1a WNV RO9750 Ro96 AF260969 1996 Culex pipiens Romania 1a WNV VLG4 Rus99a AF317203 1999 Human Volgograd, Russia 1a WNV LEIV-Vlg99-27889 Rus99b AY277252 1999 Human Volgograd, Russia 1a WNV PaH001 Tu97 AY268133 1997 Human Tunisia 1a WNV PaAn001 Fr00 AY268132 2000 Horse France 1a WNV Eg 101 Eg51 AF260968 1951 Human Egypt 1a WNV Chin-01 Chin01 AY490240 1950s ? Russia 1a WNV Kunjin MRM61C Kunjin D00246 1960 Cx. annulirostris Australia 1b WNV Sarafend Sarafend AY688948 Laboratory strain 2 WNV B956 (WNFCG) Ug37 NC_001563 1937 Human Uganda 2 WNV goshawk-Hungary/04 Hu04 DQ116961 2004 Goshawk Hungary 2 Rabensburg virus (97-103) RabV AY765264 1997 Cx. pipiens Czech R. 3? WNV LEIV-Krnd88-190 Rus98 AY277251 1998 Dermacentor marginatus Caucasus, Russia (Georgia?) 4? The nucleotide sequences of the Hungarian WNV strains goose-Hungary/03 (Hu03) and goshawk-Hungary/04 (Hu04) were submitted to the GenBank database. They are available under accession numbers DQ118127 and DQ116961, respectively. Results In this study, the complete genome sequences of WNV strains derived from a 6-week-old goose, which died in 2003 during an outbreak of encephalitis in a Hungarian goose flock (strain goose-Hungary/03), and from a goshawk, which also died from encephalitis in the same region 1 year later (strain goshawk-Hungary/04), were determined, aligned, and phylogenetically analyzed. The genome of the goose-Hungary/03 strain is composed of 10,969 nucleotides (nt) and contains 1 open reading frame between nucleotide positions 97 and 10,398, coding for a 3,433 amino acid (aa)–long putative polyprotein precursor. The complete genomic sequence of the virus was subjected to a BLAST search against gene bank databases. The highest identity rates (98% at the nucleotide and 99% at the amino acid level) were found with WNV strains isolated in 1998 in Israel and in 1999 in the United States. In addition, phylogenetic analysis was performed to indicate the relationships between the Hungarian goose–derived WNV strain and selected representatives of WNV clades and clusters. The resulting phylogenetic tree (Figure) confirmed the results of the BLAST search, i.e., the Hungarian goose–derived WNV strain is clustering close to the previously mentioned WNV strains isolated in the United States and Israel, which belong to lineage 1a of WNV. Other European WNV strains (isolated in Italy, France, and Romania) are more distant to the Hungarian strain; they form a separate cluster consisting of a Romanian/Russian and a French/Italian subcluster. The complete nucleotide sequence of the goshawk-Hungary/04 WNV strain is composed of 11,028 nt and contains 1 open reading frame between nucleotide positions 97 and 10,401, coding for a 3,434-aa putative polyprotein precursor. In BLAST search, the strain showed the highest (96% nt and 99% aa) identity to the WNV prototype strain B 956. Consequently, as the phylogram also indicates (Figure), this virus belongs to lineage 2 of WNV. Alignments of the available partial sequences from the E protein coding regions of other representatives of this cluster showed even higher identities (97%–98% nt and 100% aa) with WNV strains isolated in central Africa in 1972 (AnB3507, AF001563) and in 1983 (HB83P55, AF001557), respectively ( 15 ). More recently (in early August 2005), additional lethal cases of encephalitis occurred in birds of prey in the same place in which the goshawk died of West Nile encephalitis in 2004, involving up to a total of 3 goshawks and 2 sparrow hawks (A. nisus); 2 of the goshawks and 1 sparrow hawk died. Preliminary investigations detected WNV-specific nucleic acid in the brains of the birds. The partial nucleotide sequence of the 2005 virus (1,000 bp at the NS5´–3´-UTR regions) showed 99.9% identity with the goshawk-Hungary/04 strain (only 1 substitution at nucleotide position 9,376 [g→a] has been observed, which did not influence the putative amino acid sequence). Additional observation of the outbreak and investigations of the cases are in progress. Discussion The primary aim of our investigations was to show the genetic relatedness of the WNV strains detected in Hungary in the last 2 years and to estimate their clinical and epidemiologic impact. The phylogenetic analysis emphasizes the close genetic relationship of the goose-Hungary/03 strain with a WNV strain isolated in Israel in 1998 and the WNV strain introduced in New York in 1999, since the 3 WNVs form 1 single cluster within clade 1a of lineage 1. These strains caused outbreaks in birds, humans, and horses. Previous European WNV isolates exhibited lower identity values, e.g., the strain that was responsible for the Romanian outbreak(s) in 1996 and 1997 showed only 96% nt identity with the Hungarian goose-2003 strain, and in the phylogenetic tree the other European isolates form a separate cluster consisting of 2 subclusters (Figure). The earliest representatives of the Israel/USA/goose-Hungary/03 cluster were reported by Malkinson et al. ( 23 ) from ill and dead white storks (Ciconia ciconia) in Israel in 1998. These storks, however, had hatched in central Europe, and during their autumn migration southwards, strong winds had blown them off course, from their usual route to Africa, to southern Israel. Malkinson et al. suspected that these birds introduced the neurovirulent genotype of WNV to Israel from their hatching place. The wetlands of southeastern Hungary are foraging and nesting habitats for storks and many other wild bird species, and the goose farm, where the WNV outbreak occurred in 2003, is located in this region. These facts, together with the close phylogenetic relatedness of the Israeli/US/Hungarian WNV strains, strongly support the theory that storks carried the neurovirulent WNV strain from central Europe (that is, from Hungary) to Israel, which sheds new light on the introduction of WNV to New York. This virus could have originated in Israel (which is the generally accepted although not proven theory) or central Europe. In both cases, however, the virus seems to have its true origin in Europe. In a recent publication, Lvov et al. suggested that WNV could have been introduced into New York by ships traveling from Black Sea ports ( 24 ). When a WNV infection was detected in 2004 in a goshawk fledgling, which died from encephalitis in the same region of Hungary in which the outbreak in geese and humans occurred during the previous year, we anticipated a WNV strain more or less identical to the genotype detected there in 2003. The genomic sequence of this strain was not closely related to the sequence of the WNV strain detected in geese in the year before, however, but belonged to the group of central African lineage 2 WNV strains. A closely related strain from this cluster (ArB3573, AF001565, and AF458349) was identified as a neuroinvasive strain of WNV in a mouse model ( 14 ). To our knowledge, this report is the first on the emergence of a lineage 2 WNV strain outside Africa. Migratory birds that had overwintered in central Africa probably introduced this exotic strain to the wetlands of Hungary. On the other hand, as the goshawk is not a migratory species, and infection occurred in August, the African WNV strain must have already successfully adapted to local mosquito vectors. Consequently, this neurotropic, exotic WNV strain may become a resident pathogen in Europe with all the possible public health consequences. Our results indicate that the WNV strains that emerged in 2 consecutive years and caused avian deaths in Hungary are epidemiologically unrelated. Genetically distinct WNV strains are circulating simultaneously yet independently in local birds and thus most likely also in local mosquito populations within the same region. They cause sporadic cases of encephalitis and also raise the possibility of spreading to other European countries or even to other continents, as happened in 1999 with another WNV strain, which resulted in a public health catastrophe in America. In addition to the above 2 novel WNVs, we recently characterized another novel flavivirus of so far unknown human pathogenicity named Rabensburg virus, which has been isolated from Culex pipiens mosquitoes in 1997 and 1999 at the Czech Republic–Austria border, only a few hundred kilometers from the region where the Hungarian WNVs emerged. After the entire genome was sequenced, Rabensburg virus turned out to represent either a new (third) lineage of WNV or a novel flavivirus of the JEV group ( 18 ). Thus, several distinct WNV strains seem to circulate in central Europe. In 2001 another flavivirus of the JEV group, Usutu virus, which has never previously been observed outside Africa, emerged in Austria and resulted in deaths in several species of birds, especially Eurasian blackbirds (Turdus merula) ( 21 ). This virus became a resident pathogen in Austria and continues to disperse and cause deaths in blackbirds and other species of birds ( 25 , 26 ). The snowy winter and rainy spring of 2005 resulted in serious floods in the area in which the Hungarian WNV strains were identified. Since the floodplains and polders were under water, the conditions for mosquito development were ideal. The summer was also very rainy, which resulted in more floods in the region and continuous mosquito gradation. The most recent data imply that the lineage 2 WNV strain may have overwintered in Hungary, causing several clinical cases of encephalitis in Accipiter species in 2005 as well. The routine diagnostic techniques in most of the European public health and veterinary laboratories are designed to detect lineage 1 WNV strains. In a recent PCR external quality assurance multicenter test, <40% of the involved laboratories could detect lineage 2 WNV strains (Matthias Niedrig, pers. comm.). Therefore, a major goal of this article is to increase the scientific and public awareness of this potential public health threat for Europe and, perhaps, America. Furthermore, comprehensive investigations on the occurrence, ecology, and epidemiology of the different WNV strains circulating in central Europe, as well as the development of monitoring and surveillance programs, must be of highest priority. One may also speculate on environmental factors, such as climate change or global warming, that may have enhanced the recent emergence of viruses, which had previously been restricted to Africa, in new habitats and continents. Improved observation, reporting, and detection methods have also contributed to the apparent increasing emergence of these viruses.
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