Dynamics of West Nile Virus Persistence in House Sparrows (Passer domesticus)

West Nile Virus (WNV) is now endemic throughout North America, with annual recurrence dependent upon successful overwintering when cold temperatures drive mosquito vectors into inactivity and halt transmission. To investigate whether avian hosts may serve as an overwintering mechanism, groups of eight to ten House Sparrows were experimentally infected with a WN02 genotype of WNV and then held until necropsy at 3, 5, 7, 9, 12, 15, or 18 weeks post-infection (pi) when they were assessed for the presence of persistent infection. Blood was collected from all remaining birds every two weeks pi, and sera tested for WNV RNA and WNV neutralizing antibodies. West Nile virus RNA was present in the sera of some birds up to 7 weeks pi and all birds retained neutralizing antibodies throughout the experiment. The detection of persistently infected birds decreased with time, from 100% (n = 13) positive at 3 weeks post-infection (pi) to 12.5% (n = 8) at 18 weeks pi. Infectious virus was isolated from the spleens of birds necropsied at 3, 5, 7 and 12 weeks pi. The current study confirmed previous reports of infectious WNV persistence in avian hosts, and further characterized the temporal nature of these infections. Although these persistent infections supported the hypothesis that infected birds may serve as an overwintering mechanism, mosquito-infectious recrudescent viremias have yet to be demonstrated thereby providing proof of principle.     

Species composition of mosquitoes (Diptera, Culicidae) and possibility of the West Nile virus natural foci formation in the South of Western Siberia

In 2004 June-July collections of mosquito adults and small mammals were carried out in two areas of Novosibirsk Region (forest-steppe and steppe zones), where the West Nile virus (WNV) was for the first time recorded in birds with different migration status in 2002-2004. Seventeen species of mosquitoes were found; significant changes in their species composition and abundance, as compared with latest faunistic studies made in the sixties-seventies of the last century, are revealed. WNV markers (antigen, RNA) are found in small mammals; highly sensitive to the WNV replication mosquito species are also found. These facts allow supposing a possibility of the formation of stable West Nile virus natural foci in the South of Western Siberia, under conditions of forest-steppe and steppe zones.     

Prevalence of west Nile virus antibodies in a breeding population of American Kestrels (Falco sparverius) in Pennsylvania

West Nile virus (WNV) has been identified in nearly 300 species of wild birds, including raptors, in North America since its introduction in New York City in 1999. American Kestrels (Falco sparverius) are susceptible to WNV infection, and the numbers of these birds have declined along the Atlantic coast in recent years. We examined the population biology and WNV exposure of kestrels breeding in the area surrounding Hawk Mountain Sanctuary in Kempton, Pennsylvania, USA. The reproductive biology of kestrels in this area was studied from 1992 until 2004. The number of kestrels breeding in nestboxes in 2004 was only 44% of the 6-yr mean observed prior to 1999. During the 2004 nesting season (study period: 8 June through 22 July 2004), adult kestrels were trapped near the site of their nestboxes. Blood samples were obtained, and serum antibodies specific to WNV were quantified using a plaque reduction neutralization test. Of 22 birds tested, 21 exhibited serum antibodies to WNV, suggesting that most (95%) of the adult kestrels in the population had been exposed to WNV.     

The genotyping of the West Nile virus in birds in the far eastern region of Russia in 2002-2004

Samples from 20 species of trapped and dead birds were collected in the Far Eastern Region in 2002-2004 and were analyzed by the anti-WNV MAb-modified immunoenzyme assay for antigen detection and RT-PCR for viral RNA detection. Five positive samples from cinereous vultures (Aegypius monachus) and two positive samples from cattle egret (Bubulcus ibis) were found in both tests. The sequencing of the 322 bp fragments of protein E gene showed 99-99.67% homology with the strain WNV/LEIV-VlgOO-27924 of the WNV isolated in Volgograd, Russia, 2000. Additionally, five positive samples from birds (Pica pica, Corvus macrorhynchos, Larus crossirostris, Parus minor, Emberiza spodocephala) collected in autumn 2004 were found during screening with anti-WNV MAb-modified ELISA. These results confirm that the WNV is circulating in the Far Eastern Region of Russia and outbreaks of WN fever in humans may be possible. This demonstrates that the genotype 1a of the West Nile virus could spread in the southern regions of the Far East by migrating birds and introduction of the WNV into other southern regions of the Asian part of Russia are probably.     

Mosquito‐borne West Nile virus (WNV) surveillance in the Upper Rhine Valley,Germany

West Nile virus (WNV) could be introduced into Germany via migratory birds originating from Africa or southern Europe and subsequently transmitted to indigenous birds, humans, or horses by mosquitoes. Neither the virus itself nor antibodies against WNV have yet to be found in mosquitoes and horses, whereas antibodies have been detected in migrating birds and in humans that were in close contact with birds. At present, the West Nile virus itself has yet to be detected in Germany. This investigation was conducted primarily in major bird breeding, resting, and roosting habitats (hotspots) in the Upper Rhine Valley. Adult mosquitoes were trapped using CO₂‐baited Encephalitis Vector Surveillance (EVS)‐traps and were tested for WNV by the VecTest WNV Antigen Assay. In 2007 and 2008, a total of 11,073 host‐seeking adult female mosquitoes (13 species) were tested, and all tests were negative for WNV. Statistical calculations could be performed only where sufficient numbers of mosquitoes were trapped. For these sites, WNV infection among mosquitoes could be ruled out with 80% certainty. For the evaluation of the WNV situation in Germany, the results of this investigation are a further indication that the virus has not yet arrived.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Prevalence of neutralizing antibodies to West Nile virus in Eleonora's Falcons in the Canary Islands

Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration.     

West Nile Virus Infection in American Robins: New Insights on Dose Response

West Nile virus (WNV) is a vector-borne pathogen that was first detected in the United States in 1999. The natural transmission cycle of WNV involves mosquito vectors and avian hosts, which vary in their competency to transmit the virus. American robins are an abundant backyard species in the United States and appear to have an important role in the amplification and dissemination of WNV. In this study we examine the response of American robins to infection with various WNV doses within the range of those administered by some natural mosquito vectors. Thirty American robins were assigned a WNV dosage treatment and needle inoculated with 10^0.95 PFU, 10^1.26 PFU, 10^2.15 PFU, or 10^3.15 PFU. Serum samples were tested for the presence of infectious WNV and/or antibodies, while oral swabs were tested for the presence of WNV RNA. Five of the 30 (17%) robins had neutralizing antibodies to WNV prior to the experiment and none developed viremia or shed WNV RNA. The proportion of WNV-seronegative birds that became viremic after WNV inoculation increased in a dose dependent manner. At the lowest dose, only 40% (2/5) of the inoculated birds developed productive infections while at the highest dose, 100% (7/7) of the birds became viremic. Oral shedding of WNV RNA followed a similar trend where robins inoculated with the lower two doses were less likely to shed viral RNA (25%) than robins inoculated with one of the higher doses (92%). Viremia titers and morbidity did not increase in a dose dependent manner; only two birds succumbed to infection and, interestingly, both were inoculated with the lowest dose of WNV. It is clear that the disease ecology of WNV is a complex interplay of hosts, vectors, and viral dose delivered.     

Serologic evidence of West Nile virus infection in black bears (Ursus americanus) from New Jersey

Serum samples obtained from 51 free-ranging black bears (Ursus americanus) in northwestern New Jersey in February and March 2002 were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis virus. Three (6%) of the black bears tested positive for WNV-neutralizing antibodies. One additional sample was positive for flavivirus-neutralizing antibodies but could not be differentiated for a specific virus type. This is the first report of WNV infection in black bears.     

West nile virus antibodies in bats from New Jersey and New York

Eighty-three serum samples were obtained from big brown (Eptesicus fuscus), little brown (Myotis lucifugus), and northern long-eared (Myotis septentriotalis) bats (Chiroptera: Vespertilionidae), from New Jersey and New York (USA) between July and October 2002. Samples were analyzed for neutralizing antibodies to West Nile virus (WNV) and St. Louis encephalitis (SLE) virus. One little brown bat and one northern long-eared bat tested positive for WNV neutralizing antibodies. No bats had antibodies to SLE virus. This was the first large-scale investigation of WNV infection in bats in New Jersey. Additional work is needed to determine the effects of WNV on bat populations.     

Induction of neutralizing antibodies in reindeer (Rangifer tarandus) after administration of a killed West Nile virus vaccine

In 2002, West Nile virus (WNV) infection with clinical neurologic disease and encephalomyelitis was described in reindeer (Rangifer tarandus). The susceptibility of reindeer to WNV prompted questions concerning vaccination of reindeer to prevent WNV infection. Between January and April 2003, eleven 2-4-yr-old, castrated male reindeer, some of which had antibody titers suggestive of prior exposure to WNV, were vaccinated three times at 4-wk intervals with a commercially available vaccine approved for use in horses. No adverse reactions to vaccination were noted. All vaccinated reindeer developed high neutralizing antibody titers to WNV, as determined by the plaque reduction neutralization test. Reindeer without antibody titers from previous natural exposure to WNV required a primary vaccination and one or two booster vaccinations for development of neutralizing antibody to WNV. Protective efficacy of vaccination was not evaluated. Vaccination of reindeer for WNV may be warranted in certain circumstances combined with management practices to limit exposure to potential vectors.     

Control of arbovirus infections by a coordinated response: West Nile Virus in England and Wales

Although there is no recognized transmission of human arboviral infections in the UK, concerns about the possible spread of West Nile virus (WNV) have precipitated coordinated activities around both surveillance and response. The Department of Health has chaired a UK WNV task force since the end of 2000. This is a multidisciplinary group of senior representatives from Agencies and Government Departments involved in human and animal health, entomology and academic departments. Activities include surveillance for WNV infections in humans, and in dead birds, mosquitoes and horses. All have been negative for WNV. A WNV contingency plan was produced in 2004, and this could be used as a generic plan for an effective and coordinated response in the event of the emergence of a new vector-borne zoonotic infection.     

High Prevalence of West Nile Virus in Domestic Birds and Detection in 2 New Mosquito Species in Madagascar

West Nile virus is an arthropod-borne zoonosis transmitted by a large number of mosquito species, and birds play a key role as reservoir of the virus. Its distribution is largely widespread over Africa, Asia, the Americas and Europe. Since 1978, it has frequently been reported in Madagascar. Studies described a high seroprevalence level of the virus in humans in different areas of the island and a human fatal case of WNV infection was reported in 2011. Despite these reports, the epidemiology of WNV in Madagascar, in particular, viral circulation remains unclear. To explore the transmission of WNV in two rural human populations of Madagascar, we investigated local mosquitoes and poultry for evidence of current infections, and determined seroprevalence of candidate sentinel species among the local poultry. These 2 areas are close to lakes where domestic birds, migratory wild birds and humans coexist. Serological analysis revealed WNV antibodies in domestic birds (duck, chicken, goose, turkey and guinea fowl) sampled in both districts (Antsalova 29.4% and Mitsinjo 16.7%). West Nile virus nucleic acid was detected in one chicken and in 8 pools of mosquitoes including 2 mosquito species (Aedeomyia madagascarica and Anopheles pauliani) that have not been previously described as candidate vectors for WNV. Molecular analysis of WNV isolates showed that all viruses detected were part of the lineage 2 that is mainly distributed in Africa, and were most closely matched by the previous Malagasy strains isolated in 1988. Our study showed that WNV circulates in Madagascar amongst domestic birds and mosquitoes, and highlights the utility of poultry as a surveillance tool to detect WNV transmission in a peri-domestic setting.     

Pathology and epidemiology of natural West Nile viral infection of raptors in Georgia

Carcasses from 346 raptors found between August 2001 and December 2004 were tested for West Nile virus (WNV) using virus isolation and immunohistochemistry; 40 were positive for WNV by one or both methods. Of these 40 birds, 35 had histologic lesions compatible with WNV infection, one had lesions possibly attributable to WNV, and four had no histologic evidence of WNV. The most common histologic lesions associated with WNV infection were myocardial inflammation, necrosis, and fibrosis; skeletal muscle degeneration, inflammation, and fibrosis; and lymphoplasmacytic encephalitis. Other lesions included hepatitis, lymphoid depletion in spleen and bursa, splenic and hepatic hemosiderosis, pancreatitis, and ganglioneuritis. Gross lesions included calvarial and leptomeningeal hemorrhage, myocardial pallor, and splenomegaly. Red-tailed hawks (Buteo jamaicensis) (10/56), sharp-shinned hawks (Accipiter striatus) (8/40), and Cooper's hawks (Accipiter cooperii) (10/103) were most commonly affected. Also affected were red-shouldered hawks (Buteo lineatus) (2/43), an osprey (Pandion haliaetus) (1/5), barred owls (Strix varia) (4/27), a great horned owl (Bubo virginianus) (1/18), and eastern screech owls (Megascops asio) (4/42). Although birds were examined throughout the year, positive cases occurred only during the summer and late fall (June-December). Yearly WNV mortality rates ranged from 7-15% over the four years of the study. This study indicates trends in infection rates of WNV in raptorial species over a significant time period and supports the available information regarding pathology of WNV infection in Strigiformes and Falconiformes. Although many species tested were positive for WNV infection, severity of lesions varied among species.     

Efficacy of wildlife rehabilitation centers in surveillance and monitoring of pathogen activity: a case study with West Nile virus

Surveillance is critical for identifying and monitoring pathogen activity in wildlife populations, but often is cost- and time-prohibitive and logistically challenging. We tested the hypothesis that wildlife rehabilitation centers are useful for monitoring pathogen activity using West Nile virus (WNV) as a case study. We hypothesized that birds submitted to wildlife rehabilitation centers would have a similar prevalence of antibody to WNV as free-ranging birds. From 2008 to 2010, we collected sera from peridomestic birds submitted to the Wildlife Care Clinic (WCC), a wildlife rehabilitation center in central Iowa, and tested them for antibodies to WNV. We also collected and tested sera from free-ranging peridomestic birds in the area from which approximately 50% of WCC submissions historically originated. Prevalences of WNV antibodies in free-ranging birds and in peridomestic WCC birds were 2.3% (44/1,936) and 2.8% (2/72), respectively. However, none of the birds submitted to the WCC from the area where we captured free-ranging birds had antibodies (0/29). Our results indicate that rehabilitation facilities are not likely to be useful for monitoring WNV activity at small spatial scales or over short-time periods due to the low endemic prevalence of WNV, and low and variable submission rates. However, at larger spatial scales (ca. nine Iowa counties) WNV antibody prevalence in peridomestic birds submitted to the WCC was similar to that of free-ranging birds. Although limitations to using rehabilitation birds to monitor WNV must be considered, testing these birds could be useful for monitoring WNV activity regionally, especially with many states limiting surveillance due to budgetary constraints.     

Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.     

Seroprevalence of West Nile virus in nonhuman primates as related to mosquito abundance at two national primate research centers

West Nile virus (WNV) surfaced as an emerging infectious disease in the northeastern United States in 1999, gradually spread across the continent, and is now endemic throughout North America. Outdoor-housed nonhuman primates at the Tulane National Primate Research Center (TNPRC) in Louisiana were documented with a relatively high prevalence (36%) of antibodies to West Nile virus. We examined the prevalence of antibodies to WNV in a nonhuman primate population housed in outdoor colonies at the Yerkes National Primate Research Center Field Station located near Atlanta, Georgia. We screened rhesus macaques (Macaca mulatta) and sooty mangabeys (Cercocebus atys) that were at least 3 y old by serum neutralization for antibodies to WNV and confirmed these results by hemagglutination-inhibition assay. None of the 45 rhesus monkeys had antibodies to WNV, but 3 of the 45 mangabeys (6.6%) were positive by both serum neutralization and hemagglutination-inhibition tests. The ratio of seroprevalences in the TNPRC and Yerkes primate populations was similar to the ratio of WNV incidences in people in Louisiana and Georgia from 2002 to 2004. The difference in the exposure of nonhuman primates (and possibly humans) to WNV between these 2 regions is consistent with the difference in the abundance of mammal-biting WNV-infectious mosquitoes, which was 23 times lower near Yerkes than around TNPRC in 2003 and 33 times lower in 2004.