Persistent West Nile virus transmission and the apparent displacement St. Louis encephalitis virus in southeastern California, 2003-2006

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective "avian herd immunity" due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003-2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003-2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.     

Cross-correlation map analyses show weather variation influences on mosquito abundance patterns in Saginaw County, Michigan, 1989-2005

Weather is important determinant of mosquito abundance that, in turn, influences vectorborne disease dynamics. In temperate regions, transmission generally is seasonal as mosquito abundance and behavior varies with temperature, precipitation, and other meteorological factors. We investigated how such factors affected species-specific mosquito abundance patterns in Saginaw County, MI, during a 17-yr period. Systematic sampling was undertaken at 22 trapping sites from May to September, during 1989-2005, for 19,228 trap-nights and 300,770 mosquitoes in total. Aedes vexans (Meigen), Culex pipiens L. and Culex restuans Theobald, the most abundant species, were analyzed. Weather data included local daily maximum temperature, minimum temperature, total precipitation, and average relative humidity. In addition to standard statistical methods, cross-correlation mapping was used to evaluate temporal associations with various lag periods between weather variables and species-specific mosquito abundances. Overall, the average number of mosquitoes was 4.90 per trap-night for Ae. vexans, 2.12 for Cx. pipiens, and 1.23 for Cx. restuans. Statistical analysis of the considerable temporal variability in species-specific abundances indicated that precipitation and relative humidity 1 wk prior were significantly positively associated with Ae. vexans, whereas elevated maximum temperature had a negative effect during summer. Cx. pipiens abundance was positively influenced by the preceding minimum temperature in the early season but negatively associated with precipitation during summer and with maximum temperature in July and August. Cx. restuans showed the least weather association, with only relative humidity 2-24 d prior being linked positively during late spring-early summer. The recently developed analytical method applied in this study could enhance our understanding of the influences of weather variability on mosquito population dynamics.     

Emergence of attenuated West Nile virus variants in Texas, 2003

In order to understand how West Nile virus (WNV) has evolved since its introduction into North America, we have studied the genetic and phenotypic variation among WNV isolates collected in various areas during consecutive transmission seasons. The present report describes for the first time phenotypic changes occurring in the North American WNV population. Several isolates collected in Texas during 2003 display a small plaque (sp) and temperature sensitive (ts) phenotype, as well as reduced replication in cell culture, in comparison to isolates collected in 2002 and New York in 1999. Studies of mouse neuroinvasiveness/neurovirulence also indicate that several of these isolates were attenuated in neuroinvasiveness, but not for neurovirulence. The complete genome and deduced amino acid sequences of several of these isolates have been determined in order to map the mutations responsible for this phenotypic variation. These data indicate microevolution of WNV and the emergence of isolates exhibiting phenotypic variation.     

Drought-induced amplification and epidemic transmission of West Nile virus in southern Florida

We show that the spatial-temporal variability of human West Nile (WN) cases and the transmission of West Nile virus (WNV) to sentinel chickens are associated with the spatial-temporal variability of drought and wetting in southern Florida. Land surface wetness conditions at 52 sites in 31 counties in southern Florida for 2001-2003 were simulated and compared with the occurrence of human WN cases and the transmission of WNV to sentinel chickens within these counties. Both WNV transmission to sentinel chickens and the occurrence of human WN cases were associated with drought 2-6 mo prior and land surface wetting 0.5-1.5 mo prior. These dynamics are similar to the amplification and transmission patterns found in southern Florida for the closely related St. Louis encephalitis virus. Drought brings avian hosts and vector mosquitoes into close contact and facilitates the epizootic cycling and amplification of the arboviruses within these populations. Southern Florida has not recorded a severe, widespread drought since the introduction of WNV into the state in 2001. Our results indicate that widespread drought in the spring followed by wetting during summer greatly increase the probability of a WNV epidemic in southern Florida.     

Direct (non-vector) transmission of West Nile virus in geese

During a recent epizootic, losses due to West Nile virus (WNV) infection in young goose flocks were estimated to be far greater than expected if mosquito-borne transmission was the principal route of infection. Contact transmission was investigated experimentally as an alternative explanation. A group of 10, 3-week-old geesewere inoculated subcutaneously and placed in one insect-proof room with 20 geese of the same age. A group of10 geese were housed in an adjacent insect-proof room to serve as an environmental control. All geese in theinoculated group produced antibodies, eight became viraemic and five died between 7 and 10 days afterinfection. Virus was shed from the cloaca and oral cavity by three geese. Two of the in-contact birds died ondays 10 and 17 after infection, and WNV was recovered from another three birds. None of the environmentalcontrol group became infected. This result strongly suggests that horizontal transmission of WNV can occur incommercial flocks and may be aggravated if cannibalism and feather-picking of sick geese occur.     

Meteorological and hydrological influences on the spatial and temporal prevalence of West Nile virus in Culex mosquitoes, Suffolk County, New York

The factors determining the spatial and temporal distribution of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) activity are not well understood. Here, we explore the effects of hydrological and meteorological conditions on WNV infection among Culex genus mosquitoes collected during 2001-2009 in Suffolk County, Long Island, NY. We show that WNV infection rates in assayed pools of Culex mosquitoes are associated in both space and time with hydrological and meteorological variability. Specifically, wet winter, warm and wet spring conditions, and dry summer conditions are associated with the increased local prevalence of WNV among Culex mosquitoes during summer and fall. These findings indicate that within Suffolk County, and for a given year, areas at risk for heightened WNV activity may be identified in advance by using hydrology model estimates of land surface wetness and observed meteorological conditions.     

Multiple pathways to the attenuation of West Nile virus in South-East Texas in 2003

West Nile virus (WNV) was first detected in Texas in 2002. During 2003, several isolates exhibiting significant attenuation of mouse neuroinvasiveness, and in some cases a small plaque and temperature sensitive phenotype when compared to other North American WNV isolates, were obtained from birds and mosquitoes in South-East Texas. To determine the attenuation markers of WNV, we have sequenced the genomes of three attenuated isolates and four temporally related virulent isolates and compared the amino acid substitutions in a total of 101 isolates, including three previously published genomes of attenuated strains, to identify mutations that are potentially involved in attenuation. Surprisingly, the attenuated strains fall into three separate “groups”, suggesting that the attenuated phenotype evolved on three separate occasions in 2003. None of the groups share the same nucleotide changes or amino acid substitutions, and there are few mutations that can be clearly defined alone as being associated with attenuation.     

Mosquitoes infected with West Nile virus in the Florida Keys,Monroe County,Florida, USA

More than 30,000 mosquitoes in 22 species or species groups were collected from the Florida Keys, Monroe County, FL, USA, in dry ice-baited light and gravid traps. Dry ice-baited traps collected more mosquitoes than did gravid traps. West Nile virus was detected in pools of Anopheles atropos Dyar & Knab, Deinocerites cancer Theobald, and Ochlerotatus taeniorhynchus (Wiedemann).     

West Nile virus infection in mosquitoes in the mid-south USA, 2002-2005

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was first detected in the Tennessee Valley and in Alabama in August 2001. In summer 2002, intensive viral activity was seen, but in subsequent years, viral activity settled into an enzootic pattern. Here, we report an analysis of viral activity in the mosquito fauna in the Mid-South from 2002 (the first year viral activity was detected in mosquitoes) through 2005. Eight mosquito species were infected with WNV during 2002. However, viral activity was only detected in four species--Culex salinarius Coquillett, Culex erraticus Dyar & Knab, Coquillettidia perturbans Walker, and Aedes vexans Meigen--in multiple years. The greatest number of positive pools was in Cx. erraticus and Cx. salinarius. Despite being specifically targeted for collection, Aedes albopictus Skuse was only found to be infected during the epiornitic year (2002), suggesting that under enzootic transmission conditions its role as a bridge vector in the region may not be significant. Virus-positive pools of Cx. erraticus were identified from winter-resting and early season dry ice-baited trap collections in 2005, implicating this species in WNV overwintering in Alabama. Molecular analysis of individuals initially identified as members of the Culex pipiens L. complex suggested that alleles characteristic of Cx. pipiens predominated in mosquitoes collected in Huntsville, AL, whereas alleles in the Auburn, AL, population were predominately characteristic of Culex quinquefaciatus Say. The southern boundary of the overlap zone of the two species seems to be located primarily between Huntsville and Auburn, a distance of 350 km.     

West Nile virus investigations in South Moravia, Czechland

Seven virus isolates were obtained from 11,334 mosquitoes after the 1997 Morava River flooding in South Moravia (Czech Republic): 6 strains of Tahyna bunyavirus, California antigenic group (5 from Aedes vexans, 1 from Ae. cinereus), and 1 strain of West Nile flavivirus (WNV) from Culex pipiens. In 1999, one isolate of Tahyna virus from Ae. vexans and one isolate of WNV from Cx. pipiens were recovered from a total of 14,354 mosquitoes examined in the same area, whereas no virus was detected there in 1,179 overwintering mosquitoes (mostly Cx. pipiens) in March 2000. The infection rate of mosquitoes with arboviruses was significantly higher in 1997, the year of the flood and an enormously high population density of mosquitoes. Antibodies neutralizing WNV were detected in 13 of 619 (2.1%) hospitalized patients or persons seeking outpatient clinics of the area in 1997. Five of the seroreactors revealed clinical symptoms compatible with West Nile fever: in 2 of them (children), recent infection with WNV was confirmed by a significant increase of antibody titer between acute and convalescent serum samples.     

Diversity and evolution of West Nile virus in Illinois and the United States, 2002-2005

Evolutionary analyses of West Nile virus (WNV) have been limited by uneven sampling across geographic regions and over time. In this study, an expanded data set of 68 WNV envelope gene sequences from the Midwest (Illinois) was created and combined with published sequences to investigate spatial and temporal structuring in the United States viral population. Results indicate an overall lack of geographic structure to WNV in the United States, supporting the notion of WNV as a rapidly expanding pathogen not significantly restricted in its spread by geographic distance. However, analyses of viral genetic diversity show a steady increase in WNV nucleotide-level diversity over time. Additionally, evolutionary rate calculations indicate that WNV has evolved at approximately 0.85 x 10(-3) substitutions/site/year, largely through neutral substitution and purifying selection. Overall, these results show WNV across the United States to be a panmictic viral population that is diversifying and evolving.     

Emergence and co-infections of West Nile virus and Toscana virus in Eastern Thrace,Turkey

The objective of this study was to identify the impact of West Nile virus (WNV) and Toscana virus (TOSV) in febrile diseases of unknown aetiology in Eastern Thrace, Turkey; this study was conducted during August–October 2012, and included 18 clinical cases and 296 blood donors for local serosurveillance. Antibodies were determined via commercial assays and further tested for specificity via neutralization assays (NA). Viral RNAs were sought via specific and/or generic primers. WNV infections were diagnosed in seven patients (38.8%), detected via RNA+IgM in four, RNA in one and IgM and low avidity IgG in two cases. The most common symptom was fever (>38°C), followed by headache, malaise/fatigue, myalgia/arthralgia, muscle stiffness/lower back pain, anorexia, nausea/vomiting, diarrhoea, supraorbital/retrobulbar pain and abdominal pain. Neurological symptoms were noted in one individual. WNV strains in RNA-detectable patients were characterized as lineage 1. TOSV RNA or IgM were identified in two individuals with confirmed WNV infections and in one patient without evidence of WNV exposure. The clinical and laboratory findings in individuals with WNV/TOSV co-infection were comparable to those in WNV-induced disease. The TOSV strain in the patient with detectable viral RNA was characterized as genotype A. In local blood donors, seroreactivity for specific WNV and TOSV immunoglobulins was observed in 1.7% (5/296) and 14.4% (26/180), respectively. These findings indicate the emergence of WNV and TOSV-associated diseases in Eastern Thrace. WNV/TOSV co-infections were documented for the first time.     

Overwintering of West Nile virus in Southern California

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003-2004 and 2004-2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November-March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence i n southern California. Continued transmission andvertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.     

Persistence of West Nile virus immunoglobulin M antibodies, Greece

A major outbreak of West Nile virus (WNV) lineage 2 infections was observed in 2010 in Greece. In order to check the persistence of WNV IgM antibodies, a second serum sample taken 75-180 days after onset of the illness from 29 patients with WNV infection was tested. A third sample was obtained 181-270 days after onset of the illness from 8 of the 12 patients with IgM-positive second sample. Mixed effects linear regression analysis indicated that the approximate time at which IgM index became negative was 164 (95% confidence interval, 95% CI 99-236) days after the symptoms' onset. Persistence of IgM antibodies was observed in 12% of patients at 181-270 days of follow-up. A sharp decrease in the IgM levels was observed, mainly in patients who had high IgM index value in the acute phase. All patients were WNV IgG positive at the follow-up.     

West Nile virus and blood product safety in Germany

West Nile Virus (WNV) is a mosquito-transmitted flavivirus, widely distributed throughout Africa, Asia and the Middle East. WNV may cause epidemics of human meningoencephalitis. The unexpected emergence of WNV (New York, 1999) and its rapid spread throughout North America during the following years caused a number of blood transfusion- and organ transplant-associated transmissions of WNV. In order to estimate the potential WNV threat for Central Europe, we analyzed the anti-WNV prevalence and WNV-RNA incidence among 14,437 and 9,976 blood donors from Germany. There was a high rate of initially anti-WNV reactives (5.9%), but only a few cases (0.03%) were confirmed as anti-WNV positive by neutralization assay. No WNV-RNA positive blood donor was identified in this study. Whereas WNV-RNA was frequently detected in manufacturing plasma pools from the US, none was detected in pools of European or Asian origin. Virus inactivation steps integrated into the manufacturing process of plasma derivatives were shown to be sufficient to assure the WNV safety of plasma derivatives. A well-characterized WNV reference material was prepared, showing 340 WNV-RNA copies per infectious dose.     

Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)

Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F1 progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18 degrees C for a minimal filial infection rate (MFIR) of approximately 1.4/1,000 and four of 1,873 adult progeny reared at 26 degrees C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10(5.6) plaque-forming units (PFU)/ml (=10(5.9) PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was approximately 1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.