WN Virus Research Articles

Novel tools to study West Nile virus NS3 protease activity

West Nile Virus (WNV) belongs to a group of pathogenic viruses called flaviviruses. West Nile virus infection can be mild, causing so-called West Nile Fever (WNF) or severe neuroinvasive form of the disease (WNND), and ultimately even death. There are currently no known medications to prevent West Nile virus infection. Only symptomatic treatment is used. To date, there are no unequivocal tests enabling a quick and unambiguous assessment of WN virus infection. The aim of the research was to obtain specific and selective tools for determining the activity of the West Nile virus serine proteinase. Using the methods of combinatorial chemistry with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. The FRET ABZ-Ala-Lys-Gln-Arg-Gly-Gly-Thr-Tyr(3-NO2)–NH2 substrate was obtained, characterized by kinetic parameters (KM = 4.20 ± 0.32 × 10-5 M) as for the majority of proteolytic enzymes. The obtained sequence was used to develop and synthesize highly sensitive functionalized quantum dot-based protease probes (QD). A QD WNV NS3 protease probe was obtained to detect an increase in fluorescence of 0.05 nmol enzyme in the assay system. This value was at least 20 times lower than that observed with the optimized substrate. The obtained result may be the basis for further research on the potential use of the WNV NS3 protease in the diagnosis of West Nile virus infection.

Ecology of breeding habitats of mosquito population and screening for virus of Japanese encephalitis and West Nile in the coastal area of Kerala, India.

Japanese encephalitis (JE) and West Nile virus (WNV) are two mosquito-borne diseases of medical and veterinary importance. Climate impacts in certain ecosystems are better understood; however, coastal brackish water ecosystems and their role in vector borne viral diseases have not been well studied. Hence, the aim of this study is to evaluate the distribution, spatial pattern as well as the screening of these mosquitoes for JE and WN virus in coastal area of Alappuzha district, Kerala, India. Immature mosquitoes were collected using dipper methods along the coastal wetlands areas. Adult mosquitoes were collected in four sampling sites with light trap filled with dry ice, operating overnight. The collected mosquitoes were identified by species, using the taxonomic keys. Mosquitoes were pooled for virus detection. Water samples were collected using standard methods. The total quantity of rainfall and salinity varied from zero to 365.2 mm and from 1.5 to 28 ppt respectively. A total of 10,695 adult mosquitoes and 38,083 immatures revealed the presence of five genus and 23 species. The most predominant species was Culex tritaeniorhynchus. Immature collections Cx. sitiens was the only one species collected during the summer season and in the monsoon season the pond was replaced by fresh water mosquitoes such as Cx. tritaeniorhynchus and Cx. gelidus. A total of 186 pools were screened for JEV and WNV. However, none of the pools were found as positive for the virus. The major vector of JEV Cx. tritaeniorhynchus and Cx. gelidus have adapted to immature development in coastal brackish water habitats. The public health authorities need to recognize thepotential impact on human health of brackish water-adapted mosquito vectors that weretraditionally considered to be freshwater species, and take appropriate surveillance and control measures.

West Nile Virus: Measures against Emergence in Malaysia

West Nile virus has a potential to emerge in new areas and cause large epidemics as was witnessed in the United States following its introduction in 1999. The virus is now a global public health threat, having been detected on every continent except Antarc tica. Once restricted to Africa, its expansion beyond its natural habitat is related to some viral, vectoral, anthropologic and environmental factors. The successful establishment and spread of the virus depend in part on viral adaptations, availability of competent hosts and mosquito vectors and suitable environmental conditions. A combination of measures can be applied to minimise the likelihood of a WN virus epidemic. Available vaccines are only for veterinary use, human vaccines are still in development . Vector control, animal vaccination, targeted surveillance and strong cooperation between relevant authorities are important in preventing a WN virus epidemic in Malaysia.

THE STUDYOF THE АREA OF DISTRIBUTION OF WEST NILE VIRUS IN THE TERRITORY OF THE EUROPEAN PART OF RUSSIA; THE RESULTS OF SEROEPIDEMIOLOGICAL RESEARCH. COMMUNICATION 2: CENTRAL, PRIVOLZHSKY AND NORTH-WESTERN FEDERAL DISTRICTS

Sera of 3951 inhabitants of Central, Privolzhsky and Northwest federal districts of Russia were examined with the use of ELISA-IgG and neutralization test. Antibodies to West Nile virus (WN) were detected in the sera of residents of Voronezh (1,6% of cases), Tula (1,5%), Tambov (0,7%) and Lipetsk(0,6%) regions. Two positive samples have been detected in residents of Moscow and Moscow region, which were possibly infected, during theirs travel to endemic for WN fever regions. All sera obtained from Kaluga, Ryazan, Tver, Vologda, Ulyanovsk regions and The Republic of Tatarstan were negative. Our data, in combination with the morbidity rate of WN fever indicate on epidemiological activity of foci of this infection in the South, the North Caucasus, Privolzhsky (Saratovskaya region) and Central (the forest-steppe zone and Tula region) districts. The northern border of circulation of WN virus probably dispose of the latitude of the Tula region.

THE STUDYOF THE АREA OF DISTRIBUTION OF WEST NILE VIRUS IN THE TERRITORY OF THE EUROPEAN PART OF RUSSIA; THE RESULTS OF SEROEPIDEMIOLOGICAL RESEARCH. COMMUNICATION 2: CENTRAL, PRIVOLZHSKY AND NORTH-WESTERN FEDERAL DISTRICTS

Sera of 3951 inhabitants of Central, Privolzhsky and Northwest federal districts of Russia were examined with the use of ELISA-IgG and neutralization test. Antibodies to West Nile virus (WN) were detected in the sera of residents of Voronezh (1,6% of cases), Tula (1,5%), Tambov (0,7%) and Lipetsk(0,6%) regions. Two positive samples have been detected in residents of Moscow and Moscow region, which were possibly infected, during theirs travel to endemic for WN fever regions. All sera obtained from Kaluga, Ryazan, Tver, Vologda, Ulyanovsk regions and The Republic of Tatarstan were negative. Our data, in combination with the morbidity rate of WN fever indicate on epidemiological activity of foci of this infection in the South, the North Caucasus, Privolzhsky (Saratovskaya region) and Central (the forest-steppe zone and Tula region) districts. The northern border of circulation of WN virus probably dispose of the latitude of the Tula region.

ELISA diagnostic test systems, serodiagnosis of arboviral infections

There are developed and tested by ourselves ELISA kits for serodiagnostics of most endemic to Russia arboviral infections and some imported tropical diseases. Their application allowed in 1999 to set the value WN virus in the etiology of outbreaks ofWN fever in the southern region of Russia, then in Voronezh and Tula regions. From 1999 to 2014, according to the data of Russian Service for Surveillance on Consumer Rights Protection and Human Wellbeing in the Russia there were diagnosed 2284 cases of WNfevers and 1725 cases of CCHF. In 2012-2014 specific IgG and neutralizing antibodies to the WN virus were detected in the population of the Southern and Black earth regions of Russia. Data of the examination of residents ’ sera from Tatarstan, Tver, Ryazan, Kaluga, Kursk and Vologda regions were proved to be negative. In 2000 there were revealedfirst cases of CCHF in the Volgograd region, Kalmykia and Dagestan also. The application of ELISA-IgM kits allowed to reveal cases of Sindbis, Batai, Tahyna, Inkoo, sandfly fevers, Uukuniemi and Bhanja. During the period of2009-2014 there were verified 190 imported cases of dengue fever, Chikungunya, WNfever, sandfly fevers, and Japanese encephalitis among persons returning from travel in tropical countries.

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009–10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.

Vector Competence of Culex neavei and Culex quinquefasciatus (Diptera: Culicidae) from Senegal for Lineages 1, 2, Koutango and a Putative New Lineage of West Nile Virus

West Nile virus (WN virus) is one of the most widespread arbovirus and exhibits a great genetic diversity with 8 lineages, at least 4 (1, 2, Koutango, and putative new) are present in Africa. In West Africa, Culex neavei and Culex quinquefasciatus are considered as potential vectors for WN virus transmission in sylvatic or urban context. We analyzed the vector competence of these Culex species from Senegal for African lineages and envelope proteins sequences of viral strains used. We showed that lineage 1 is transmitted by both Culex mosquitoes, whereas the putative new lineage 8 is transmitted only by Cx. neavei. Our findings suggest that genetic variability can affect vector competence and depend on mosquito. However, when considering the infective life rate, the mosquito population seems to be inefficient for WN virus transmission in the field and could explain the low impact of WN virus in Africa.

Mapping the Serological Prevalence Rate of West Nile fever in Equids, Tunisia

West Nile fever (WNF) is a viral disease of wild birds transmitted by mosquitoes. Humans and equids can also be affected and suffer from meningoencephalitis. In Tunisia, two outbreaks of WNF occurred in humans in 1997 and 2003; sporadic cases were reported on several other years. Small-scale serological surveys revealed the presence of antibodies against WN virus (WNV) in equid sera. However, clinical cases were never reported in equids, although their population is abundant in Tunisia. This study was achieved to characterize the nationwide serological status of WNV in Tunisian equids. In total, 1189 sera were collected in 2009 during a cross-sectional survey. Sera were tested for IgG antibodies, using ELISA and microneutralization tests. The estimated overall seroprevalence rate was 28%, 95% confidence interval [22; 34]. The highest rates were observed (i) in the north-eastern governorates (Jendouba, 74%), (ii) on the eastern coast (Monastir, 64%) and (iii) in the lowlands of Chott El Jerid and Chott el Gharsa (Kebili, 58%; Tozeur, 52%). Environmental risk factors were assessed, including various indicators of wetlands, wild avifauna, night temperature and chlorophyllous activity (normalized difference vegetation index: NDVI). Multimodel inference showed that lower distance to ornithological sites and wetlands, lower night-time temperature, and higher NDVI in late spring and late fall were associated with higher serological prevalence rate. The model-predicted nationwide map of WNF seroprevalence rate in Tunisian equids highlighted different areas with high seroprevalence probability. These findings are discussed in the perspective of implementing a better WNF surveillance system in Tunisia. This system might rely on (i) a longitudinal survey of sentinel birds in high-risk areas and time periods for WNV transmission, (ii) investigations of bird die-offs and (iii) syndromic surveillance of equine meningoencephalitis.

Assay of Several Inactivation Steps on West Nile Virus and H7N1 Highly Pathogenic Avian Influenza Virus Suspensions

Objectives: In any research laboratory, precautionary measures must be taken in order to reduce or eliminate the potential risk of accidental infection by biosafety level 3 (BSL3) pathogens, as Highly Pathogenic Avian Influenza Virus (HPAI) or West Nile Virus (WNV). Appropriate virus inactivation procedures have to be set up to allow necessary further processing of specimens outside BSL3 facilities. Methods: To study the elimination of WN and HPAI virus infectivity, the effect of different chemical and physical inactivation procedures on viral suspensions were investigated. A proper cell culture assay for each virus was performed to verify several treatments, which are commonly performed preceding transfer of materials outside of biocontainment, still allowing further investigations like sequencing or genome amplification. Results: Chemical inactivation with AVL buffer (Qiagen), Trizol® Reagent or Phenol chloroform: isoamylic alcohol treatment, as well as physical treatment (heat at two temperatures and three contact times) reduced viral infectivity in the viral suspension below the detection limit. Conclusion: Thermal treatments, but also Trizol® Reagent and AVL buffer (Qiagen), are suitable to produce noninfectious specimens for further use in molecular biology techniques

Temporal distribution and spatial pattern of abundance of the Rift Valley fever and West Nile fever vectors in Barkedji, Senegal

The temporal distribution and spatial pattern of abundance of mosquito vectors of Rift Valley fever (RVf) and West Nile fever (WNf) were studied during the 2005 and 2006 rainy seasons at Barkedji, Senegal. Mosquitoes were collected every two weeks with CDC light traps with dry ice at 79 sites including temporary ponds, barren, shrubby savannah, wooded savannah, steppes, and villages at different distances (between 0 and 600 m) from the nearest pond. The temporal distributions of these vectors varied between 2005 and 2006 and were positively correlated with rainfall for Aedes (Aedimorphus) vexans Patton, with rainfall after a lag time of one month for Culex (Culex) poicilipes (Theobald) and Culex (Culex) neavei Theobald. All the vectors had their highest abundances and parity rates between September and November. The highest vector abundances were observed in the barren and temporary ponds. The distance of trap location to the nearest ponds was negatively correlated to the abundance of the vectors. Taking into account the linear regression equations, it was predicted that mosquitoes would not disperse and be collected by the light trap, up to 1,500 m to the nearest ponds. The implications of these findings in the epidemiology and control of RVF and WNF at Barkedji are discussed.

Induction of virus-specific neutralizing immune response against West Nile and Japanese encephalitis viruses by chimeric peptides representing T-helper and B-cell epitopes

West Nile virus (WNV) and Japanese encephalitis virus (JEV), the members of JEV serocomplex group are pathogens of global health concern. The co-circulation of these viruses poses challenges in effective diagnostics due to antigenic similarity between the E-protein of these viruses. The present study aimed to design chimeric peptides and study the immune response against the same. B-cell epitopes were predicted on structural proteins of WNV and JEV based on bioinformatics tools. The peptides representing to these B-cell epitopes were synthesized and subjected to ELISA. Two peptides, one each from WNV (named WE147) and JEV (named JE40) E-protein, showed virus-specific and strong reactivity to the immune mice sera and human clinical samples. The chimeric peptides for WNV and JEV were constructed by synthesizing the B-cell epitope of WNV (WE147) or JEV (JE40) with T-helper epitope (JM17) separated by diglycine spacer in between. The immune response generated against these chimeric peptides was found to be specific to the respective B-cell epitopes. The anti-peptide sera showed virus-specific reactivity in ELISA and in immunofluorescence assay with no cross-reactivity. Also, the anti-peptide sera could neutralize JE and WN viruses in an in vitro virus neutralization assay. The B-cell epitopes identified in the present study may be used as diagnostic markers for differentiating between WN and JE virus infections. The present study can form a basis for future design of vaccines.

Phylodynamics, vectorial competence and genetic diversity of West Nile virus in Africa: implications for global emergence of West Nile

West Nile virus (WNV) is a flavivirus (Flaviviridae family) and its transmission cycle involves Culex spp. mosquitoes and birds as reservoirs host whereas humans and horses are dead-end hosts. Clinical symptoms of WN human infections range from asymptomatic or mild influenza like disease to severe neurological and meningoencephalitis syndromes. West Nile is a neglected emerging disease with major breakthrough in 1999 with the introduction of WN virus (WNV) in New York City and the subsequent spread to whole northern America over the last decade causing massive human and animals infection leading to some fatal cases. In Eastern Europe, circulation of WNV with recurrent emergences impacting human and animal health since 1996 is similar to the situation in the USA. Strikingly, in Africa WN appears to have a minor effect despite regular isolations from mosquitoes and vertebrates hosts. In addition, WNV exhibited a great diversity with eight different lineages among which only one (lineage 1) is found worldwide and 4 are present in Africa. In order to understand factors underlying the different patterns of transmission and processes involved in the emergence of WN in the different contexts, genetic diversity, phylodynamics and vectorial competence of WNV have been studied in Africa. Phylogenetic analysis based on partial and complete genome suggests an interconnection of zoonotic amplifications in Africa with emergence in Europe as well as replacement between lineages over time. Vectorial competence of lineages circulating in Africa for a domestic mosquitoes Culex quinquefasciatus showed significant differences between strains of various lineages tested for infections, dissemination and transmission rates. Indeed the different strains can be classified as low, intermediary and high infection profile. Analysis of the transmission patterns with sequences of the strains suggest that glysoylation of the envelope protein of WNV, a key player in the virus entry in the cell, may play an important role. The implications of our findings are discussed in the context of global emergence of WN.

Identification of Critical Molecular Determinants of West Nile Virus PrM Protein: A Potential Site for Antiviral Targeting

get. This involves both oligomerization of the capsid (C) protein and packaging of the viral RNA. The RNA binding properties of the C protein is poorly understood. This study aims to characterize the C and RNA interaction. Method: Co-localization study was performed by transfecting in vitro transcribed WN virus RNA and C protein clones into BHK cells and visualized under fluorescence microscopy. RNA binding properties of C protein were further investigated with a Northwestern Blot assay and RNA pull-down assay. Synthesized C protein peptides were used to map out the RNA binding regions on C protein. In addition, C protein immunopurified from BHK cells were used to investigate the effect of phosphorylation of C protein on its RNA binding properties. Results: RNA and C protein have failed to show colocalization in BHK cells by immuno-fluorescence but interactions were observed at the molecular level. It showed that the first 465 and last 693 nucleotides of the WN virus RNA had specific affinity for the full length C protein. In addition, the aminoand carboxy-terminal of the C protein were shown to bind to the virus RNA. It was also found that the C protein had affinity for viral anti-sense RNA. Phosphorylated peptides of C protein and C protein expressed in BHK cells show attenuated binding to viral RNA. Conclusion: C protein interaction with anti-sense indicates that its interaction with viral sense RNA may not be specific. Phosphorylation of C protein could play a role in regulating C and RNA interaction and allow time for viral assembly. Understanding the interaction of C protein with viral RNA and role of phosphorylation in nucleocapsid assembly could help develop anti-viral strategies aimed at disrupting viral assembly.

Epidemiology of West Nile infection in Volgograd, Russia, in relation to climate change and mosquito (Diptera: Culicidae) bionomics

In 1999, there was the large outbreak of West Nile fever (WNF) in Southern Russia (>500 cases in the Volgograd Province). In 2000-2004, the WNF incidence rate decreased steadily to zero, but a new outbreak occurred in 2007 (64 cases). The analysis of historical climate data for Volgograd from 1900 to present showed that the years 1999 and 2007 were the hottest ones due to a very mild "winter" (Dec.-Mar.) and a hot "summer" (June-Sep.). There are up to 15 potential WNF vectors in Volgograd, but only Culex pipiens and Culex modestus are abundant in late summer, both in urban and rural settings. Only these species are naturally attracted to and feed on both humans and birds. The RNA of pathogenic WN virus genovariant was found by reverse transcriptase polymerase chain reaction only in Culex mosquitoes at the infection rate of about 0.04%. So these species may be considered as potential WNF "bridge vectors" between birds and humans as well as main vectors in sylvatic avain cycle. Their abundance in an epidemic season was higher in the years with a mild winter and a hot summer, so this phenomenon may serve as a connecting link between a climate and WNF epidemiology. These findings give some hints on the predisposing factors for WNF epidemic as well as the possibility to predict WNF outbreaks in the temperate climate zones.

Size matters: West Nile Virus neutralizing antibodies in resident and migratory birds in Spain

The rapid range expansion of West Nile Virus has raised interest in understanding the population dynamics and dispersal patterns of emerging infectious diseases by wildlife. We analyzed different ecological and evolutionary factors related to West Nile Virus neutralizing antibody prevalence in 72 bird species sampled in southern Spain. Prevalence of antibodies reached its maximum during the autumn and winter in comparison to summer months. Prevalence of antibodies was directly related to body mass and migratory behaviour. The greater prevalence of antibodies observed in summer migrants can be explained, among other factors, by the diversity of localities involved in their life cycles or the geographic areas visited during their migrations. Greater prevalence in larger species was explained by their longevity because the relationship was already significant when analyzing only first year birds, and probably also involved a high attraction to vectors by larger hosts. Coloniality and winter gregarism were unrelated to the prevalence of antibodies against this highly host generalist pathogen. Evolutionary relationships between species were unrelated to differences in the prevalence of antibodies. Our results suggest larger species as good candidates for easy, faster and cheaper monitoring of local, seasonal and annual changes in WN virus serology.

Seroprevalence study on the diffusion of the West Nile Virus among blood donors, healthcare workers, jockeys, grooms and fowlers, veterinary surgeons and hunters in Messina (Italy)

West Nile virus (WNV) is a mosquito-transmitted flavivirus widely distributed in Africa, Middle East, Asia, Southern Europe and in 1999 was first identified in the United States as a cause of disease in New York City. It mainly circulates among birds, but can infect many species of mammals. Epidemics can occur in rural as well as urban areas. 1,280 sera were collected during 2006 from 80 stable workers, as jockey and grooms, 100 fowlers, 500 blood donors, 600 healthcare workers, 100 veterinary surgeons and 100 hunters in the Messina province to evaluate the prevalence of the WNV infection, by ELISA test, in relation to risk exposure or not. None of the 1280 subjects examined has shown positive for antibodies anti WN virus. Among the strategies of control and surveillance, finally, in our opinion, are and will be indispensable the programs of continuous antibody survey in all the risk categories and in the general population in order to succeed to preview which effects could have the presence of infections from WNV, also imported from other zones where the virus is constantly present, in future and which it could be the impact of geographic factors on the epidemic spread of the disease.

Validation of a Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.

The DE loop of the domain III of the envelope protein appears to be associated with West Nile virus neutralization

The envelope (E) protein of WNV plays an important role in the virus neutralization. Using a mAb 5E8, a neutralizing epitope on the domain III of the E of the New York strain of WN virus was characterized. Results from neutralization-escape mutants and site-directed mutagenesis revealed that the 5E8 epitope is a highly conformation dependent epitope consisting of at least residues E330, E332 and E367 on the domain III. Besides known critical neutralizing epitopes E330 and E332, our results indicate that residue E367, a component of DE loop on the domain III, appeared to be associated with neutralization but little with neuroinvasion of the virus, as reported previously (Beasley et al., 2002).

γδ T Cells Facilitate Adaptive Immunity against West Nile Virus Infection in Mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.