West Nile Virus Genes Research Articles

Genotypic and phenotypic characterization of West Nile virus NS5 methyltransferase mutants

Although West Nile virus (WNV) causes annual cases of neurological disease and deaths in humans, a vaccine has not been licensed for human use. Several WNV genes have been targeted for mutagenesis in attempts to generate live attenuated vaccine candidates, including the non-structural protein NS5. Specifically, mutation of WNV NS5-K61A or NS5-E218A in the catalytic tetrad of the methyltransferase decreases enzyme activity of the NS5 protein and correspondingly attenuates the virus in mice. In this report, NS5-K61A, NS5-E218A, and a double mutant encoding both mutations (NS5-K61A/E218A) were compared both in vitro and in vivo. Each single mutant was strongly attenuated in highly susceptible outbred mice, whereas the double mutant unexpectedly was not attenuated. Sequencing analysis demonstrated that the double mutant was capable of reversion at both residues NS5-61 and NS5-218, whereas the genotype of the single mutants did not show evidence of reversion. Overall, either NS5-K61A or NS5-E218A methyltransferase mutations could be potential mutations to include in a candidate live WNV vaccine; however, multiple mutations in the catalytic tetrad of the methyltransferase are not tolerated.

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression.

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome.

Genetic Variability of West Nile Virus in U.S. Blood Donors from the 2012 Epidemic Season.

West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4–5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999–2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Overall, these analyses showed that following a brief contraction in 2008–2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S. history.

Functional Analysis of West Nile Virus Proteins in Human Cells.

West Nile Virus (WNV) lineage 2 strains have been responsible for large outbreaks of neuroinvasive disease in the United States and Europe between 1999 and 2012. Different strains in this lineage have previously been shown to produce either severe or mild neuroinvasive disease in mice. Phylogenetic and amino acid comparisons between highly or less virulent lineage 2 strains have demonstrated that the nonstructural (NS) gene(s) were most variable. However, the roles of some of the NS proteins in virus life cycle are unknown. The aim of this chapter is to describe simple computational and experimental approaches that can be used to: (1) explore the possible roles of the NS proteins in virus life cycle and (2) test whether the subtle amino acid changes in WNV NS gene products contributed to the evolution of more virulent strains. The computational approaches include methods based on: (1) sequence similarity, (2) sequence motifs, and (3) protein membrane topology predictions. Highlighted experimental procedures include: (1) isolation of viral RNA from WNV-infected cells, (2) cDNA synthesis and PCR amplification of WNV genes, (3) cloning into GFP expression vector, (4) bacterial transformation, (5) plasmid isolation and purification, (6) transfection using activated dendrimers (Polyfect), and (7) immunofluorescence staining of transfected mammalian cells.

Substitution of the premembrane and envelope protein genes of Modoc virus with the homologous sequences of West Nile virus generates a chimeric virus that replicates in vertebrate but not mosquito cells.

BackgroundMost known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified.MethodsFusion polymerase chain reaction (PCR) was used to replace the capsid (C), premembrane (prM) and envelope (E) genes and the prM-E genes of a full-length MODV infectious cDNA clone with the corresponding regions of WNV and CxFV. Fusion products were directly transfected into baby hamster kidney-derived cells that stably express T7 RNA polymerase. At 4 days post-transfection, aliquots of each supernatant were inoculated onto vertebrate (BHK-21 and Vero) and mosquito (C6/36) cells which were then assayed for evidence of viral infection by reverse transcription-PCR, Western blot and plaque assay.ResultsChimeric virus was recovered in cells transfected with the fusion product containing the prM-E genes of WNV. The virus could infect vertebrate but not mosquito cells. The in vitro replication kinetics and yields of the chimeric virus were similar to MODV but the chimeric virus produced larger plaques. Chimeric virus was not recovered in cells transfected with any of the other fusion products.ConclusionsOur data indicate that genetic elements outside of the prM-E gene region of MODV condition its vertebrate-specific phenotype.

Evolutionary Dynamics of West Nile Virus in the United States, 1999–2011: Phylogeny, Selection Pressure and Evolutionary Time-Scale Analysis

West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10−4 substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.

A detailed comparative analysis on the overall codon usage patterns in West Nile virus

West Nile virus (WNV) is a member of the family Flaviviridae and its genome consists of an 11-kb single-stranded, positive-sense RNA. WNV is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans, which serve as incidental dead-end hosts. Understanding the extent and causes of biases in codon usage is essential to the comprehension of viral evolution. In this study, we performed a comprehensive analysis of 449 WNV strains, for which complete genome sequences are available. Effective number of codons (ENC) indicates that the overall codon usage among WNV strains is only slightly biased. Codon adaptation index (CAI) values found for WNV genes are different from the CAI values found for human genes. The relative synonymous codon usage among WNV strains isolated from birds, equines, humans and mosquitoes are roughly similar and are influenced by the relative dinucleotide frequencies. Taking together, the results of this work suggest that WNV genomic biases are the result of the evolution of genome composition, the need to escape the antiviral cell responses and a dynamic process of mutation and selection to re-adapt its codon usage to different environments.

Development and evaluation of a novel TaqMan fluorescence probe-based real-time reverse transcriptase polymerase chain reaction assay for detection and quantification of West Nile virus

In order to improve and accelerate the detection of West Nile virus (WNV), a rapid and specific real-time reverse transcription polymerase chain reaction (rtRT-PCR) was established. Primers and probe were designed according to the conservative sequence of capsid protein gene of WNV. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of rtRT-PCR. The amplifying curve showed that this method could successfully amplify 101 copies/μl WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. The assay system showed high reproducibility with coefficient of variation (CV) < 2%. The detection of WNV can be completed within 2 to 3 h. By detecting cDNA samples (n = 55) with rtRT-PCR and the conventional PCR assay, the established rtRT-PCR showed 96.36% (37 + 16/55) coincidence rate with the conventional PCR. All the results showed that the newly established rtRT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV. Key words: West Nile virus, capsid protein gene, real-time RT-PCR.

A Novel Approach for the Rapid Mutagenesis and Directed Evolution of the Structural Genes of West Nile Virus

Molecular clone technology has proven to be a powerful tool for investigating the life cycle of flaviviruses, their interactions with the host, and vaccine development. Despite the demonstrated utility of existing molecular clone strategies, the feasibility of employing these existing approaches in large-scale mutagenesis studies is limited by the technical challenges of manipulating relatively large molecular clone plasmids that can be quite unstable when propagated in bacteria. We have developed a novel strategy that provides an extremely rapid approach for the introduction of mutations into the structural genes of West Nile virus (WNV). The backbone of this technology is a truncated form of the genome into which DNA fragments harboring the structural genes are ligated and transfected directly into mammalian cells, bypassing entirely the requirement for cloning in bacteria. The transfection of cells with this system results in the rapid release of WNV that achieves a high titer (∼10(7) infectious units/ml in 48 h). The suitability of this approach for large-scale mutagenesis efforts was established in two ways. First, we constructed and characterized a library of variants encoding single defined amino acid substitutions at the 92 residues of the "pr" portion of the precursor-to-membrane (prM) protein. Analysis of a subset of these variants identified a mutation that conferred resistance to neutralization by an envelope protein-specific antibody. Second, we employed this approach to accelerate the identification of mutations that allow escape from neutralizing antibodies. Populations of WNV encoding random changes in the E protein were produced in the presence of a potent monoclonal antibody, E16. Viruses resistant to neutralization were identified in a single passage. Together, we have developed a simple and rapid approach to produce infectious WNV that accelerates the process of manipulating the genome to study the structure and function of the structural genes of this important human pathogen.

Analyses of mutations selected by passaging a chimeric flavivirus identify mutations that alter infectivity and reveal an interaction between the structural proteins and the nonstructural glycoprotein NS1

We previously described a single-cycle dengue vaccine (RepliVAX D2) engineered from a capsid (C) gene-deleted West Nile virus (WNV) expressing dengue virus serotype 2 (DENV2) prM/E genes in place of the corresponding WNV genes. That work demonstrated that adaptation of RepliVAX D2 to grow in WNV C-expressing cells resulted in acquisition of non-synonymous mutations in the DENV2 prM/E and WNV NS2A/NS3 genes. Here we demonstrate that the prM/E mutations increase the specific infectivity of chimeric virions and the NS2A/NS3 mutations independently enhance packaging. Studies with the NS2A mutant demonstrated that it was unable to produce a larger form of NS1 (NS1'), suggesting that the mutation had been selected to eliminate a ribosomal frame-shift “slippage site” in NS2A. Evaluation of a synonymous mutation at this slippage site confirmed that genomes that failed to make NS1' were packaged more efficiently than WT genomes supporting a role for NS1/NS1' in orchestrating virion assembly.

Establishment and evaluation of real-time PCR for West Nile virus detection

AbstractA rapid real-time polymerase chain reaction (RT-PCR) for detecting West Nile virus (WNV) was established. Primers were designed according to the sequence of the capsid protein gene of WNV by Primer Premier 5.0. In this way, an inexpensive assay using the intercalating dye SYBR Green I was developed and validated. The amplifying curve showed that this method could successfully amplify 102 copies/μl of the WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay system showed high reproducibility with coefficient of variation (CV) &lt;2%. Thus the newly established RT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.

An Outbreak of West Nile Virus Infection in Captive Lesser Scaup (Aythya affinis) Ducklings

This report describes West Nile virus (WNV)-associated mortality in captive lesser scaup (Aythya affinis) ducklings that occurred in Saskatchewan, Canada, in July and August 2007. There were no clinical signs or gross necropsy findings suggestive of the cause of death; however, microscopic lesions were consistent with WNV infection, including nonsuppurative encephalitis and myocardial, pancreatic, and splenic necrosis. Necrosis of the thymus and thyroid was also observed in some birds, which has not previously been reported in association with WNV infection. Immunohistochemistry revealed WNV antigen in multiple tissues, including thymus and thyroid, and reverse transcription polymerase chain reaction resulted in the identification of WNV gene sequence in all of the ducklings that were tested. This outbreak is of interest because waterfowl (Anseriformes) are not thought to be particularly susceptible to WNV, and there is little information about WNV infection in prefledging birds. The apparent susceptibility of lesser scaup to WNV demonstrated in this study may have implications for declining lesser scaup populations in the wild.

Microarray-based assay for the detection of genetic variations of structural genes of West Nile virus

Adaptation through fixation of spontaneous mutations in the viral genome is considered to be one of the important factors that enable recurrent West Nile virus (WNV) outbreaks in the U.S. Genetic variations can alter viral phenotype and virulence, and degrade the performance of diagnostic and screening assays, vaccines, and potential therapeutic agents. A microarray assay was developed and optimized for the simultaneous detection of any nucleotide mutations in the entire structural region of WNV in order to facilitate public health surveillance of genetic variation of WNV. The DNA microarray consists of 263 oligonucleotide probes overlapping at half of their lengths which have been immobilized on an amine-binding glass slide. The assay was validated using 23 WNV isolates from the 2002–2005 U.S. epidemics. Oligonucleotide-based WNV arrays detected unambiguously all mutations in the structural region of each one of the isolates identified previously by sequencing analysis, serving as a rapid and effective approach for the identification of mutations in the WNV genome.

Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep

We describe the development and preliminary characterization of a recombinant canarypox virus vectored vaccine for protective immunization of ruminants against bluetongue virus (BTV) infection. Sheep ( n = 6) immunized with recombinant canarypox virus vector (BTV-CP) co-expressing synthetic genes encoding the two outer capsid proteins (VP2 and VP5) of BTV serotype 17 (BTV-17) developed high titers (40–160) of virus-specific neutralizing antibodies and were resistant to challenge with a field strain of BTV-17. In contrast, sheep ( n = 5) immunized with a commercial recombinant canarypox virus vector expressing the E and preM genes of West Nile virus were seronegative to BTV and developed pyrexia, lymphopenia, and extended, high-titered viremias following challenge exposure to the field strain of BTV-17. These data confirm that the BTV-CP vaccine may be useful for the protective immunization of ruminants against bluetongue, and it may avoid the problems inherent to live-attenuated (LA) BTV vaccines.

Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge

The safety and efficacy of a canarypox vector expressing PrM and E genes of West Nile virus (WNV) (ALVAC-WNV) was evaluated in dogs and cats. One group of 17 dogs (vaccinated with 10 5.6 TCID 50) and two groups of cats (groups 1 [ n = 14] vaccinated with 10 7.5 TCID 50 and 2 [ n = 8] 10 5.6 TCID 50) were vaccinated twice at 28-day intervals. Fifteen dogs and eleven cats served as negative controls. The cats and dogs were challenged 120 and 135 days after the second immunization, respectively via the bites of Aedes albopictus mosquitoes infected with WNV. The first dose of vaccine induced a detectable antibody response in four dogs and five cats (one immunized with low and four with high doses). After the second dose, all the vaccinated dogs and all of the cats, immunized with high dose had detectable antibody titers, whereas only four of eight cats in the low dose group were seropositive. None of the vaccinated dogs and one vaccinated cat developed viremia following the WNV mosquito-challenge. In contrast, 14 of the 15 control dogs and 9 of the 11 control cats developed viremia. The experimental vaccine described in this study may be of value in the prevention of WNV infection in dogs and cats.

Expression and evolutionary analysis of West Nile virus (Merion Strain)

The authors report a new strain of West Nile virus (WNV) with the expression analysis of its individual open reading frames. Since its sudden appearance in the summer of 1999 in New York City, the virus has spread rapidly across the continental United States into Canada and Mexico. Besides, its rapid transmission by various vectors, the spread of this virus through organ transplantation, blood transfusion, and mother-child transmission through breast milk is of concern. In order to understand molecular variations of WNV in North America and to generate new tools for understanding WNV biology, a complete clone of WNV has been constructed. Investigations so far have focused only on half of its genes products and a detailed molecular and cell biological aspects on all of WNV gene have yet to be clearly established. The open reading frames of WNV were recovered through an reverse transcriptase-polymerase chain reaction (RT-PCR)-PCR using brain tissue from a dead crow collected in Merion, PA, and cloned into a mammalian expression vector. The deduced amino acid sequences of individual open reading frames were analyzed to determine various structural motifs and functional domains. Expression analysis shows that in neuronal cells, C, NS1, and NS5 proteins are nuclear localized whereas the rest of the antigens are confined to the cytoplasm when they are expressed in the absence of other viral antigens. This is the first report that provides an expression analysis as well as intracellular distribution pattern for all of WNV gene products, cloned from an infected bird. Evolutionary analysis of Merion strain sequences indicates that this strain is distinct phylogenetically from the previously reported WNV strains.

Emergence and Control of Zoonotic Viral Encephalitides

The viral encephalitides of Eastern, Western, and Venezuelan equine encephalitis viruses have been of public health concern for years. Over the last decade, several outbreaks caused by emerging zoonotic viral encephalitides, such as West Nile virus in North America and Nipah virus in Malaysia and Singapore in 1999, resulted in serious illnesses and deaths in persons, domesticated food animals, and wildlife. The Institute of Medicine has cited a number of factors that have led to these and other emerging disease outbreaks: 1) a growing human population that is moving into habitats of wildlife and domesticated livestock and poultry; 2) global climate changes that have caused changes in arthropod vector and rodent reservoir populations; 3) rapid travel and movement of people and animals worldwide; and 4) changing human behaviors (1). and Control of Zoonotic Viral Encephalitides is a timely book that gives an overview of agent, host, environmental, and other factors that have led to the emergence and transmission of several zoonotic viral encephalitides, including flaviviruses, alphaviruses, and rabies virus. The book also details important avenues for their control. This book is a special issue of the Archives of Virology, and its 244 pages comprise 21 presentations that were made at a symposium on Emergence and Control of Zoonotic Viral Encephalitides. The symposium was held April 6–8, 2003, in Les Pensieres, Veyrier du Lac, France. The first presentation gives an overview of the emergence of zoonotic viruses maintained by wildlife reservoir hosts and describes a conceptual model of processes that would account for the transmission of viruses among species. The second presentation describes the role of disease surveillance in polio eradication and the identification of emerging viral encephalitides. The third presentation gives an overview of the mechanisms of genetic changes and neurovirulence of encephalitogenic arboviruses. The following 13 presentations include overviews of molecular determinants of virulence of West Nile virus in North America, genetic determinants of Venezuelan equine encephalitis virus, evolution and dispersal of encephalitic flaviviruses, and West Nile and other zoonotic viruses in Russia. Presentations that follow address lyssaviruses and henipaviruses transmitted by frugivorous bats, host-management strategies of novel viral encephalitides associated with bats, regulation of transcription and the nature of the cell receptor with regard to henipaviruses, and entry machinery of flaviviruses. Also included are presentations on persistent infection and suppression of host response by alphaviruses, subversive neuroinvasive strategy of rabies virus, neurovirulence and host factors in flavivirus encephalitis, regulation of apoptosis by viruses infecting insects, and Semliki Forest virus infection of laboratory mice as models to study the pathogenesis of viral encephalitis. The book finishes with presentations on a novel principle of attenuation for developing new generation live flavivirus vaccines, on tick-borne encephalitis, and on a recombinant vaccine developed from a canarypox virus carrying the prM/E genes of West Nile virus that will protect horses against a West Nile virus–mosquito challenge, and on diagnosis of zoonotic viral encephalitis. The book will be worthwhile to virologists and other infectious disease researchers and practitioners interested in the biology, virulence, and genetic evolution of viral encephalitides, and the factors involved in their emergence.

West Nile virus surveillance: A simple method for verifying the integrity of RNA in mosquito (Diptera: Culicidae) pools.

In a West Nile virus (WNV) -free ecosystem, it is essential to verify the integrity of RNA before concluding that RNA extracted from mosquito specimens is negative for WNV gene sequences. The primary objective of our study was to develop a rapid molecular assay to rapidly screen mosquitoes for the presence of 18S RNA and WNV gene sequences. Mosquitoes, collected from multiple sites on the island of O'ahu, were pooled into groups of 1-50 mosquitoes according to capture site, date, and species. Using primer design software and the GenBank database, generic oligonucleotide primer pairs were designed to amplify mosquito18S rRNA gene sequences from different species. RNA was extracted from mosquito pools, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the presence of mosquito18S rRNA and WNV gene sequences. Three of the seven primer pairs successfully detected 18S rRNA sequences for both Aedes and Culex by RT-PCR, and one primer pair successfully amplified 18S rRNA sequences for 15 different mosquito species. All 64 mosquito pools from 10 different sites on the island of Oahu, Hawaii, were negative for WNV nonstructural protein-5 gene sequences. This simple, one-step RT-PCR method for screening mosquito pools for arboviruses will become an increasingly valuable tool as WNV becomes endemic throughout the Americas.

Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge.

An ALVAC (canarypoxvirus)-based recombinant (vCP2017) expressing the prM and E genes derived from a 1999 New York isolate of West Nile virus (WNV) was constructed and assessed for its protective efficacy in horses in two different experiments. In the first trial, a dose titration study was conducted to evaluate both serum neutralising antibody responses to WNV and duration of immunity. In the second trial the onset of protection was determined. Twenty-eight adult horses received two doses of vCP2017 administered intramuscularly at 5-week intervals and sixteen horses comprised age-matched non-vaccinated controls. Individual sera were taken periodically and tested for neutralising antibodies against WNV. Horses were challenged by allowing WNV-infected Aedes albopictus mosquitoes to feed on them two weeks (second trial) or one year (first trial) after the second vaccination. After challenge, horses were monitored for clinical signs of disease, and blood samples were collected for detection of WNV viremia and antibody. In both trials, all vaccinated horses developed neutralising antibodies against WNV. None of the vaccinated or control horses developed clinical signs of WNV disease upon challenge. None of the nine horses challenged 2 weeks after primary vaccination and only one of the ten vaccinated horses challenged 1 year after vaccination developed detectable viremia after challenge, whereas more than 80% of the controls became infected. Results from these studies demonstrated that a primary course of two doses of vCP2017 provides both antibody response and an early immunity in horses against WNV viremia.

Prospects for development of a vaccine against the West Nile virus.

Vaccination provides the ultimate measure for personal protection against West Nile disease. The development of a West Nile vaccine for humans is justified by the uncertainty surrounding the size and frequency of future epidemics. At least two companies (Acambis Inc. and Baxter/immuno) have initiated research and development on human vaccines. West Nile encephalitis has also emerged as a significant problem for the equine industry. One major veterinary vaccine manufacturer (Ft. Dodge) is developing formalin-inactivated and naked DNA vaccines. The advantages and disadvantages of formalin-inactivated whole virion vaccines, Japanese encephalitis vaccine for cross-protection, naked DNA, and live attenuated vaccines are described. A novel technology platform for live, attenuated recombinant vaccines (ChimeriVax) represents a promising approach for rapid development of a West Nile vaccine. This technology uses yellow fever 17D as a live vector for envelope genes of the West Nile virus. Infectious clone technology is used to replace the genes encoding the prM and E structural proteins of yellow fever 17D vaccine virus with the corresponding genes of West Nile virus. The resulting virion has the protein coat of West Nile, containing all antigenic determinants for neutralization and one or more epitopes for cytotoxic T lymphocytes. The genes encoding the nucleocapsid protein, nonstructural proteins, and untranslated terminal regions responsible for replication remain those of the original yellow fever 17D virus. The chimeric virus replicates in the host like yellow fever 17D but immunizes specifically against West Nile virus.