West Nile Virus Proteins Research Articles

Monoclonal antibodies to the West Nile virus NS5 protein map to linear and conformational epitopes in the methyltransferase and polymerase domains

The West Nile virus (WNV) NS5 protein contains a methyltransferase (MTase) domain involved in RNA capping and an RNA-dependent RNA polymerase (RdRp) domain essential for virus replication. Crystal structures of individual WNV MTase and RdRp domains have been solved; however, the structure of full-length NS5 has not been determined. To gain more insight into the structure of NS5 and interactions between the MTase and RdRp domains, we generated a panel of seven monoclonal antibodies (mAbs) to the NS5 protein of WNV (Kunjin strain) and mapped their binding sites using a series of truncated NS5 proteins and synthetic peptides. Binding sites of four mAbs (5D4, 4B6, 5C11 and 6A10) were mapped to residues 354-389 in the fingers subdomain of the RdRp. This is consistent with the ability of these mAbs to inhibit RdRp activity in vitro and suggests that this region represents a potential target for RdRp inhibitors. Using a series of synthetic peptides, we also identified a linear epitope (bound by mAb 5H1) that mapped to a 13 aa stretch surrounding residues 47 and 49 in the MTase domain, a region predicted to interact with the palm subdomain of the RdRp. The failure of one mAb (7G6) to bind both N- and C-terminally truncated NS5 recombinants indicates that the antibody recognizes a conformational epitope that requires the presence of residues in both the MTase and RdRp domains. These data support a structural model of the full-length NS5 molecule that predicts a physical interaction between the MTase and the RdRp domains.

Effects of the number of amino acid residues in the signal segment upstream or downstream of the NS2B-3 cleavage site on production and secretion of prM/M-E virus-like particles of West Nile virus

Expression of genes for precursor M (prM) and envelope (E) proteins of West Nile virus (WNV) leads to the production of small, capsidless, and non-infectious virus-like particles (VLPs) possessing the E antigen which is responsible for viral entry and immune protection. It has been reported that processing of the secretion signal affects viral release. We examined the secretion efficiency of VLPs into the culture medium from RK13 or 293 T cells transfected with expression vectors for prM and E proteins of WNV which were constructed to comprise different lengths of signal peptides upstream of the prM-E domain. The number of amino acid residues present in the segment markedly affected the production, processing, and secretion of VLPs. Secreted VLPs possessed both the processed M protein and the glycosylated E protein. In addition, immunization with VLPs induced neutralizing antibodies in C3H/HeN mice. These results indicate that the number of amino acid residues comprising the N-terminus of the signal segment controls the efficiency of assembly, maturation, and release of VLPs in the absence of viral protease, which in turn indicates the potential of VLPs as a candidate for an effective WNV subunit vaccine.

Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 microg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 microg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-microg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.

A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West Nile virus (WNV) that neutralizes infection even after virus has spread to the central nervous system. Herein, we define its mechanism of inhibition. E16 blocks infection primarily at a post-attachment step as antibody-opsonized WNV enters permissive cells but cannot escape from endocytic compartments. These cellular experiments suggest that E16 blocks the acid-catalyzed fusion step that is required for nucleocapsid entry into the cytoplasm. Indeed, E16 directly inhibits fusion of WNV with liposomes. Additionally, low-pH exposure of E16–WNV complexes in the absence of target membranes did not fully inactivate infectious virus, further suggesting that E16 prevents a structural transition required for fusion. Thus, a strongly neutralizing anti–WNV MAb with therapeutic potential is potently inhibitory because it blocks viral fusion and thereby promotes clearance by delivering virus to the lysosome for destruction.

Conservation and Variability of West Nile Virus Proteins

West Nile virus (WNV) has emerged globally as an increasingly important pathogen for humans and domestic animals. Studies of the evolutionary diversity of the virus over its known history will help to elucidate conserved sites, and characterize their correspondence to other pathogens and their relevance to the immune system. We describe a large-scale analysis of the entire WNV proteome, aimed at identifying and characterizing evolutionarily conserved amino acid sequences. This study, which used 2,746 WNV protein sequences collected from the NCBI GenPept database, focused on analysis of peptides of length 9 amino acids or more, which are immunologically relevant as potential T-cell epitopes. Entropy-based analysis of the diversity of WNV sequences, revealed the presence of numerous evolutionarily stable nonamer positions across the proteome (entropy value of ≤1). The representation (frequency) of nonamers variant to the predominant peptide at these stable positions was, generally, low (≤10% of the WNV sequences analyzed). Eighty-eight fragments of length 9–29 amino acids, representing ∼34% of the WNV polyprotein length, were identified to be identical and evolutionarily stable in all analyzed WNV sequences. Of the 88 completely conserved sequences, 67 are also present in other flaviviruses, and several have been associated with the functional and structural properties of viral proteins. Immunoinformatic analysis revealed that the majority (78/88) of conserved sequences are potentially immunogenic, while 44 contained experimentally confirmed human T-cell epitopes. This study identified a comprehensive catalogue of completely conserved WNV sequences, many of which are shared by other flaviviruses, and majority are potential epitopes. The complete conservation of these immunologically relevant sequences through the entire recorded WNV history suggests they will be valuable as components of peptide-specific vaccines or other therapeutic applications, for sequence-specific diagnosis of a wide-range of Flavivivirus infections, and for studies of homologous sequences among other flaviviruses.

Evidence of a humoral response to a novel protein WARF4 embedded in the West Nile virus NS4B gene encoded by an alternative open reading frame

West Nile virus (WNV) is a flavivirus that is maintained in a bird-mosquito transmission cycle. Humans, horses and other non-avian vertebrates are usually incidental hosts. However, WNV is a neurotropic virus, which requires an efficient humoral response for the control of a neuroinvasive infection. The WNV genome encodes three structural (capsid, premembrane/membrane and envelope) and seven non-structural proteins. Bioinformatic analysis performed on the WNV genomes detected a conserved alternative open reading frame restricted to the lineage I virus. To quickly verify the existence of this putative protein, entitled West Nile Alternative Reading Frame 4 (WARF4), we produced a prokaryotic recombinant source of WARF4 and verified its immunogenicity in vivo by analyzing 43 horse serum samples, of which 15 were positive for antibodies to WNV premembrane and envelope (prM-E) proteins. Specific antibodies to WARF4 were significantly detected in 5 out of the 15 serum samples testing positive for antibodies to prM-E WNV proteins. Our findings provide evidence of a significant antibody response to the WARF4 protein in the serum of the horse testing positive for antibodies to prM-E proteins, thus indicating that this antigen might be a potential tool for further characterization of the immune response of WNV infections in humans as well.

Interferon antagonist function of Japanese encephalitis virus NS4A and its interaction with DEAD-box RNA helicase DDX42

The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-α/β. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.

The Two-component NS2B-NS3 Proteinase Represses DNA Unwinding Activity of the West Nile Virus NS3 Helicase

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.

Comprehensive Analysis of West Nile Virus–Specific T Cell Responses in Humans

Cellular responses have been shown to play a role in immune control and clearance of West Nile virus (WNV) in murine models. However, little is known about the immunogenic regions of the virus or the phenotype of responding T cells in human infection. Frozen peripheral blood mononuclear cells (PBMCs) from 35 WNV-infected blood donors were screened for virus-specific T cell responses by an interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot assay that used 452 overlapping peptides spanning all WNV proteins. More-detailed phenotypic studies were performed on subjects with high-magnitude T cell responses. In individuals with identified responses, the total number of recognized WNV peptides ranged from 1 to 9 (median, 2 peptides), and the overall magnitude of responses ranged from 50 to 4210 spot-forming cells (SFCs) per 10(6) PBMCs (median, 130 SFCs/10(6) PBMCs). A subset of 8 frequently recognized peptides from the regions of the genome encoding membrane, envelope, and nonstructural 3 and 4b proteins was identified. Phenotypic study of the highest magnitude WNV-specific T cell responses revealed that most were mediated by CD8+ cells that expressed perforin and/or granzyme B. These findings are the first to define the breadth and characteristics of the human T cell response to WNV and have implications for candidate vaccine design and evaluation.

West Nile Virus Envelope Protein Inhibits dsRNA-Induced Innate Immune Responses

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.

Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (Corvus brachyrhynchos)

BackgroundBased on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection.MethodsVarious combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed.ResultsForty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody.ConclusionThe most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows.

Co-immunization with West Nile DNA and inactivated vaccines provides synergistic increases in their immunogenicities in mice

West Nile virus is now distributed throughout many temperate, subtropical and tropical areas: vaccines need to be developed that are affordable for developed and developing countries. Here, we constructed and evaluated a DNA vaccine expressing the premembrane and envelope proteins of West Nile virus (pcWNME). Mice immunized twice with 100 or 10 μg of pcWNME developed high or moderate levels of neutralizing antibodies, respectively. These mice were protected from viremia and death after lethal challenge. Mice immunized with a mixture of 1 μg of pcWNME and a small amount (1/10 dose) of a commercial inactivated vaccine developed moderate levels of neutralizing antibodies, whereas immunization with pcWNME or the inactivated vaccine alone induced only low or undetectable levels: co-immunization with the DNA and protein vaccines synergistically increased their own immunogenicities. The synergism reduced the amount of DNA sufficient to induce neutralizing antibodies: a single immunization with doses as low as 0.1 μg induced a titer of 1:40 at a 90% plaque reduction 6 or 9 weeks after immunization. Both IgG1 and IgG2a antibodies were induced in mice by co-immunization with the DNA and protein vaccines.

565. Complex Adenovirus-Mediated Expression of C, PreM, E, and NS1 Proteins of West Nile Virus Induces Both Humoral and Cellular Immune Responses

565. Complex Adenovirus-Mediated Expression of C, PreM, E, and NS1 Proteins of West Nile Virus Induces Both Humoral and Cellular Immune Responses

Immunochemical properties of the West Nile virus prM protein and the C-terminal fragment of the M protein

The cDNA fragments 466–966 and 878–1088 coding for the precursor M (prM) protein and polypeptide M31–75-E1–30 of the Russian strain LEIV-Vlg99-27889-human of the West Nile virus (WNV) were synthesized and cloned. The corresponding prM and M31–75-E1–30 recombinant polypeptides were purified by affinity chromatography. The prM polypeptide interacted with a polyclonal serum against WNV in ELISA and immunoblotting, demonstrating the immunochemical similarity of the recombinant polypeptide and the native WNV prM protein. Six species-specific monoclonal antibodies (mAbs) against the prM recombinant polypeptide recognized at least four epitopes on the recombinant polypeptides. In addition, mAb 7D11 displayed a virus-neutralizing activity. The patterns of mAb interactions with the prM, M31–75-E1–30, E1–180, and E260–466 recombinant polypeptides revealed cross-reacting epitopes in regions 260–466 of the E protein and 31–75 of the M31–75-E1–30 polypeptide and the WNV prM protein. A spatial model revealed structural similarity of the C-terminal regions of the E and M proteins of WNV, supporting the results of immunochemical experiments. Based on virus neutralization by mAb 7D11, which recognized an epitope mapping to region 31–75 of the WNV M protein, an important function in virus penetration into the cell was assumed for the C-terminal region of the M protein.

The Location of Asparagine-linked Glycans on West Nile Virions Controls Their Interactions with CD209 (Dendritic Cell-specific ICAM-3 Grabbing Nonintegrin)

Mammalian cell-derived West Nile virus preferentially infects cells expressing the C-type lectin CD209L (dendritic cellspecific ICAM-3 grabbing nonintegrin-related protein; liver- and lymph node-specific ICAM-3 grabbing nonintegrin) but not cells expressing CD209 (dendritic cell-specific ICAM-3 grabbing nonintegrin). In contrast, Dengue virus infection is enhanced in cells expressing either attachment factor. The West Nile virus envelope (E) protein contains a single N-linked glycosylation site at residue 154, whereas Dengue virus E contains sites at residues 153 and 67. We introduced a glycosylation site at position 67 into West Nile virus E. Reporter virus particles pseudotyped with this E protein infected cells using either CD209 or CD209L. We also introduced glycosylation sites at several novel positions. All sites allowed CD209L-mediated infection, but only a subset promoted CD209 use. As seen for other viruses, mannose-rich glycans on West Nile virus were required for its interactions with CD209. Surprisingly, however, mannose-rich glycans were not required for CD209L-mediated infection. Complex glycans, particularly N-acetylglucosamine-terminated structures, were able to mediate reporter virus particle interactions with CD209L. We propose that CD209L recognizes glycosylated flaviviruses with broad specificity, whereas CD209 is selective for flaviviruses bearing mannose-rich glycans. The location of the N-linked glycosylation sites on a virion determines the types of glycans incorporated, thus controlling viral tropism for CD209-expressing cells.

The glycosylation site in the envelope protein of West Nile virus (Sarafend) plays an important role in replication and maturation processes

The complete genome of West Nile (Sarafend) virus [WN(S)V] was sequenced. Phylogenetic trees utilizing the complete genomic sequence, capsid gene, envelope gene and NS5 gene/3' untranslated region of WN(S)V classified WN(S)V as a lineage II virus. A full-length infectious clone of WN(S)V with a point mutation in the glycosylation site of the envelope protein (pWNS-S154A) was constructed. Both growth kinetics and the mode of maturation were affected by this mutation. The titre of the pWNS-S154A virus was lower than the wild-type virus. This defect was corrected by the expression of wild-type envelope protein in trans. The pWNS-S154A virus matured intracellularly instead of at the plasma membrane as shown for the parental WN(S)V.

Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge

Chimeric (D2/WN) viruses containing the pre-membrane (prM) and envelope (E) proteins of West Nile virus (WN virus) and the capsid (C) and non-structural proteins of dengue 2 (DEN2) virus were used to evaluate the protective immunity elicited by either the flaviviral E protein or non-structural proteins. AG129 interferon-deficient mice, previously shown to be protected against lethal DEN1 or DEN2 viral infection after vaccination with a wild-type or candidate vaccine strain of DEN1 or DEN2 virus, respectively, were immunized with chimeric D2/WN virus and then challenged with DEN2 virus. D2/WN chimeric viruses were non-pathogenic in AG129 mice. These viruses elicited little anti-DEN E antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Only 15% of D2/WN-immunized mice survived challenge with DEN2 virus. However, their mean survival time increased by 11-14 days over non-immunized controls. These results suggest that, whilst the non-structural proteins were able to enhance mean survival times of AG129 mice, this protection was not as effective as protection mediated by the E protein.

Quantifying the Specific Binding between West Nile Virus Envelope Domain III Protein and the Cellular Receptor αVβ3 Integrin

A previous study has illustrated that the alphaVbeta3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor. In this study, the specificity and affinity binding of WNV E DIII protein to alphaVbeta3 integrin was confirmed with co-immunoprecipitation and receptor competition assay. Binding of WNV E DIII protein to alphaVbeta3 integrin induced the phosphorylation of focal adhesion kinase that is required to mediate ligand-receptor internalization into cells. A novel platform was then developed using the atomic force microscopy to measure this specific binding force between WNV E DIII protein and the cellular receptor, alphaVbeta3 integrin. The single protein pair-interacting force measured was in the range of 45 +/- 5 piconewtons. This interacting force was highly specific as minimal force was measured in the WNV E DIII protein interaction with alphaVbeta5 integrin molecules and heparan sulfate. These experiments provided an insight to quantitate virus-receptor interaction. Force measurement using atomic force microscopy can serve to quantitatively analyze the effect of candidate drugs that modulate virus-host receptor affinity.

Replication of West Nile virus in equine peripheral blood mononuclear cells

A cell model of primary monocytes and other mononuclear cells isolated from equine blood was used to study the kinetics of West Nile virus (WNV) replication in a natural host. West Nile virus has emerged on the North American continent as a significant cause of morbidity and mortality in a wide range of avian and mammalian species. While other flaviviruses are known to infect monocytes and lymphocytes, the ability of WNV to productively replicate in specific immune cells of peripheral blood has not been assessed. In this study, enriched populations of monocytes and lymphocytes as well as purified monocytes, CD4+, CD8+ and B lymphocytes were obtained from equine blood. Productive WNV replication was demonstrated by viral growth curves, quantitative RT-PCR for WNV RNA, and indirect immunofluorescence detection of a non-structural WNV protein. Enriched and purified monocytes consistently supported productive viral replication in blood from nine of nine horses tested while a minor subset of CD4+ lymphocytes supported productive replication in cells from three of the nine horses tested. Peak viral titers of 3.2–6.6 log 10 PFU/ml were reached at 6 days post-inoculation (p.i.) and titers were maintained through 10–15 days p.i. Activation of monocytes with bacterial lipopolysaccharide, which resulted in activation of nuclear transcription factor κB (NF-κB) plus elevation of nitric oxide and type I interferon levels, reduced or eliminated WNV replication. These results suggest that immune cells of the peripheral blood may serve as target cells for initial replication of WNV and may play a role in subsequent viral dissemination. Furthermore, primary equine immune cell cultures represent a potentially useful model of a natural WNV host when testing compounds such as antivirals for use in WNV treatment.

Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus

West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.