Cache Valley virus: an emerging arbovirus of public and veterinary health importance.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus (Bunyavirales: Peribunyaviridae) that has been identified as a teratogen in ruminants causing fetal death and severe malformations during epizootics in the U.S. CVV has recently emerged as a viral pathogen causing severe disease in humans. Despite its emergence as a public health and agricultural concern, CVV has yet to be significantly studied by the scientific community. Limited information exists on CVV's geographic distribution, ecological cycle, seroprevalence in humans and animals, and spectrum of disease, including its potential as a human teratogen. Here, we present what is known of CVV's virology, ecology, and clinical disease in ruminants and humans. We discuss the current diagnostic techniques available and highlight gaps in our current knowledge and considerations for future research.

Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics.

California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA.IMPORTANCEOrthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics.

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

Exposing cryptic epitopes on the Venezuelan equine encephalitis virus E1 glycoprotein prior to treatment with alphavirus cross-reactive monoclonal antibody allows blockage of replication early in infection.

Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.

The Specificity of the Persistent IgM Neutralizing Antibody Response in Zika Virus Infections among Individuals with Prior Dengue Virus Exposure.

Dengue viruses (DENV) and Zika virus (ZIKV) are related mosquito-borne flaviviruses with similar disease manifestations, vector ecologies, and geographic ranges. The ability to differentiate these viruses serologically is vital due to the teratogenic nature of ZIKV and the potential confounding of preexisting cross-reactive anti-DENV antibodies. Here, we illustrate the kinetics of the IgM neutralizing antibody (NAb) response using longitudinal samples ranging from acute ZIKV infection to late convalescence from individuals with evidence of prior DENV infection. By serially depleting antibody isotypes prior to the neutralization assay, we determined that IgM contributes predominantly to ZIKV neutralization and is less cross-reactive than the IgG NAb. The IgM NAb peaked around 14 days (95% confidence interval [95% CI], 13 to 15) and had a median duration of 257 days (95% CI, 133 to 427). These results demonstrate the persistence of IgM NAb after ZIKV infection and imply its potential role in diagnosis, vaccine evaluation, serosurveillance, and research on flavivirus-host interactions.

Development of diagnostic microsphere-based immunoassays for Heartland virus.

BACKGROUND: Heartland virus (HRTV), a recently reclassified member of the genus Bandavirus, family Phenuiviridae, was first isolated in 2009 from a Missouri farmer exhibiting leukopenia and thrombocytopenia with suspected ehrlichiosis. Since then, more HRTV cases have been diagnosed, and firstline laboratory diagnostic assays are needed to identify future infections Objectives. We sought to develop rapid and reliable IgM and IgG microsphere immunoassays (MIAs) to test sera of patients suspected of having HRTV infection, and to distinguish between recent and past infections. STUDY DESIGN: Heartland virus antigen was captured by an anti-HRTV monoclonal antibody covalently bound to microspheres. Antibodies in human sera from confirmed HRTV-positive and negative cases were reacted with the microsphere complexes and detected using a BioPlex® 200 instrument. Assay cutoffs were determined by receiver operator characteristic analysis of the normalized test output values, equivocal zones for each assay were defined, and sensitivities, specificities, accuracies, and imprecision values were calculated. RESULTS: Sensitivities, specificities and accuracies of the IgM and IgG MIAs were all >95 %. Both tests were precise within and between assay plates, and cross-reactivity with other arboviruses was not observed. CONCLUSIONS: HRTV IgM and IgG MIAs are accurate and rapid first-line methods to serologically identify recent and past HRTV infections.

A Monoclonal Antibody Specific for Japanese Encephalitis Virus with High Neutralizing Capability for Inclusion as a Positive Control in Diagnostic Neutralization Tests.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern because of its recent geographic expansion. Although commercial vaccines are available and used in some endemic countries, JEV continues to cause illness, with more than 60,000 cases reported annually. To develop a reproducible positive control antibody useable in diagnosis of JEV infections, murine hybridomas were developed from mice inoculated with a combination of IXIARO JEV vaccine and JEV domain III of the envelope protein (E-DIII). Monoclonal antibodies (MAbs) were characterized for their ability to neutralize virus in vitro. Monoclonal antibody 17BD3-2 was found to be JEV specific and highly neutralizing, with a plaque reduction neutralization test (PRNT)90 endpoint titer of 1.25 µg/mL. The functional epitopes were mapped using virus neutralization escape variants to amino acid residues S309, K312, and G333 in E-DIII. This MAb may be substituted for human immune sera used as a positive control in PRNT for distribution to public health laboratories worldwide in potential future outbreaks of JEV.

Incorporation of IgG Depletion in a Neutralization Assay Facilitates Differential Diagnosis of Zika and Dengue in Secondary Flavivirus Infection Cases.

Zika virus (ZIKV) has emerged as a major global public health concern due to its link as a causative agent of human birth defects. Laboratory diagnosis of suspected ZIKV infections by serological testing of specimens collected a week or more after symptom onset primarily relies on detection of anti-ZIKV-specific IgM antibodies by enzyme-linked immunosorbent assay coupled with detection of ZIKV-specific neutralizing antibody by neutralization tests. A definitive diagnosis based on serological assays is possible during primary ZIKV infections; however, due to the cross-reactivity of antibodies elicited during flaviviral infections, a definitive diagnosis is not always possible, especially among individuals who have previously been exposed to closely related flaviviruses, such as dengue virus (DENV). Here, we investigated the neutralizing IgM antibody profiles of 33 diagnostic specimens collected from individuals with suspected primary and secondary flaviviral infections acquired when visiting areas experiencing active ZIKV transmission in 2015 and 2016. Specimens collected between 1 day and 3 months postexposure were tested for ZIKV and dengue virus type 1 (DENV1) and type 2 (DENV2) by the plaque reduction neutralization test (PRNT) before and after IgG depletion. We found that IgG depletion prior to neutralization testing had little effect in differentiating samples from individuals with secondary infections taken less than 3 weeks postexposure; however, IgG depletion significantly reduced the cross-reactive neutralizing antibody titers and increased the percentage of cases discernible by PRNT from 15.4% (95% confidence interval [CI], 4.3 to 42.2%) to 76.9% (95% CI, 49.7 to 91.8%) for samples collected between roughly 3 and 12 weeks postexposure. These results highlight the potential of IgG depletion to improve the specificity of PRNT for better confirmation and differential diagnosis of flavivirus infections.

Ability To Serologically Confirm Recent Zika Virus Infection in Areas with Varying Past Incidence of Dengue Virus Infection in the United States and U.S. Territories in 2016.

Cross-reactivity within flavivirus antibody assays, produced by shared epitopes in the envelope proteins, can complicate the serological diagnosis of Zika virus (ZIKAV) infection. We assessed the utility of the plaque reduction neutralization test (PRNT) to confirm recent ZIKAV infections and rule out misleading positive immunoglobulin M (IgM) results in areas with various levels of past dengue virus (DENV) infection incidence. We reviewed PRNT results of sera collected for diagnosis of ZIKAV infection from 1 January through 31 August 2016 with positive ZIKAV IgM results, and ZIKAV and DENV PRNTs were performed. PRNT result interpretations included ZIKAV, unspecified flavivirus, DENV infection, or negative. For this analysis, ZIKAV IgM was considered false positive for samples interpreted as a DENV infection or negative. In U.S. states, 208 (27%) of 759 IgM-positive results were confirmed to be ZIKAV compared to 11 (21%) of 52 in the U.S. Virgin Islands (USVI), 15 (15%) of 103 in American Samoa, and 13 (11%) of 123 in Puerto Rico. In American Samoa and Puerto Rico, more than 80% of IgM-positive results were unspecified flavivirus infections. The false-positivity rate was 27% in U.S. states, 18% in the USVI, 2% in American Samoa, and 6% in Puerto Rico. In U.S. states, the PRNT provided a virus-specific diagnosis or ruled out infection in the majority of IgM-positive samples. Almost a third of ZIKAV IgM-positive results were not confirmed; therefore, providers and patients must understand that IgM results are preliminary. In territories with historically higher rates of DENV transmission, the PRNT usually could not differentiate between ZIKAV and DENV infections.

Rapid colorimetric detection of Zika virus from serum and urine specimens by reverse transcription loop-mediated isothermal amplification (RT-LAMP).

Zika virus (ZIKV) has emerged as a major global public health concern in the last two years due to its link as a causative agent of human birth defects. Its rapid expansion into the Western Hemisphere as well as the ability to be transmitted from mother to fetus, through sexual transmission and possibly through blood transfusions has increased the need for a rapid and expansive public health response to this unprecedented epidemic. A non-invasive and rapid ZIKV diagnostic screening assay that can be performed in a clinical setting throughout pregnancy is vital for prenatal care of women living in areas of the world where exposure to the virus is possible. To meet this need we have developed a sensitive and specific reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay to detect ZIKV RNA in urine and serum with a simple visual detection. RT-LAMP results were shown to have a limit of detection 10-fold higher than qRT-PCR. As little as 1.2 RNA copies/µl was detected by RT-LAMP from a panel of 178 diagnostic specimens. The assay was shown to be highly specific for ZIKV RNA when tested with diagnostic specimens positive for dengue virus (DENV) and chikungunya virus (CHIKV). The assay described here illustrates the potential for a fast, reliable, sensitive and specific assay for the detection of ZIKV from urine or serum that can be performed in a clinical or field setting with minimal equipment and technological expertise.

Vertebrate Host Susceptibility to Heartland Virus.

Heartland virus (HRTV) is a recently described phlebovirus initially isolated in 2009 from 2 humans who had leukopenia and thrombocytopenia. Serologic assessment of domestic and wild animal populations near the residence of 1 of these persons showed high exposure rates to raccoons, white-tailed deer, and horses. To our knowledge, no laboratory-based assessments of viremic potential of animals infected with HRTV have been performed. We experimentally inoculated several vertebrates (raccoons, goats, chickens, rabbits, hamsters, C57BL/6 mice, and interferon-α/ß/γ receptor-deficient [Ag129]) mice with this virus. All animals showed immune responses against HRTV after primary or secondary exposure. However, neutralizing antibody responses were limited. Only Ag129 mice showed detectable viremia and associated illness and death, which were dose dependent. Ag129 mice also showed development of mean peak viral antibody titers >8 log10 PFU/mL, hemorrhagic hepatic lesions, splenomegaly, and large amounts of HRTV antigen in mononuclear cells and hematopoietic cells in the spleen.

A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

Molecular, serological and in vitro culture-based characterization of Bourbon virus, a newly described human pathogen of the genus Thogotovirus.

BACKGROUND: In June of 2014, a previously healthy man from Kansas with a recent history of tick exposure died from complications related to an illness marked by fever, thrombocytopenia and leukopenia. An isolate was derived from the blood of this patient during the course of diagnostic testing. This isolate was subsequently identified as a novel orthomyxovirus of the genus Thogotovirus by next generation sequencing and was named Bourbon virus after the patient's county of residence. OBJECTIVES: To support research and diagnostic aims, we provide a basic description of Bourbon virus at both the molecular and serological levels. Furthermore, to preliminarily identify potential host and vector range associations we have characterized the growth kinetics of Bourbon virus in a variety of vertebrate and invertebrate cell lines. STUDY DESIGN: Bourbon virus was subjected to next generation-high throughput sequencing, phylogenetic, and basic structural protein analyses as well as 2-way plaque reduction neutralization assays. Also, we inoculated a variety of cell types with Bourbon virus and evaluated the growth kinetics by determining viral titers in the supernatants taken from infected cells over time. RESULTS: Bourbon virus possesses 24-82% identity at the amino acid sequence level and low serological cross-reactivity with other Thogotoviruses. In vitro growth kinetics reveal robust replication of Bourbon virus in mammalian and tick cells. CONCLUSIONS: Molecular and serological characterizations identify Bourbon virus as a novel member of the genus Thogotovirus. Results from cell culture analyses suggest an association between Bourbon virus and mammalian and tick hosts.

Development and Characterization of Monoclonal Antibodies Directed Against the Nucleoprotein of Heartland Virus.

Heartland virus (HRTV), a phlebovirus first isolated from two Missouri farmers in 2009, has been proposed to be transmitted to humans by the bite of infected Amblyomma americanum ticks. It is closely related to severe fever with thrombocytopenia syndrome virus (SFTSV) from China, another previously unrecognized phlebovirus that has subsequently been associated with hundreds of cases of severe disease in humans. To expand diagnostic capacity to detect HRTV infections, 20 hybridoma clones secreting anti-HRTV murine monoclonal antibodies (MAbs) were developed using splenocytes from HRTV-inoculated AG129 alpha/beta and gamma interferon receptor-deficient mice. Nine of these MAbs were characterized herein for inclusion in future HRTV diagnostic assay development. All of the MAbs developed were found to be non-neutralizing and reactive to linear epitopes on HRTV nucleocapsid protein. MAb 2AF11 was found to be cross-reactive with SFTSV.

Development of a small animal peripheral challenge model of Japanese encephalitis virus using interferon deficient AG129 mice and the SA14-14-2 vaccine virus strain.

Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern due to its recent geographic expansion. While commercial vaccines are available and used in some endemic countries, JEV continues to be a public health problem, with 50,000 cases reported annually. Research with virulent JEV in mouse models to develop new methods of prevention and treatment is restricted to BSL-3 containment facilities, confining these studies to investigators with access to these facilities. We have developed an adult small animal peripheral challenge model using interferon-deficient AG129 mice and the JEV live-attenuated vaccine SA14-14-2, thus requiring only BSL-2 containment. A low dose of virus (10PFU/0.1ml) induced 100% morbidity in infected mice. Increased body temperatures measured by implantable temperature transponders correlated with an increase in infectious virus and viral RNA in serum, spleen and brain as well as an increase in pro-inflammatory markers measured by a 58-biomarker multi-analyte profile (MAP) constructed during the course of infection. In the future, the MAP measurements can be used as a baseline for comparison in order to better assess the inhibition of disease progression by other prophylactic and therapeutic agents. The use of the AG129/JEV SA14-14-2 animal model makes vaccine and therapeutic studies feasible for laboratories with limited biocontainment facilities.

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.

Mutations in the West Nile prM protein affect VLP and virion secretion in vitro.

Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion.

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, but only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.

Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein.

Hybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection.