The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Pei Yong Shi - One of the best experts on this subject based on the ideXlab platform.
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cyclosporine inhibits Flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Min Qing, Zhiming Yuan, Bo Zhang, Feng Yang, Gang Zou, John M Robida, Hengli Tang, Pei Yong ShiAbstract:Although Flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in Flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting Flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits Flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits Flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of Flavivirus replication complex and could be targeted for potential antiviral development.
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a virus type specific serological diagnosis of Flavivirus infection using virus like particles
Virologica Sinica, 2009Co-Authors: Min Qing, Zhiming Yuan, Pei Yong ShiAbstract:Many Flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of Flavivirus infections in human. Because antibodies generated during Flavivirus infections cross-react with other Flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting Flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many Flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed Flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (i) VLPs from a panel of Flaviviruses are incubated with Flavivirus-infected sera at 37°C for 1 h; (ii)the neutralized VLPs are used to infect Vero cells; and (iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the “gold standard” of the classic PRNT; importantly, it shortens the assay time from >10 days to <1 day, and can be performed in biosafety level-2 facility.
Michael S. Diamond - One of the best experts on this subject based on the ideXlab platform.
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An mRNA Vaccine Protects Mice against Multiple Tick-Transmitted Flavivirus Infections
Elsevier, 2018Co-Authors: Laura A. Vanblargan, Gregory D Ebel, Theodore C Pierson, Sunny Himansu, Bryant M. Foreman, Michael S. DiamondAbstract:Summary: Powassan virus (POWV) is an emerging tick-transmitted Flavivirus that circulates in North America and Russia. Up to 5% of deer ticks now test positive for POWV in certain regions of the northern United States. Although POWV infections cause life-threatening encephalitis, there is no vaccine or countermeasure available for prevention or treatment. Here, we developed a lipid nanoparticle (LNP)-encapsulated modified mRNA vaccine encoding the POWV prM and E genes and demonstrated its immunogenicity and efficacy in mice following immunization with one or two doses. The POWV mRNA vaccine induced high titers of neutralizing antibody and sterilizing immunity against lethal challenge with different POWV strains. The mRNA vaccine also induced cross-neutralizing antibodies against multiple other tick-borne Flaviviruses and protected mice against the distantly related Langat virus. These data demonstrate the utility of the LNP-mRNA vaccine platform for the development of vaccines with protective activity against multiple Flaviviruses. : VanBlargan et al. demonstrate a lipid nanoparticle-encapsulated mRNA vaccine against Powassan virus, an emerging tick-borne Flavivirus, is highly immunogenic in mice and protects against lethal Powassan virus infection. Furthermore, the vaccine induces a cross-reactive antibody response against other tick-borne Flavivirus that is protective against disease caused by Langat virus infection in mice. Keywords: Flavivirus, vaccine, pathogenesis, encephabilitis, mouse model
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enhancing dengue virus maturation using a stable furin over expressing cell line
Virology, 2016Co-Authors: Swati Mukherjee, Devika Sirohi, Zhenguo Chen, Richard J Kuhn, Kimberly A Dowd, Michael S. Diamond, Theodore C PiersonAbstract:Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the Flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature Flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for Flavivirus neutralization and structural studies.
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non structural protein 1 is required for west nile virus replication complex formation and viral rna synthesis
Virology Journal, 2013Co-Authors: Soonjeon Youn, Rebecca L Ambrose, Jason M Mackenzie, Michael S. DiamondAbstract:Background Flavivirus NS1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. Despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and Golgi vesicles, NS1 is as an essential gene for Flavivirus replication. How NS1 modulates infection remains uncertain given that the viral RNA replication complex localizes to the cytosolic face of the endoplasmic reticulum.
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a fusion loop antibody enhances the infectious properties of immature Flavivirus particles
Journal of Virology, 2011Co-Authors: Izabela A Rodenhuiszybert, Michael S. Diamond, Bastiaan Moesker, Julia M Da Silva Voorham, Heidi Van Der Endemetselaar, Jan Wilschut, Jolanda M SmitAbstract:Flavivirus-infected cells secrete a mixture of mature, partially immature, and fully immature particles into the extracellular space. Although mature virions are highly infectious, prM-containing fully immature virions are noninfectious largely because the prM protein inhibits the cell attachment and fusogenic properties of the virus. If, however, cell attachment and entry are facilitated by anti-prM antibodies, immature Flavivirus becomes infectious after efficient processing of the prM protein by the endosomal protease furin. A recent study demonstrated that E53, a cross-reactive monoclonal antibody (MAb) that engages the highly conserved fusion-loop peptide within the Flavivirus envelope glycoprotein, preferentially binds to immature Flavivirus particles. We investigated here the infectious potential of fully immature West Nile virus (WNV) and dengue virus (DENV) particles opsonized with E53 MAb and observed that, like anti-prM antibodies, this anti-E antibody also has the capacity to render fully immature Flaviviruses infectious. E53-mediated enhancement of both immature WNV and DENV depended on efficient cell entry and the enzymatic activity of the endosomal furin. Furthermore, we also observed that E53-opsonized immature DENV particles but not WNV particles required a more acidic pH for efficient cleavage of prM by furin, adding greater complexity to the dynamics of antibody-mediated infection of immature Flavivirus virions.
Min Qing - One of the best experts on this subject based on the ideXlab platform.
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cyclosporine inhibits Flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Min Qing, Zhiming Yuan, Bo Zhang, Feng Yang, Gang Zou, John M Robida, Hengli Tang, Pei Yong ShiAbstract:Although Flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in Flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting Flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits Flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits Flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of Flavivirus replication complex and could be targeted for potential antiviral development.
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a virus type specific serological diagnosis of Flavivirus infection using virus like particles
Virologica Sinica, 2009Co-Authors: Min Qing, Zhiming Yuan, Pei Yong ShiAbstract:Many Flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of Flavivirus infections in human. Because antibodies generated during Flavivirus infections cross-react with other Flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting Flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many Flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed Flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (i) VLPs from a panel of Flaviviruses are incubated with Flavivirus-infected sera at 37°C for 1 h; (ii)the neutralized VLPs are used to infect Vero cells; and (iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the “gold standard” of the classic PRNT; importantly, it shortens the assay time from >10 days to <1 day, and can be performed in biosafety level-2 facility.
Karin Stiasny - One of the best experts on this subject based on the ideXlab platform.
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pre existing yellow fever immunity impairs and modulates the antibody response to tick borne encephalitis vaccination
NPJ vaccines, 2019Co-Authors: Victoria Bradt, Stefan Malafa, Amrei Von Braun, Johanna Jarmer, Georgios Tsouchnikas, Iris Medits, Kerstin Wanke, Urs Karrer, Karin StiasnyAbstract:Flaviviruses have an increasing global impact as arthropod-transmitted human pathogens, exemplified by Zika, dengue, yellow fever (YF), West Nile, Japanese encephalitis, and tick-borne encephalitis (TBE) viruses. Since all Flaviviruses are antigenically related, they are prone to phenomena of immunological memory ('original antigenic sin'), which can modulate immune responses in the course of sequential infections and/or vaccinations. In our study, we analyzed the influence of pre-existing YF vaccine-derived immunity on the antibody response to TBE vaccination. By comparing samples from YF pre-vaccinated and Flavivirus-naive individuals, we show that YF immunity not only caused a significant impairment of the neutralizing antibody response to TBE vaccination but also a reduction of the specific TBE virus neutralizing activities (NT/ELISA-titer ratios). Our results point to a possible negative effect of pre-existing cross-reactive immunity on the outcome of Flavivirus vaccination that may also pertain to other combinations of sequential Flavivirus infections and/or vaccinations.
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Flaviviruses and Flavivirus vaccines
Vaccine, 2012Co-Authors: Franz X Heinz, Karin StiasnyAbstract:Several human-pathogenic Flaviviruses (including yellow fever, dengue, Japanese encephalitis, West Nile and tick-borne encephalitis viruses) have a significant public health impact in different parts of the world and the potential of emerging in previously non-endemic regions. For some viruses, the structure of the most important immunogen, the envelope protein E, has been determined to atomic resolution by X-ray crystallography, and the architecture of virus particles has been resolved by cryo-electron microscopy. Through the combination of structural and immunological investigations, we now have a detailed understanding of the mechanisms of virus neutralization and antibody-dependent enhancement (ADE) of infectivity at a molecular level. The latter phenomenon has been proposed to play an important role in the immunopathology of severe forms of dengue virus infections (hemorrhagic dengue fever and dengue shock syndrome) and is therefore of special relevance in the context of dengue vaccines. Effective human vaccines are in use for the prophylaxis of yellow fever (live attenuated), Japanese encephalitis (live attenuated and inactivated whole virus), and tick-borne encephalitis (inactivated whole virus). Although dengue is the most important Flavivirus with respect to global disease incidence, the development and use of vaccines has been hampered so far by the theoretical risk of vaccine-related adverse events such as immune enhancement of infection and the requirement to induce a long-lasting protective immune response against all four dengue serotypes simultaneously. Currently, several kinds of dengue vaccines are in development, but only one of these candidates (a chimeric dengue-yellow fever live attenuated vaccine) has reached the stage of phase 3 clinical trials.
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correlation between elisa hemagglutination inhibition and neutralization tests after vaccination against tick borne encephalitis
Journal of Medical Virology, 1996Co-Authors: Heidemarie Holzmann, Michael Kundi, Karin Stiasny, Jan Clement, P Mckenna, Christian Kunz, F X HeinzAbstract:The significance of IgG antibody levels determined by a binding assay (ELISA) was investigated as a surrogate marker for the presence of neutralizing and hemagglutination inhibiting antibodies in sera from individuals vaccinated against tick-borne encephalitis (TBE). To assess the extent of interference by Flavivirus cross-reactive antibodies, sera from persons with a proven or suspected history of other Flavivirus infections and/or vaccinations were also examined. An excellent and highly significant correlation was found between ELISA IgG units and the antibody titers obtained by the hemagglutination inhibition (HI) as well as by the neutralization test (NT), provided that there was no other exposure to Flavivirus antigens except TBE vaccination. Yellow fever vaccination and/or dengue virus infections induced significant levels of antibodies reactive in the TBE ELISA and HI test, which did not exhibit, however, neutralizing activity against TBE virus. The phenomenon and problem of “original antigenic sin” was demonstrated in a TBE vaccinee with a history of previous Flavivirus infections. TBE vaccination first induced a booster reaction resulting in a rise in the level of cross-reactive antibodies only, whereas TBE virus-neutralizing antibodies became detectable only after the third vaccination. It is concluded that the level of IgG antibodies determined by ELISA is a good marker for predicting the presence of neutralizing antibodies after TBE vaccination, but only in the absence of Flavivirus cross-reactive antibodies. Otherwise, a neutralization assay is necessary for assessing immunity. © 1996 Wiley-Liss, Inc.
Zhiming Yuan - One of the best experts on this subject based on the ideXlab platform.
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transmembrane domains of ns2b contribute to both viral rna replication and particle formation in japanese encephalitis virus
Journal of Virology, 2016Co-Authors: Xiaodan Li, Zhiming Yuan, Cheng Lin Deng, Han Qing Ye, Hong Lei Zhang, Qiu Yan Zhang, Dong Dong Chen, Pan Tao Zhang, Bo ZhangAbstract:ABSTRACT Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that Flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the Flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, Flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. IMPORTANCE Many Flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the Flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent Flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of Flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein.
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inhibition of japanese encephalitis virus infection by Flavivirus recombinant e protein domain iii
Virologica Sinica, 2013Co-Authors: Jingjing Fan, Yi Liu, Xuping Xie, Bo Zhang, Zhiming YuanAbstract:Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus closely related to the human pathogens including yellow fever virus, dengue virus and West Nile virus. There are currently no effective antiviral therapies for all of the Flavivirus and only a few highly effective vaccines are licensed for human use. In this paper, the E protein domain III (DIII) of six heterologous Flaviviruses (DENV1-4, WNV and JEV) was expressed in Escherichia coli successfully. The proteins were purified after a solubilization and refolding procedure, characterized by SDS-PAGE and Western blotting. Competitive inhibition showed that all recombinant Flavivirus DIII proteins blocked the entry of JEV into BHK-21 cells. Further studies indicated that antibodies induced by the soluble recombinant Flavivirus DIII partially protected mice against lethal JEV challenge. These results demonstrated that recombinant Flavivirus DIII proteins could inhibit JEV infection competitively, and immunization with proper folding Flavivirus DIII induced cross-protection against JEV infection in mice, implying a possible role of DIII for the cross-protection among Flavivirus as well as its use in antigens for immunization in animal models.
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cyclosporine inhibits Flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: Min Qing, Zhiming Yuan, Bo Zhang, Feng Yang, Gang Zou, John M Robida, Hengli Tang, Pei Yong ShiAbstract:Although Flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in Flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting Flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits Flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits Flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of Flavivirus replication complex and could be targeted for potential antiviral development.
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a virus type specific serological diagnosis of Flavivirus infection using virus like particles
Virologica Sinica, 2009Co-Authors: Min Qing, Zhiming Yuan, Pei Yong ShiAbstract:Many Flaviviruses are emerging and reemerging pathogens, such as West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV), and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of Flavivirus infections in human. Because antibodies generated during Flavivirus infections cross-react with other Flavivirus members, plaque reduction neutralization test (PRNT) is the only available assay to determine the infecting Flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety level-3 or level-4 containment for many Flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed Flavivirus viral-like particles (VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps: (i) VLPs from a panel of Flaviviruses are incubated with Flavivirus-infected sera at 37°C for 1 h; (ii)the neutralized VLPs are used to infect Vero cells; and (iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen (as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the “gold standard” of the classic PRNT; importantly, it shortens the assay time from >10 days to <1 day, and can be performed in biosafety level-2 facility.