scholarly journals Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice

2020 ◽  
Vol 8 (12) ◽  
pp. 1890
Author(s):  
Pham-Tue-Hung Tran ◽  
Naveed Asghar ◽  
Urban Höglund ◽  
Olivia Larsson ◽  
Lars Haag ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
Matrix Protein ◽  
Particle System ◽  
Nonstructural Protein ◽  
Stable Cell Line ◽  
Reporter Virus ◽  
Structural Genes ◽  
West Nile ◽  
Nonstructural Protein 1

Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.

scholarly journals Nonstructural Protein 1-Specific Immunoglobulin M and G Antibody Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Flaviviral Infections in Humans

2014 ◽  
Vol 53 (2) ◽  
pp. 557-566 ◽  
Author(s):  
Day-Yu Chao ◽  
Jedhan Ucat Galula ◽  
Wen-Fan Shen ◽  
Brent S. Davis ◽  
Gwong-Jen J. Chang
Keyword(s):  
West Nile Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Igg Antibody ◽  
West Nile ◽  
Dengue Viruses ◽  
Nonstructural Protein 1 ◽  
Antibody Capture

IgM antibody- and IgG antibody-capture enzyme-linked immunosorbent assays (MAC/GAC-ELISAs) targeted at envelope protein (E) of dengue viruses (DENV), West Nile virus, and Japanese encephalitis virus (JEV) are widely used as serodiagnostic tests for presumptive confirmation of viral infection. Antibodies directed against the flavivirus nonstructural protein 1 (NS1) have been proposed as serological markers of natural infections among vaccinated populations. The aim of the current study is to optimize an IgM and IgG antibody-capture ELISA (MAC/GAC-ELISA) to detect anti-NS1 antibodies and compare it with anti-E MAC/GAC-ELISA. Plasmids to express premembrane/envelope (prM/E) or NS1 proteins of six medically important flaviviruses, including dengue viruses (DENV-1 to DENV-4), West Nile virus (WNV), and Japanese encephalitis virus (JEV), were constructed. These plasmids were used for the production of prM/E-containing virus-like particles (VLPs) and secreted NS1 (sNS1) from COS-1 cells. Archived clinical specimens from patients with confirmed DENV, JEV, and WNV infections, along with naive sera, were subjected to NS1-MAC/GAC-ELISAs before or after depletion of anti-prM/E antibodies by preabsorption with or without VLPs. Human serum specimens from previously confirmed DENV infections showed significantly enhanced positive-to-negative (P/N) ratios for NS1-MAC/GAC-ELISAs after the depletion of anti-prM/E antibodies. No statistical differences in sensitivities and specificities were found between the newly developed NS1- and VLP-MAC/GAC-ELISAs. Further application of the assays to WNV- and JEV-infected serum panels showed similar results. A novel approach to perform MAC/GAC-ELISAs for NS1 antibody detection was successfully developed with great potential to differentiate antibodies elicited by the tetravalent chimeric yellow fever-17D/dengue vaccine or DENV infection.

scholarly journals Differential Effects of Mutations in NS4B on West Nile Virus Replication and Inhibition of Interferon Signaling

2007 ◽  
Vol 81 (21) ◽  
pp. 11809-11816 ◽  
Author(s):  
Jared D. Evans ◽  
Christoph Seeger
Keyword(s):  
West Nile Virus ◽  
Nonstructural Protein ◽  
Signal Transduction Pathway ◽  
Janus Kinase ◽  
Transduction Pathway ◽  
Interferon Signaling ◽  
Structural Genes ◽  
West Nile ◽  
Tyrosine Kinase 2 ◽  
Janus Kinase 1

ABSTRACT West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350, 2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in the inhibition of the IFN signaling pathway during WNV infections.

Levels of circulating NS1 impact West Nile virus spread to the brain

2021 ◽  
Author(s):  
Alex W. Wessel ◽  
Kimberly A. Dowd ◽  
Scott B. Biering ◽  
Ping Zhang ◽  
Melissa A. Edeling ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Endothelial Dysfunction ◽  
Cell Surface ◽  
Virus Replication ◽  
Nonstructural Protein ◽  
Disease Outcome ◽  
Ns1 Protein ◽  
West Nile ◽  
Nonstructural Protein 1 ◽  
The Brain

Dengue (DENV) and West Nile (WNV) viruses are arthropod-transmitted flaviviruses that respectively cause systemic vascular leakage and encephalitis syndromes in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity of the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. As exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcome. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here we assessed West Nile virus (WNV) NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in circulation facilitate WNV dissemination to the brain and disease outcome. Our findings help understand the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

scholarly journals Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication

2003 ◽  
Vol 77 (23) ◽  
pp. 12901-12906 ◽  
Author(s):  
Michael K. Lo ◽  
Mark Tilgner ◽  
Pei-Yong Shi
Keyword(s):  
West Nile Virus ◽  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Health Priorities ◽  
Stable Cell Line ◽  
West Nile ◽  
Subgenomic Replicon ◽  
Genes Encoding ◽  
High Throughput Assay

ABSTRACT Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

scholarly journals West Nile Virus NS1 Antagonizes Interferon Beta Production by Targeting RIG-I and MDA5

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Hong-Lei Zhang ◽  
Han-Qing Ye ◽  
Si-Qing Liu ◽  
Cheng-Lin Deng ◽  
Xiao-Dan Li ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
West Nile Virus ◽  
Viral Replication ◽  
Signaling Pathway ◽  
Interferon Beta ◽  
Nonstructural Protein ◽  
Innate Immune ◽  
West Nile ◽  
Nonstructural Protein 1 ◽  
Inducible Gene

ABSTRACT West Nile virus (WNV) is a mosquito-borne flavivirus that causes epidemics of encephalitis and viscerotropic disease worldwide. This virus has spread rapidly and has posed a significant public health threat since the outbreak in New York City in 1999. The interferon (IFN)-mediated antiviral response represents an important component of virus-host interactions and plays an essential role in regulating viral replication. Previous studies have suggested that multifunctional nonstructural proteins encoded by flaviviruses antagonize the host IFN response via various means in order to establish efficient viral replication. In this study, we demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes IFN-β production, most likely through suppression of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In a dual-luciferase reporter assay, WNV NS1 significantly inhibited the activation of the IFN-β promoter after Sendai virus infection or poly(I·C) treatment. NS1 also suppressed the activation of the IFN-β promoter when it was stimulated by interferon regulatory factor 3 (IRF3)/5D or its upstream molecules in the RLR signaling pathway. Furthermore, NS1 blocked the phosphorylation and nuclear translocation of IRF3 upon stimulation by various inducers. Mechanistically, WNV NS1 targets RIG-I and melanoma differentiation-associated gene 5 (MDA5) by interacting with them and subsequently causing their degradation by the proteasome. Furthermore, WNV NS1 inhibits the K63-linked polyubiquitination of RIG-I, thereby inhibiting the activation of downstream sensors in the RLR signaling pathway. Taken together, our results reveal a novel mechanism by which WNV NS1 interferes with the host antiviral response. IMPORTANCE WNV Nile virus (WNV) has received increased attention since its introduction to the United States. However, the pathogenesis of this virus is poorly understood. This study demonstrated that the nonstructural protein 1 (NS1) of WNV antagonizes the induction of interferon beta (IFN-β) by interacting with and degrading retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are crucial viral sensors in the host innate immune system. Further experiments suggested that NS1-mediated inhibition of the RIG-I-like receptor (RLR) signaling pathway involves inhibition of RIG-I K63-linked polyubiquitination and that the proteasome plays a role in RIG-I degradation. This study provides new insights into the regulation of WNV NS1 in the RLR signaling pathway and reveals a novel mechanism by which WNV evades the host innate immune response. The novel findings may guide us to discover new therapeutic targets and develop effective vaccines for WNV infections.

scholarly journals Serological Evaluation of Suspected West Nile Virus Human Cases following Its Introduction during a Dengue Outbreak in Puerto Rico in 2007

2011 ◽  
Vol 18 (6) ◽  
pp. 978-983 ◽  
Author(s):  
Elizabeth Hunsperger ◽  
Manuela Beltran ◽  
Luz Nereida Acosta ◽  
Jorge Jordan-Munoz ◽  
Jomil Torres ◽  
...  
Keyword(s):  
Puerto Rico ◽  
West Nile Virus ◽  
Nonstructural Protein ◽  
Diagnostic Testing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Serum Samples ◽  
West Nile ◽  
Nonstructural Protein 1 ◽  
Testing Algorithm

ABSTRACTA laboratory testing algorithm was evaluated to confirm West Nile virus (WNV) infection in human serum following the introduction of the virus in Puerto Rico in 2007. This testing algorithm used two standard diagnostic assays, the IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) and real-time reverse transcriptase PCR (RT-PCR), along with two nonconventional assays, the nonstructural protein 1 (NS1) ELISA and a 90%-plaque-reduction neutralization test (PRNT90) with IgG depletion for dengue virus (DENV) and WNV. A total of 2,321 serum samples from suspected WNV human cases were submitted for testing. Approximately one-third (867, 37%) were cross-reactive for DENV and WNV by MAC ELISA and had negative RT-PCR results for both viruses. Of a subset of 43 samples tested, 31 (72%) of these cases were identified as positive for DENV in the PRNT90with IgG depletion and 8 (19%) were positive in the DENV NS1 antigen ELISA. These two assays combined differentiated 36 (84%) of the samples that could not be diagnosed using the standard diagnostic testing methods.

scholarly journals Development of Monoclonal Antibodies to West Nile Virus and Their Application in Immunohistochemistry

2012 ◽  
Vol 19 (11) ◽  
pp. 1853-1858 ◽  
Author(s):  
Jiro Hirota ◽  
Shinya Shimizu ◽  
Tomoyuki Shibahara ◽  
Takashi Isobe ◽  
Manabu Yamada ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
West Nile ◽  
Nonstructural Protein 1 ◽  
Formalin Fixed Paraffin Embedded

ABSTRACTWest Nile virus (WNV) is endemic throughout Africa, Eurasia, America, and Australia and has important implications for avian, horse, and human health. In these regions, dead birds are monitored for the presence of WNV through immunohistochemistry (IHC) and PCR. However, a number of the tools for IHC are inadequate owing to their cross-reactivity to other Japanese encephalitis serogroup viruses. Here we have established eight monoclonal antibodies (MAbs) to WNV. Four of them bound to the envelope protein, three of them bound to nonstructural protein 1 (NS1), and one bound to precursor membrane protein (prM), as shown by Western blot analysis. The anti-NS1 MAbs and the anti-prM MAb did not cross-react with Japanese encephalitis virus (JEV), Murray valley encephalitis virus, or St. Louis encephalitis virus in an indirect enzyme-linked immunosorbent assay. One NS1-specific MAb, SHW-32B1, and the previously reported NS1-specific MAb, SHW-7A11, were shown by IHC to specifically detect the cytoplasm of degenerated cells in the heart and brain of a WNV-infected goose. Neither of these MAbs were shown by IHC to cross-react with degenerated cells in the brain of a JEV-infected pig. These MAbs are the first reported anti-NS1 MAbs that can be used for WNV-specific IHC using formalin-fixed, paraffin-embedded sections. They may be useful for WNV research and surveillance.

scholarly journals New Sensitive Competitive Enzyme-Linked Immunosorbent Assay Using a Monoclonal Antibody against Nonstructural Protein 1 of West Nile Virus NY99

2011 ◽  
Vol 19 (2) ◽  
pp. 277-283 ◽  
Author(s):  
Jiro Hirota ◽  
Yoshihiro Shimoji ◽  
Shinya Shimizu
Keyword(s):  
Monoclonal Antibody ◽  
West Nile Virus ◽  
Neutralization Test ◽  
Nonstructural Protein ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
The Other ◽  
Enzyme Linked Immunosorbent Assay ◽  
West Nile ◽  
Nonstructural Protein 1

ABSTRACTAn anti-West Nile virus (anti-WNV) monoclonal antibody, SHW-7A11, was developed for competitive enzyme-linked immunosorbent assays (c-ELISAs). SHW-7A11 reacted with nonstructural protein 1 in Western blot analysis. SHW-7A11 was relatively specific for the WNV strain NY99 and recognized Kunjin and Eg101 strains in indirect ELISAs. Two c-ELISAs were developed for sera diluted 10 and 100 times and named c-ELISA10 and c-ELISA100, respectively. Both c-ELISAs detected antibodies against WNV NY99 and Kunjin strains. Little cross-reactivity was observed for antibodies against Japanese encephalitis virus and St. Louis encephalitis virus in these assays. Using the cutoff point for the St. Louis encephalitis virus, all WNV-infected chickens were found to be positive on day 21 after infection in both c-ELISAs. On the other hand, all infected chickens were found to be positive on day 35 after infection in a virus neutralization test. Our newly developed SHW-7A11-based c-ELISA can detect WNV infection with sera diluted 10 to 100 times. Therefore, this c-ELISA can be used for WNV serosurveillance of chickens and wild birds.

Identification of anti-premembrane antibody as a serocomplex-specific marker to discriminate Zika, dengue and West Nile virus infections

2021 ◽  
Author(s):  
Szu-Chia Hsieh ◽  
Wen-Yang Tsai ◽  
Jih-Jin Tsai ◽  
Mars Stone ◽  
Graham Simmons ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Nonstructural Protein ◽  
Denv Infection ◽  
Antibody Responses ◽  
Severe Dengue ◽  
Specific Marker ◽  
Virus Infections ◽  
West Nile ◽  
Serological Tests ◽  
Nonstructural Protein 1

Although transmission of Zika virus (ZIKV) in the Americas has greatly declined since late 2017, recent reports of reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection underscore a critical need for serological tests that can discriminate past ZIKV, DENV and/or other flavivirus infections and improve our understanding of the immune interactions between these viruses and vaccine strategy in endemic regions. As serological tests for ZIKV primarily focus on envelope (E) and nonstructural protein 1 (NS1), antibodies to other ZIKV proteins have not been explored. Here we employed Western blot analysis using antigens of 6 flaviviruses from 3 serocomplexes to investigate antibody responses following reverse-transcription-polymerase-chain reaction-confirmed ZIKV infection. Panels of 20 primary ZIKV and 20 ZIKV with previous DENV infection recognized E proteins of all 6 flaviviruses and NS1 protein of ZIKV with some cross-reactivity to DENV. While the primary ZIKV panel recognized only the premembrane (prM) protein of ZIKV, the ZIKV with previous DENV panel recognized both ZIKV and DENV prM proteins. Analysis of antibody responses following 42 DENV and 18 West Nile virus infections revealed similar patterns of recognition by anti-E and anti-NS1 antibodies, whereas both panels recognized prM protein of homologous serocomplex but not others. The specificity was further supported by analysis of sequential samples. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be used to delineate current and past flavivirus infections in endemic areas. IMPORTANCE Despite a decline in Zika virus (ZIKV) transmission since late 2017, questions regarding its surveillance, potential re-emergence, and interactions with other flaviviruses in endemic regions remain unanswered. Recent studies have reported reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection, highlighting a need for better serological tests to discriminate past ZIKV, DENV and/or other flavivirus infections and improved understanding of the immune interactions and vaccine strategy for these viruses. As most serological tests for ZIKV focused on envelope and nonstructural protein 1, antibodies to other ZIKV proteins including potentially specific antibodies remain understudied. We employed Western blot analysis using antigens of 6 flaviviruses to study antibody responses following well-documented ZIKV, DENV and West Nile virus infections and identified anti-premembrane antibody as a flavivirus serocomplex-specific marker to delineate current and past flavivirus infections in endemic areas.

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