scholarly journals Conserved amino acids 193–324 of non-structural protein 3 are a dominant source of peptide determinants for CD4+ and CD8+ T cells in a healthy Japanese encephalitis virus-endemic cohort

2004 ◽  
Vol 85 (5) ◽  
pp. 1131-1143 ◽  
Author(s):  
Priti Kumar ◽  
Paramadevanapalli Sulochana ◽  
Gejjehalli Nirmala ◽  
Maganti Haridattatreya ◽  
Vijaya Satchidanandam
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cd8 T Cells ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Parent Protein ◽  
Ns3 Protein ◽  
Conserved Amino Acids

Our earlier identification of the non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) as a dominant CD4+ as well as CD8+ T cell-eliciting antigen in a healthy JEV-endemic cohort with a wide HLA distribution implied the presence of several epitopes dispersed over the length of the protein. Use of various truncated versions of NS3 in lymphocyte stimulation and interferon (IFN)-γ secretion assays revealed that amino acids (aa) 193–324 of NS3 were comparable with, if not superior to, the full-length protein in evoking Th1 responses. The potential of this 14·4 kDa stretch to stimulate IFN-γ production from both subtypes of T cells in a manner qualitatively and quantitatively similar to the 68 kDa parent protein suggested the presence within it of both class I and II epitopes and demonstrated that the entire immunogenicity of NS3 was focused on aa 193–324. Interestingly, this segment contained five of the eight helicase motifs of NS3. Analysis of variability of the NS3 protein sequence across 16 JEV isolates revealed complete identity of aa 219–318, which is contained within the above segment, suggesting that NS3-specific epitopes tend to cluster in relatively conserved regions that harbour functionally critical domains of the protein.

scholarly journals Japanese encephalitis virus–primed CD8+ T cells prevent antibody-dependent enhancement of Zika virus pathogenesis

2020 ◽  
Vol 217 (9) ◽  
Author(s):  
Dong Chen ◽  
Zhiliang Duan ◽  
Wenhua Zhou ◽  
Weiwei Zou ◽  
Shengwei Jin ◽  
...  
Keyword(s):  
T Cells ◽  
Viral Replication ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cd8 T Cells ◽  
Zika Virus ◽  
Encephalitis Virus ◽  
Protective Effects ◽  
Zikv Infection ◽  
Lethal Effects

Cross-reactive anti-flaviviral immunity can influence the outcome of infections with heterologous flaviviruses. However, it is unclear how the interplay between cross-reactive antibodies and T cells tilts the balance toward pathogenesis versus protection during secondary Zika virus (ZIKV) and Japanese encephalitis virus (JEV) infections. We show that sera and IgG from JEV-vaccinated humans and JEV-inoculated mice cross-reacted with ZIKV, exacerbated lethal ZIKV infection upon transfer to mice, and promoted viral replication and mortality upon ZIKV infection of the neonates born to immune mothers. In contrast, transfer of CD8+ T cells from JEV-exposed mice was protective, reducing the viral burden and mortality of ZIKV-infected mice and abrogating the lethal effects of antibody-mediated enhancement of ZIKV infection in mice. Conversely, cross-reactive anti-ZIKV antibodies or CD8+ T cells displayed the same pathogenic or protective effects upon JEV infection, with the exception that maternally acquired anti-ZIKV antibodies had no effect on JEV infection of the neonates. These results provide clues for developing safe anti-JEV/ZIKV vaccines.

scholarly journals Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein

2003 ◽  
Vol 84 (10) ◽  
pp. 2795-2805 ◽  
Author(s):  
Chun-Tang Chiou ◽  
Chih-Chi Andrew Hu ◽  
Pi-Hsin Chen ◽  
Ching-Len Liao ◽  
Yi-Ling Lin ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Assembly ◽  
Susceptibility Gene ◽  
Immunogold Labelling ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Ns3 Protein ◽  
Associated Proteins ◽  
Golgi Network

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B–NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting -1 PRF and the NS3 Protein

2021 ◽  
Author(s):  
Du Yu ◽  
Yundi Zhao ◽  
Junhui Pan ◽  
Xingmiao Yang ◽  
Zhenjie Liang ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Replication ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Ns3 Protein

scholarly journals ZBTB38 in Porcine Alveolar Macrophages Positively Regulates the Proliferation of Japanese Encephalitis Virus

2021 ◽  
Author(s):  
Yi Zheng ◽  
Yu-Yong Zhou ◽  
Chun-Xia Chai ◽  
San-Jie Cao ◽  
Qi-Gui Yan ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Alveolar Macrophages ◽  
Encephalitis Virus ◽  
Porcine Alveolar Macrophage ◽  
Envelope Gene ◽  
Interacting Protein ◽  
Btb Domain ◽  
New Information ◽  
Ns3 Protein

Abstract Background Japanese encephalitis (JE) is an important zoonotic disease caused by Japanese encephalitis virus (JEV), and pigs are intermediate host of this disease. Previous studies have confirmed that JEV can proliferate in the respiratory tract of mice and spread through it. Therefore, this study aimed to screen the proteins interacting with JEV on porcine alveolar macrophage cell and verify its role in the proliferation of JEV.Methods and results Porcine alveolar macrophages cell line 3D4/21 were infected with JEV, and obvious cytopathic effect (CPE) was observed. Zinc finger and BTB domain containing 38 (ZBTB38) was screened out as an interacting protein using co-immunoprecipitation assay and validated through knockout and overexpression of ZBTB38 in 3D4/21 cells. The results demonstrated that loss of ZBTB38 function basically had no effect on the attachment and entry processes of JEV, while the transcription level of JEV envelope gene, the expression level of NS3 protein and the number of virions were all significantly down-regulated in the subsequent infection stage. Conclusion Overall, one core conclusion was drawn in this paper that ZBTB38 promotes the proliferation of JEV especially in the middle and late stages of infection. This study provides new information for understanding the pathogenic mechanism of JEV, especially the respiratory transmission caused by JEV infection.

scholarly journals Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain

2004 ◽  
Vol 85 (2) ◽  
pp. 471-482 ◽  
Author(s):  
Priti Kumar ◽  
Venkatramana D. Krishna ◽  
Paramadevanapalli Sulochana ◽  
Gejjehalli Nirmala ◽  
Maganti Haridattatreya ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Structural Proteins ◽  
Healthy Children ◽  
Immunodeficiency Virus

Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4+ and CD8+ T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

scholarly journals Structure-Based Mutational Analysis of Several Sites in the E Protein: Implications for Understanding the Entry Mechanism of Japanese Encephalitis Virus

2015 ◽  
Vol 89 (10) ◽  
pp. 5668-5686 ◽  
Author(s):  
Haibin Liu ◽  
Yi Liu ◽  
Shaobo Wang ◽  
Yanjun Zhang ◽  
Xiangyang Zu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Japanese Encephalitis Virus ◽  
Membrane Fusion ◽  
Japanese Encephalitis ◽  
Receptor Binding ◽  
Viral Entry ◽  
Envelope Protein ◽  
Encephalitis Virus ◽  
Entry Pathway ◽  
Entry Mechanism

ABSTRACTJapanese encephalitis virus (JEV), which causes viral encephalitis in humans, is a serious risk to global public health. The JEV envelope protein mediates the viral entry pathway, including receptor-binding and low-pH-triggered membrane fusion. Utilizing mutagenesis of a JEV infectious cDNA clone, mutations were introduced into the potential receptor-binding motif or into residues critical for membrane fusion in the envelope protein to systematically investigate the JEV entry mechanism. We conducted experiments evaluating infectious particle, recombinant viral particle, and virus-like particle production and found that most mutations impaired virus production. Subcellular fractionation confirmed that five mutations—in I0, ij, BC, and FG and the R9A substitution—impaired virus assembly, and the assembled virus particles of another five mutations—in kl and the E373A, F407A, L221S, and W217A substitutions—were not released into the secretory pathway. Next, we examined the entry activity of six mutations yielding infectious virus. The results showed N154 and the DE loop are not the only or major receptor-binding motifs for JEV entry into BHK-21 cells; four residues, H144, H319, T410, and Q258, participating in the domain I (DI)-DIII interaction or zippering reaction are important to maintain the efficiency of viral membrane fusion. By continuous passaging of mutants, adaptive mutations from negatively charged amino acids to positively charged or neutral amino acids, such as E138K and D389G, were selected and could restore the viral entry activity.IMPORTANCERecently, there has been much interest in the entry mechanism of flaviviruses into host cells, including the viral entry pathway and membrane fusion mechanism. Our study provides strong evidence for the critical role of several residues in the envelope protein in the assembly, release, and entry of JEV, which also contributes to our understanding of the flaviviral entry mechanism. Furthermore, we demonstrate that the H144A, H319A, T410A, and Q258A mutants exhibit attenuated fusion competence, which may be used to develop novel vaccine candidates for flaviviruses.

scholarly journals Fusion PCR generated Japanese encephalitis virus/dengue 4 virus chimera exhibits lack of neuroinvasiveness, attenuated neurovirulence, and a dual-flavi immune response in mice

2004 ◽  
Vol 85 (9) ◽  
pp. 2503-2513 ◽  
Author(s):  
Edward Gitau Matumbi Mathenge ◽  
Maria del Carmen Parquet ◽  
Yasutomo Funakoshi ◽  
Seiji Houhara ◽  
Pooi Fong Wong ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Line ◽  
Japanese Encephalitis ◽  
Secondary Infection ◽  
Neuronal Cell ◽  
Protein S ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Rapid Preparation

The first flavivirus chimera encoding dengue 4 virus (D4) PrM and E structural proteins in a Japanese encephalitis virus (JEV) backbone was successfully generated using the long-PCR based cDNA-fragment stitching (LPCRcFS) technique, demonstrating the technique's applicability for rapid preparation of flavivirus chimeras. The JEV/D4 chimera multiplied at levels equal to JEV and D4 in the mosquito cell line C6/36, while in a mouse neuronal cell line (N2a) JEV replicated efficiently, but JEV/D4 and D4 did not. In mouse challenge experiments, JEV/D4 showed a lack of neuroinvasiveness similar to D4 when inoculated intraperitoneally, but demonstrated attenuated neurovirulence (LD50=3·17×104 f.f.u.) when inoculated intracranially. It was also noted that mice receiving intraperitoneal challenge with JEV/D4 possessed D4-specific neutralization antibody and in addition clearly showed resistance to JEV intraperitoneal challenge (at 100×LD50). This suggests that immunity to anti-JEV non-structural protein(s) offers protection against JEV infection in vivo. Dengue secondary infection was also simulated by challenging mice pre-immunized with dengue 2 virus, with D4 or JEV/D4. Mice showed higher secondary antibody response to challenge with JEV/D4 than to D4, at 210 000 and 37 000 averaged ELISA units, respectively. Taken together, aside from demonstrating the LPCRcFS technique, it could be concluded that the PrM and E proteins are the major determinant of neuroinvasiveness for JEV. It is also expected that the JEV/D4 chimera with its pathogenicity in mice and atypical immune profile, could have applications in dengue prophylactic research, in vivo efficacy assessment of dengue vaccines and development of animal research on models of dengue secondary infection.

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