scholarly journals Sindbis Vectors Suppress Secretion of Subviral Particles of Japanese Encephalitis Virus from Mammalian Cells Infected with SIN-JEV Recombinants

Virology ◽  
1995 ◽  
Vol 209 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Konstantin V. Pugachev ◽  
Peter W. Mason ◽  
Teryl K. Frey
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Encephalitis Virus ◽  
Subviral Particles

scholarly journals Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3448-3458 ◽  
Author(s):  
Yoshio Mori ◽  
Tamaki Okabayashi ◽  
Tetsuo Yamashita ◽  
Zijiang Zhao ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Cell Lines ◽  
Japanese Encephalitis ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Core Protein ◽  
Encephalitis Virus ◽  
Wild Type ◽  
The Core

ABSTRACT Japanese encephalitis virus (JEV) core protein was detected in both the nucleoli and cytoplasm of mammalian and insect cell lines infected with JEV or transfected with the expression plasmid of the core protein. Mutation analysis revealed that Gly42 and Pro43 in the core protein are essential for the nuclear and nucleolar localization. A mutant M4243 virus in which both Gly42 and Pro43 were replaced by Ala was recovered by plasmid-based reverse genetics. In C6/36 mosquito cells, the M4243 virus exhibited RNA replication and protein synthesis comparable to wild-type JEV, whereas propagation in Vero cells was impaired. The mutant core protein was detected in the cytoplasm but not in the nucleus of either C6/36 or Vero cell lines infected with the M4243 virus. The impaired propagation of M4243 in mammalian cells was recovered by the expression of wild-type core protein in trans but not by that of the mutant core protein. Although M4243 mutant virus exhibited a high level of neurovirulence comparable to wild-type JEV in spite of the approximately 100-fold-lower viral propagation after intracerebral inoculation to 3-week-old mice of strain Jcl:ICR, no virus was recovered from the brain after intraperitoneal inoculation of the mutant. These results indicate that nuclear localization of JEV core protein plays crucial roles not only in the replication in mammalian cells in vitro but also in the pathogenesis of encephalitis induced by JEV in vivo.

scholarly journals A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

2015 ◽  
Vol 90 (5) ◽  
pp. 2676-2689 ◽  
Author(s):  
Mélissanne de Wispelaere ◽  
Cécile Khou ◽  
Marie-Pascale Frenkiel ◽  
Philippe Desprès ◽  
Nathalie Pardigon
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Encephalitis Virus ◽  
Mutant Virus ◽  
M Protein ◽  
Single Amino Acid ◽  
Progeny Virus

ABSTRACTJapanese encephalitis virus (JEV) membrane (M) protein plays important structural roles in the processes of fusion and maturation of progeny virus during cellular infection. The M protein is anchored in the viral membrane, and its ectodomain is composed of a flexible N-terminal loop and a perimembrane helix. In this study, we performed site-directed mutagenesis on residue 36 of JEV M protein and showed that the resulting mutation had little or no effect on the entry process but greatly affected virus assembly in mammalian cells. Interestingly, this mutant virus had a host-dependent phenotype and could develop a wild-type infection in insect cells. Experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the JEV mutant expresses structural proteins but fails to form infectious particles in mammalian cells. Using a mouse model for JEV pathogenesis, we showed that the mutation conferred complete attenuationin vivo. The production of JEV neutralizing antibodies in challenged mice was indicative of the immunogenicity of the mutant virusin vivo. Together, our results indicate that the introduction of a single mutation in the M protein, while being tolerated in insect cells, strongly impacts JEV infection in mammalian hosts.IMPORTANCEJEV is a mosquito-transmitted flavivirus and is a medically important pathogen in Asia. The M protein is thought to be important for accommodating the structural rearrangements undergone by the virion during viral assembly and may play additional roles in the JEV infectious cycle. In the present study, we show that a sole mutation in the M protein impairs the JEV infection cycle in mammalian hosts but not in mosquito cells. This finding highlights differences in flavivirus assembly pathways among hosts. Moreover, infection of mice indicated that the mutant was completely attenuated and triggered a strong immune response to JEV, thus providing new insights for further development of JEV vaccines.

scholarly journals Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

2014 ◽  
Vol 95 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Sankar Bhattacharyya ◽  
Utsav Sen ◽  
Sudhanshu Vrati
Keyword(s):  
Japanese Encephalitis Virus ◽  
Unfolded Protein Response ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Rnase Activity ◽  
Unfolded Protein ◽  
Protein Response

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

scholarly journals Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced by Spodoptera frugiperda Cells for Use as Vaccine and Diagnostic Antigens

2010 ◽  
Vol 17 (10) ◽  
pp. 1560-1566 ◽  
Author(s):  
Miwa Kuwahara ◽  
Eiji Konishi
Keyword(s):  
Dengue Virus ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Spodoptera Frugiperda ◽  
Mammalian Cells ◽  
Encephalitis Virus ◽  
Commercial Application ◽  
Sf9 Cells ◽  
High Level ◽  
Level Production

ABSTRACT New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda-derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.

scholarly journals Cell Type-Dependent RNA Recombination Frequency in the Japanese Encephalitis Virus

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Wei-Wei Chiang ◽  
Ching-Kai Chuang ◽  
Mei Chao ◽  
Wei-June Chen
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Viral Evolution ◽  
Cell Types ◽  
Encephalitis Virus ◽  
Viral Rna ◽  
Rna Recombination ◽  
Mosquito Cells ◽  
Rna Fragments

Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

scholarly journals NS1′ protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs

2014 ◽  
Vol 95 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Yuki Takamatsu ◽  
Kenta Okamoto ◽  
Duc Tuan Dinh ◽  
Fuxun Yu ◽  
Daisuke Hayasaka ◽  
...  
Keyword(s):  
Protein Expression ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Encephalitis Virus ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Chicken Eggs ◽  
Embryonated Chicken

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1′ protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a −1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1′ protein through the production of JEV NS1′-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1′ protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1′ protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1′ enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

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