scholarly journals Persistence of Japanese Encephalitis Virus Is Associated with Abnormal Expression of the Nonstructural Protein NS1 in Host Cells

Virology ◽  
1996 ◽  
Vol 217 (1) ◽  
pp. 220-229 ◽  
Author(s):  
LI-KUANG CHEN ◽  
CHING-LEN LIAO ◽  
CHING-GONG LIN ◽  
SZU-CHIA LAI ◽  
CHIU-I LIU ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Abnormal Expression

scholarly journals TIM-1 Promotes Japanese Encephalitis Virus Entry and Infection

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 630 ◽  
Author(s):  
Jichen Niu ◽  
Ya Jiang ◽  
Hao Xu ◽  
Changjing Zhao ◽  
Guodong Zhou ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Point Mutation ◽  
Japanese Encephalitis ◽  
Virus Entry ◽  
A549 Cells ◽  
Binding Pocket ◽  
Cytoplasmic Domain ◽  
Encephalitis Virus ◽  
Host Cells ◽  
Attachment Factor

Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus, the leading cause of viral-induced encephalitis. Several host molecules have been identified as the JEV attachment factor; however, the molecules involved in JEV entry remain poorly understood. In the present study, we demonstrate that TIM-1 is important for efficient infection by JEV. Firstly, three TIM-1 variants (V1, V2, and V3) were cloned from A549 cells, and we revealed that only ectopically TIM-1 V2 expression in 293T cells significantly promotes JEV attachment, entry and infection. Point mutation of phosphatidylserine (Ptdser) binding pocket in the TIM-1 IgV domain dampened JEV entry, indicating that TIM-1-mediated JEV infection is Ptdser-dependent. Furthermore, we found the cytoplasmic domain of TIM-1 is also required for enhancing JEV entry. Additionally, knock down of TIM-1 expression in A549 cells impaired JEV entry and infection, but not attachment, suggesting that additional factors exist in A549 cells that allow the virus to bind. In conclusion, our findings demonstrate that TIM-1 promotes JEV infection as an entry cofactor, and the polymorphism of TIM-1 is associated with JEV susceptibility to host cells.

scholarly journals Review of Emerging Japanese Encephalitis Virus: New Aspects and Concepts about Entry into the Brain and Inter-Cellular Spreading

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 111 ◽  
Author(s):  
Filgueira ◽  
Lannes
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Human Case ◽  
Encephalitis Virus ◽  
The Body ◽  
Host Cells ◽  
Asia Pacific ◽  
Latently Infected ◽  
The Impact ◽  
The Brain

Japanese encephalitis virus (JEV) is an emerging flavivirus of the Asia-Pacific region. More than two billion people live in endemic or epidemic areas and are at risk of infection. Recently, the first autochthonous human case was recorded in Africa, and infected birds have been found in Europe. JEV may spread even further to other continents. The first section of this review covers established and new information about the epidemiology of JEV. The subsequent sections focus on the impact of JEV on humans, including the natural course and immunity. Furthermore, new concepts are discussed about JEV’s entry into the brain. Finally, interactions of JEV and host cells are covered, as well as how JEV may spread in the body through latently infected immune cells and cell-to-cell transmission of virions or via other infectious material, including JEV genomic RNA.

scholarly journals NS1′ of Flaviviruses in the Japanese Encephalitis Virus Serogroup Is a Product of Ribosomal Frameshifting and Plays a Role in Viral Neuroinvasiveness

2009 ◽  
Vol 84 (3) ◽  
pp. 1641-1647 ◽  
Author(s):  
Ezequiel Balmori Melian ◽  
Edward Hinzman ◽  
Tomoko Nagasaki ◽  
Andrew E. Firth ◽  
Norma M. Wills ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Innate Immune ◽  
Site Directed Mutagenesis ◽  
Sequence Elements ◽  
Rna Pseudoknot ◽  
Frameshift Event

ABSTRACT Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1′, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1′ is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1′ is the product of a −1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1′ plays a role in viral neuroinvasiveness.

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 Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

2007 ◽  
Vol 15 (1) ◽  
pp. 88-94 ◽  
Author(s):  
Eiji Konishi ◽  
Yoko Kitai ◽  
Takashi Kondo
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
Test Method ◽  
Assay Method ◽  
Specific Antibodies ◽  
Nonstructural Protein 1 ◽  
Complement Dependent Cytotoxicity ◽  
Low Levels

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

scholarly journals Rab5 and Rab11 Are Required for Clathrin-Dependent Endocytosis of Japanese Encephalitis Virus in BHK-21 Cells

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Chun-Chun Liu ◽  
Yun-Na Zhang ◽  
Zhao-Yao Li ◽  
Jin-Xiu Hou ◽  
Jing Zhou ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Drug Targets ◽  
Encephalitis Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Rab Proteins ◽  
Endosomal Trafficking ◽  
Rab Gtpases

ABSTRACT During infection Japanese encephalitis virus (JEV) generally enters host cells via receptor-mediated clathrin-dependent endocytosis. The trafficking of JEV within endosomes is controlled by Rab GTPases, but which Rab proteins are involved in JEV entry into BHK-21 cells is unknown. In this study, entry and postinternalization of JEV were analyzed using biochemical inhibitors, RNA interference, and dominant negative (DN) mutants. Our data demonstrate that JEV entry into BHK-21 cells depends on clathrin, dynamin, and cholesterol but not on caveolae or macropinocytosis. The effect on JEV infection of dominant negative (DN) mutants of four Rab proteins that regulate endosomal trafficking was examined. Expression of DN Rab5 and DN Rab11, but not DN Rab7 and DN Rab9, significantly inhibited JEV replication. These results were further tested by silencing Rab5 or Rab11 expression before viral infection. Confocal microscopy showed that virus particles colocalized with Rab5 or Rab11 within 15 min after virus entry, suggesting that after internalization JEV moves to early and recycling endosomes before the release of the viral genome. Our findings demonstrate the roles of Rab5 and Rab11 on JEV infection of BHK-21 cells through the endocytic pathway, providing new insights into the life cycle of flaviviruses. IMPORTANCE Although Japanese encephalitis virus (JEV) utilizes different endocytic pathways depending on the cell type being infected, the detailed mechanism of its entry into BHK-21 cells is unknown. Understanding the process of JEV endocytosis and postinternalization will advance our knowledge of JEV infection and pathogenesis as well as provide potential novel drug targets for antiviral intervention. With this objective, we used systematic approaches to dissect this process. The results show that entry of JEV into BHK-21 cells requires a low-pH environment and that the process occurs through dynamin-, actin-, and cholesterol-dependent clathrin-mediated endocytosis that requires Rab5 and Rab11. Our work provides a detailed picture of the entry of JEV into BHK-21 cells and the cellular events that follow.

scholarly journals The C Terminus of the Core β-Ladder Domain in Japanese Encephalitis Virus Nonstructural Protein 1 Is Flexible for Accommodation of Heterologous Epitope Fusion

2015 ◽  
Vol 90 (3) ◽  
pp. 1178-1189 ◽  
Author(s):  
Li-Chen Yen ◽  
Jia-Teh Liao ◽  
Hwei-Jen Lee ◽  
Wei-Yuan Chou ◽  
Chun-Wei Chen ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Virus Replication ◽  
Japanese Encephalitis ◽  
Protective Immunity ◽  
Nonstructural Protein ◽  
Encephalitis Virus ◽  
The Core ◽  
Nonstructural Protein 1 ◽  
C Terminus ◽  
Infected Cells

ABSTRACTNS1 is the only nonstructural protein that enters the lumen of the endoplasmic reticulum (ER), where NS1 is glycosylated, forms a dimer, and is subsequently secreted during flavivirus replication as dimers or hexamers, which appear to be highly immunogenic to the infected host, as protective immunity can be elicited against homologous flavivirus infections. Here, by using atrans-complementation assay, we identified the C-terminal end of NS1 derived from Japanese encephalitis virus (JEV), which was more flexible than other regions in terms of housing foreign epitopes without a significant impact on virus replication. This mapped flexible region is located in the conserved tip of the core β-ladder domain of the multimeric NS1 structure and is also known to contain certain linear epitopes, readily triggering specific antibody responses from the host. Despite becoming attenuated, recombinant JEV with insertion of a neutralizing epitope derived from enterovirus 71 (EV71) into the C-terminal end of NS1 not only could be normally released from infected cells, but also induced dual protective immunity for the host to counteract lethal challenge with either JEV or EV71 in neonatal mice. These results indicated that the secreted multimeric NS1 of flaviviruses may serve as a natural protein carrier to render epitopes of interest more immunogenic in the C terminus of the core β-ladder domain.IMPORTANCEThe positive-sense RNA genomes of mosquito-borne flaviviruses appear to be flexible in terms of accommodating extra insertions of short heterologous antigens into their virus genes. Here, we illustrate that the newly identified C terminus of the core β-ladder domain in NS1 could be readily inserted into entities such as EV71 epitopes, and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability, and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

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.

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