Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth.

Vrati, Sudhanshu; Agarwal, Vinita; Malik, Poonam; Wani, S.A.; Saini, Manisha

doi:10.1099/0022-1317-80-7-1665

Cited by 61 publications

(40 citation statements)

References 22 publications

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“…Significantly, the RGG box of the RNA binding protein hnRNP U 44 also displayed a random-coil secondary structure in this region. Hence in addition to envelope playing an important role in the slow release of RNA into the cytoplasm following viral entry for GP78 as hypothesized, 46 the capsid may also contribute to this defect by binding strongly to the viral RNA.…”

Section: Resultsmentioning

confidence: 99%

Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent.

Uchil¹,

Satchidanandam²

2001

The American Journal of Tropical Medicine and Hygiene

150

108

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Abstract. We report the analysis of the complete nucleotide sequence for the Indian isolate (P20778; Genbank Accession number AF080251) of Japanese encephalitis virus (JEV). The phylogenetic tree topology obtained using thirteen complete genome sequences of JEV was reproduced with the envelope, NS1, NS3, and NS5 genes and revealed extensive divergence between the two Indian strains included. A more exhaustive analysis of JEV evolution using 107 envelope sequences available for isolates from different geographic locations worldwide revealed five distinct genotypes of JEV, displaying a minimum nucleotide divergence of 7% with high bootstrap support values. The tree also revealed overall clustering of strains based on geographic location, as well as multiple introductions of JEV into the Indian subcontinent. Nonsynonymous nucleotide divergence rates of the envelope gene estimated that the ancestor common to all JEV genotypes arose within the last three hundred years.

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Section: Resultsmentioning

confidence: 99%

Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent.

Uchil¹,

Satchidanandam²

2001

The American Journal of Tropical Medicine and Hygiene

150

108

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“…After 24 h, the cells were lysed in TRIzol reagent, and JEV RNA levels were determined by qRT-PCR. Virus titers in the supernatant of infected cells were estimated by plaque assays (73).…”

Section: Methodsmentioning

confidence: 99%

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

Nain

Mukherjee

Karmakar

et al. 2017

J Virol

123

126

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Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

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“…The culture supernatant was harvested when 75% of the cells showed a cytopathic effect, usually 36 to 48 h after infection, and was clarified by centrifugation at 1,000 ϫ g for 30 min at 4°C. Virus titers were determined using monolayers of PS cells as described earlier (37). Virus was further purified over a 20% sucrose cushion in a Beckman Coulter ultracentrifuge (Optima L-100K) at 80,000 ϫ g for 4 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism

Kalia

Khasa

Sharma

et al. 2013

J Virol

121

104

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Japanese encephalitis virus (JEV) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes.

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Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth.

Cited by 61 publications

References 22 publications

Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent.

Phylogenetic analysis of Japanese encephalitis virus: envelope gene based analysis reveals a fifth genotype, geographic clustering, and multiple introductions of the virus into the Indian subcontinent.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

Japanese Encephalitis Virus Infects Neuronal Cells through a Clathrin-Independent Endocytic Mechanism

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