scholarly journals Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

2003 ◽  
Vol 77 (11) ◽  
pp. 6450-6465 ◽  
Author(s):  
Sang-Im Yun ◽  
Seok-Yong Kim ◽  
Charles M. Rice ◽  
Young-Min Lee
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Reverse Genetics ◽  
Heterologous Gene Expression ◽  
High Titer ◽  
Single Point ◽  
Encephalitis Virus ◽  
Full Length ◽  
Plaque Morphology ◽  
Single Point Mutation

ABSTRACT Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5′ and 3′ ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>106 PFU/μg), and these cells rapidly generated a high titer of synthetic viruses (>5 × 106 PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

scholarly journals Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone

2005 ◽  
Vol 86 (8) ◽  
pp. 2209-2220 ◽  
Author(s):  
Zijiang Zhao ◽  
Tomoko Date ◽  
Yuhua Li ◽  
Takanobu Kato ◽  
Michiko Miyamoto ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Vero Cells ◽  
Encephalitis Virus ◽  
Cell Interactions ◽  
Full Length ◽  
Plaque Morphology ◽  
Large Plaque ◽  
Rna Transfection ◽  
Infected Cells

A stable plasmid DNA, pMWJEAT, was constructed by using full-length Japanese encephalitis virus (JEV) cDNA isolated from the wild-type strain JEV AT31. Recombinant JEV was obtained by synthetic RNA transfection into Vero cells and designated rAT virus. JEV rAT exhibited similar large-plaque morphology and antigenicity to the parental AT31 strain. Mutant clone pMWJEAT-E138K, containing a single Glu-to-Lys mutation at aa 138 of the envelope (E) protein, was also constructed to analyse the mechanisms of viral attenuation arising from this mutation. Recombinant JEV rAT-E138K was also recovered and displayed a smaller-plaque morphology and lower neurovirulence and neuroinvasiveness than either AT31 virus or rAT virus. JEV rAT-E138K exhibited greater plaque formation than rAT virus in virus–cell interactions under acidic conditions. Heparin or heparinase III treatment inhibited binding to Vero cells more efficiently for JEV rAT-E138K than for rAT virus. Inhibition of virus–cell interactions by using wheatgerm agglutinin was more effective for JEV rAT than for rAT-E138K on Vero cells. About 20 % of macropinoendocytosis of JEV rAT for Vero cells was inhibited by cytochalasin D treatment, but no such inhibition occurred for rAT-E138K virus. Furthermore, JEV rAT was predominantly secreted from infected cells, whereas rAT-E138K was more likely to be retained in infected cells. This study demonstrates clearly that a single Glu-to-Lys mutation at aa 138 of the envelope protein affects multiple steps of the viral life cycle. These multiple changes may induce substantial attenuation of JEV.

scholarly journals Development of a reverse genetics system for Japanese encephalitis virus strain SA14-14-2

Virus Genes ◽  
2019 ◽  
Vol 55 (4) ◽  
pp. 550-556
Author(s):  
Guohua Li ◽  
Hongli Jin ◽  
Xin Nie ◽  
Yongkun Zhao ◽  
Na Feng ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Virus Strain ◽  
Reverse Genetics ◽  
Encephalitis Virus

scholarly journals A Single N-Linked Glycosylation Site in the Japanese Encephalitis Virus prM Protein Is Critical for Cell Type-Specific prM Protein Biogenesis, Virus Particle Release, and Pathogenicity in Mice

2008 ◽  
Vol 82 (16) ◽  
pp. 7846-7862 ◽  
Author(s):  
Jeong-Min Kim ◽  
Sang-Im Yun ◽  
Byung-Hak Song ◽  
Youn-Soo Hahn ◽  
Chan-Hee Lee ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Glycosylation Site ◽  
Encephalitis Virus ◽  
Plaque Morphology ◽  
Virus Release ◽  
Cell Type ◽  
Human Neuroblastoma ◽  
Virus Growth ◽  
Prm Protein

ABSTRACT The prM protein of Japanese encephalitis virus (JEV) contains a single potential N-linked glycosylation site, N15-X16-T17, which is highly conserved among JEV strains and closely related flaviviruses. To investigate the role of this site in JEV replication and pathogenesis, we manipulated the RNA genome by using infectious JEV cDNA to generate three prM mutants (N15A, T17A, and N15A/T17A) with alanine substiting for N15 and/or T17 and one mutant with silent point mutations introduced into the nucleotide sequences corresponding to all three residues in the glycosylation site. An analysis of these mutants in the presence or absence of endoglycosidases confirmed the addition of oligosaccharides to this potential glycosylation site. The loss of prM N glycosylation, without significantly altering the intracellular levels of viral RNA and proteins, led to an ≈20-fold reduction in the production of extracellular virions, which had protein compositions and infectivities nearly identical to those of wild-type virions; this reduction occurred at the stage of virus release, rather than assembly. This release defect was correlated with small-plaque morphology and an N-glycosylation-dependent delay in viral growth. A more conservative mutation, N15Q, had the same effect as N15A. One of the four prM mutants, N15A/T17A, showed an additional defect in virus growth in mosquito C6/36 cells but not human neuroblastoma SH-SY5Y or hamster BHK-21 cells. This cell type dependence was attributed to abnormal N-glycosylation-independent biogenesis of prM. In mice, the elimination of prM N glycosylation resulted in a drastic decrease in virulence after peripheral inoculation. Overall, our findings indicate that this highly conserved N-glycosylation motif in prM is crucial for multiple stages of JEV biology: prM biogenesis, virus release, and pathogenesis.

scholarly journals Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

2014 ◽  
Vol 95 (4) ◽  
pp. 806-815 ◽  
Author(s):  
Xiao-Dan Li ◽  
Xiao-Feng Li ◽  
Han-Qing Ye ◽  
Cheng-Lin Deng ◽  
Qing Ye ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Cdna Clone ◽  
Infectious Clone ◽  
Encephalitis Virus ◽  
Full Length ◽  
Large Plaque ◽  
Adaptive Mutations ◽  
A Genome

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

scholarly journals Molecular characterization of the first Australian isolate of Japanese encephalitis virus, the FU strain

2000 ◽  
Vol 81 (10) ◽  
pp. 2471-2480 ◽  
Author(s):  
David T. Williams ◽  
Lin-Fa Wang ◽  
Peter W. Daniels ◽  
John S. Mackenzie
Keyword(s):  
Amino Acid ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Encephalitis Virus ◽  
Full Length ◽  
Gene Analysis ◽  
Australian Isolate ◽  
Korean Isolate ◽  
Gene Sequences ◽  
E Gene

The complete genomic and predicted amino acid sequence of the Japanese encephalitis virus (JEV) FU strain, a human isolate recovered from the first outbreak of Japanese encephalitis in Australian territory, was determined. Comparison of the FU genome with 15 fully sequenced JEV genomes revealed high levels of sequence identity, ranging from 88·7% (GP78) to 89·7% (K94P05) for nucleotides and 96·8% (K94P05) to 98·0% (JaGAr01) for amino acid sequences. A total of 39 unique amino acid differences were found in the FU strain polyprotein. Phylogenetic analyses were performed on all available full-length JEV genomes and a selection of 64 E gene sequences from temporally and geographically diverse JEV strains. For comparison with the E gene phylogeny, phylogenetic analysis using cognate prM gene sequences was also carried out. The FU strain was found to be most closely related to Korean isolate K94P05 in the full-length analysis and to Southeast Asian strains in the E and prM gene analyses. The E gene analysis corresponded well with the prM gene analysis and with previous genotyping studies using the prM gene. The epidemiological implications of this investigation are discussed.

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