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

Xiao Dan Li, Xiao Feng Li, Han Qing Ye, Cheng Lin Deng, Qing Ye, Chao Shan, Bao Di Shang, Lin Lin Xu, Shi Hua Li, Sheng Bo Cao, Zhi Ming Yuan, Pei Yong Shi, Cheng Feng Qin, Bo Zhang

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)806-815
Number of pages10
JournalJournal of General Virology
Volume95
Issue numberPART 4
DOIs
StatePublished - 2014

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ASJC Scopus subject areas

  • Virology

Cite this

Li, X. D., Li, X. F., Ye, H. Q., Deng, C. L., Ye, Q., Shan, C., Shang, B. D., Xu, L. L., Li, S. H., Cao, S. B., Yuan, Z. M., Shi, P. Y., Qin, C. F., & Zhang, B. (2014). Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A. Journal of General Virology, 95(PART 4), 806-815. https://doi.org/10.1099/vir.0.061838-0