RT Journal Article SR Electronic(1) A1 Li, Xiao-Dan A1 Li, Xiao-Feng A1 Ye, Han-Qing A1 Deng, Cheng-Lin A1 Ye, Qing A1 Shan, Chao A1 Shang, Bao-Di A1 Xu, Lin-Lin A1 Li, Shi-Hua A1 Cao, Sheng-Bo A1 Yuan, Zhi-Ming A1 Shi, Pei-Yong A1 Qin, Cheng-Feng A1 Zhang, BoYR 2014 T1 Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A JF Journal of General Virology, VO 95 IS 4 SP 806 OP 815 DO https://doi.org/10.1099/vir.0.061838-0 PB Microbiology Society, SN 1465-2099, AB 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., UL https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.061838-0