Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A Li, Xiao-Dan and Li, Xiao-Feng and Ye, Han-Qing and Deng, Cheng-Lin and Ye, Qing and Shan, Chao and Shang, Bao-Di and Xu, Lin-Lin and Li, Shi-Hua and Cao, Sheng-Bo and Yuan, Zhi-Ming and Shi, Pei-Yong and Qin, Cheng-Feng and Zhang, Bo,, 95, 806-815 (2014), doi = https://doi.org/10.1099/vir.0.061838-0, publicationName = Microbiology Society, issn = 0022-1317, 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., language=, type=