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

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

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