title: Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A.
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.
authors.affiliation: University of Chinese Academy of Sciences, Beijing 100049, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Xiao-Dan
authors.initials: XD
authors.lastname: Li
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Li
authors.affiliation: Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Han-Qing
authors.initials: HQ
authors.lastname: Ye
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Cheng-Lin
authors.initials: CL
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.affiliation: University of Chinese Academy of Sciences, Beijing 100049, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Chao
authors.initials: C
authors.lastname: Shan
authors.affiliation: University of Chinese Academy of Sciences, Beijing 100049, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Bao-Di
authors.initials: BD
authors.lastname: Shang
authors.affiliation: University of Chinese Academy of Sciences, Beijing 100049, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Shi-Hua
authors.initials: SH
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China.
authors.forename: Sheng-Bo
authors.initials: SB
authors.lastname: Cao
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Yuan
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
authors.forename: Cheng-Feng
authors.initials: CF
authors.lastname: Qin
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.|Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000220:Adaptation, Biological; D000818:Animals; D001921:Brain / Q000821:virology; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000502:physiology*; D004672:Encephalitis, Japanese / Q000473:pathology / Q000821:virology; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D059386:Reverse Genetics; D012692:Serial Passage; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014774:Virulence; D014779:Virus Replication
chemical_list: D050505:Mutant Proteins; C080102:NS2B protein, flavivirus; C080106:NS4A protein, flavivirus; D017361:Viral Nonstructural Proteins