A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation.
Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Qing
Q
Ye
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Xiao-Feng
XF
Li
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Hui
H
Zhao
Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Shi-Hua
SH
Li
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Yong-Qiang
YQ
Deng
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Rui-Yuan
RY
Cao
School of Medicine Jinan University, Guangzhou, 510632, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Ke-Yu
KY
Song
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Hong-Jiang
HJ
Wang
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, PR China.
Rong-Hong
RH
Hua
National Institutes for Food and Drug Control, Beijing 100050, PR China.
Yong-Xin
YX
Yu
State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China.
Xi
X
Zhou
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
E-De
ED
Qin
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.|Department of Virology, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, PR China.
Cheng-Feng
CF
Qin
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004664:Encephalitis Virus, Japanese / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D004672:Encephalitis, Japanese / Q000821:virology*; D006801:Humans; D022321:Japanese Encephalitis Vaccines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017354:Point Mutation; D016415:Sequence Alignment; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
D022321:Japanese Encephalitis Vaccines; C100315:NS1 protein, Flavivirus; D014613:Vaccines, Attenuated; D017361:Viral Nonstructural Proteins