RT Journal Article SR Electronic(1) A1 Ye, Qing A1 Li, Xiao-Feng A1 Zhao, Hui A1 Li, Shi-Hua A1 Deng, Yong-Qiang A1 Cao, Rui-Yuan A1 Song, Ke-Yu A1 Wang, Hong-Jiang A1 Hua, Rong-Hong A1 Yu, Yong-Xin A1 Zhou, Xi A1 Qin, E-De A1 Qin, Cheng-FengYR 2012 T1 A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1’ formation and contributes to attenuation JF Journal of General Virology, VO 93 IS 9 SP 1959 OP 1964 DO https://doi.org/10.1099/vir.0.043844-0 PB Microbiology Society, SN 1465-2099, AB 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., UL https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.043844-0