A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1' formation and contributes to attenuation

J Gen Virol. 2012 Sep;93(Pt 9):1959-1964. doi: 10.1099/vir.0.043844-0. Epub 2012 Jun 27.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Encephalitis Virus, Japanese / chemistry
  • Encephalitis Virus, Japanese / genetics*
  • Encephalitis Virus, Japanese / metabolism
  • Encephalitis Virus, Japanese / pathogenicity
  • Encephalitis, Japanese / virology*
  • Humans
  • Japanese Encephalitis Vaccines / chemistry
  • Japanese Encephalitis Vaccines / genetics
  • Japanese Encephalitis Vaccines / metabolism
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Point Mutation*
  • Sequence Alignment
  • Vaccines, Attenuated / chemistry
  • Vaccines, Attenuated / genetics
  • Vaccines, Attenuated / metabolism
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virulence

Substances

  • Japanese Encephalitis Vaccines
  • NS1 protein, Flavivirus
  • Vaccines, Attenuated
  • Viral Nonstructural Proteins