Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro

Virology. 1996 Sep 1;223(1):79-88. doi: 10.1006/viro.1996.0457.


Using gamma-ray irradiation, a pair of virulent (RP-9) and attenuated (RP-2ms) variants of Japanese encephalitis virus (JEV) were generated from a Taiwanese isolate, NT109. The two variants differed in plaque morphology, virus adsorption, and growth properties in BHK-21 cells: (i) RP-2ms produced smaller plaques than RP-9; (ii) RP-2ms adsorbed less efficiently to host cells but yielded a higher virus titer (burst size); and (iii) RP-2ms virions were mostly accumulated intracellularly, whereas RP-9 was released extracellularly. In addition, in an in vitro binding assay, the envelope (E) protein of RP-9, but not that of RP-2ms, bound specifically to a cellular protein of 57-kDa derived from BHK-21 cells. When injected into mice intracerebrally, RP-2ms was much less virulent than RP-9, with 50% lethal doses of > 10(7) and 0.4 plaque forming units, respectively. Moreover, when inoculated intraperitoneally, their organ tropism differed in that the main target organ for RP-2ms was liver, whereas that for RP-9 was brain. These results suggest that RP-2ms was less neurovirulent and less neuroinvasive from peripheral routes. Molecular analysis of the virus structural proteins detected only two differences between RP-9 and RP-2ms: one in E protein, Glu-138 in RP-9 and Lys-138 in RP-2ms, and the other in prM, Tyr-43 in RP-9 and His-43 in RP-2ms. Since the N-terminal 92 amino acids of prM are cleaved and not present in mature JEV virions, the single-amino-acid change of the E protein at position 138 may account for the difference between the mutants in the in vitro binding assay. Such mutation in E protein, or perhaps in conjunction with the prM mutation, may be responsible, in part, for the phenotypic differences observed in vitro and in vivo between the two mutants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aedes / cytology
  • Animals
  • Cell Line
  • Cricetinae
  • Culex / virology
  • Encephalitis Virus, Japanese / genetics
  • Encephalitis Virus, Japanese / pathogenicity
  • Encephalitis Virus, Japanese / physiology*
  • Encephalitis, Japanese / virology
  • Female
  • Gamma Rays
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred ICR
  • Mutation
  • Organ Specificity
  • Peritoneum
  • Viral Envelope Proteins / chemistry
  • Viral Envelope Proteins / genetics
  • Viral Plaque Assay
  • Virulence / genetics
  • Virus Replication


  • Membrane Glycoproteins
  • Viral Envelope Proteins
  • glycoprotein E, Japanese encephalitis virus
  • prM protein, Flavivirus