Significance and preM Sequence Analysis of Different Mutant Japanese Encephalitis Virus Strains in Persis- tently Infected KN73 Cells

The significance and preM sequence analysis of two different mutant strains of Japanese encephalitis virus during persistent infection was studied. Two wild strains of Japanese encephalitis viruses (JaGAr-01 and Nakayama) were used to infect a human hepatoma cell line. Persistent infection was established after the cells were subcultured several times. Mutant viruses in persistently infected cells were collected by freezing and thawing of the cells. Viral titers were examined by plaque assys in BHK cells. The preM coding region of four Japanese encephalitis strains, two wild-type and two mutant viruses from infected cells were amplified by RT-PCR. The PCR products were sequenced and the preM sequences of the four strains were compared. The results showed that there was a single aminot acid mutation (E9 Leu→Arg) in the JaGAr-01 persistently infecting mutant in comparison to wild-type JaGAr-01. Two amino acid replacements (E9 Leu→Arg and E13 Val→Ile) were noted in the persistently infecting Nakayama and its wild-type strains. The amino acid sequences of the mutant strains JaGAr-01 and Nakayama were completely the same. We inferred here that genotypic variation existed in preM region of all mutant viruses and that genotypic variation of protein encoded by preM region may play a role in persistent infections and may contribute to the maintenance of the characters of Japanese encephalitis virus and replication.