Phenotypic and genotypic characteristics of Japanese encephalitis attenuated live vaccine virus SA14-14-2 and their stabilities.

A novel Japanese encephalitis (JE) attenuated live vaccine virus SA14-14-2 was licensed for commercial application in China in 1989. Since then this vaccine has been widely used in China and other countries in Asia, and no vaccine associated encephalitis case was reported. The neurovirulence of the SA14-14-2 was tested in JE susceptible laboratory animals, such as mice, monkeys, hamsters and athymic nude mice. The results showed that the attenuated virus strain was avirulent to these animals by intracerebral inoculation (i.c.) or intraperitoneal inoculation (i.p.). Studies on the neuroattenuation stability revealed that no reversion after 17 times tissue culture passages or one i.c. sucking mice passage. Mosquito infection studies indicated that after one mosquito intrathoracical passage, the progeny viruses in the infected mosquitoes were unable to cause sucking mice or weanling mice disease. Molecules characteristics' studies of the SA14-14-2 virus strain showed that there are 57-61 nucleotide changes and 24-31 amino acid substitutions, eight substitutions in the E protein gene are the critical amino acid related to the virus attenuation. The E gene sequence studies have showed that the 8 critical amino acids were not changed after 22 passages in tissue cultures or one passage in mosquitoes. Comparison of the full-length sequence to the parental SA14 virus has revealed that after 22 passages in the tissue cultures, only 8 nucleotides changed leading to 4 amino acid substitutions. However they were not the reverse mutation and none of the 8 critical residues changed. The homology of the nucleotide and amino acid between the virus of passage 22 and the primary seed virus in Genbank was 99.93% and 99.88% respectively. The above results demonstrated that the SA14-14-2 virus is highly attenuated for the various JE susceptible animals. The attenuated phenotypes and the genetic characteristics of the SA14-14-2 strain are highly stable after multiple in vitro passages or mosquitoes infection. Therefore the safety of the live JE vaccine is due to a high degree of neuroattenuation and a number of stable phenotypically and genetically characteristics, suggesting that reversion to neurovirulence of the vaccine strain would be highly unlikely.