Studies On The Biological And Molecular Characteristics Of Japanese Encephalitis Virus Strains Newly Isolated In China

Japanese encephalitis (JE) is an epidemic viral encephalitis which is caused byJapanese encephalitis virus (JEV). The morbidity of JE is about 10 per 100,000 andthe mortality is 10%～30%. 30%～50% patients can lead to persistent psychologicaland neurological sequelae. In China, about 6,000 cases are infected every year and JEis a severe challenge to public health.The mouse has been shown to be a good animal model for studyingpathogenicity of JE virus. Mice less than 3 weeks old are highly susceptible to lethalinfection by all routes of inoculation with JE virus. Although there is no muchvariation by the ic. route of inoculation, there are great differences in the virulence,when virus was inoculated subcutaneously.Virus isolation and biological research in China were mostly performed in 1950s.Since then no more work has been done. In recent years, researchers of our countryhave isolated a lot of JE virus strains country-wide for the study of molecularepidemiology of JEV in China. Based on the virus strains they isolated, we selected12 representative virus strains from them and four strains isolated in 1950s as controlfor the studies of their biological characteristics.Firstly, virus titer was test on BHK₂₁ cell and virulence was tested by threeinoculation routes (ip.,sc.,ic.) in 9-11g mice and sc. route in 12-14g mice. Plaque sizesshowed divergent among different virus strains. Most were in middle size but somestrains showed larger or smaller plaques. Neurovirulence (ic.) of all the virus wassimilar with each other but the neuroinvasiveness (ip. or sc. inoculation) was divergent especially by the inoculation in 12-14g mice. Most of the strains, thevirulence of neuroinvasiveness were moderate, except a few strains with high orlow LD50. Viruses from most areas had similar virulence. But we found three isolatesfrom Heilongjiang province showed relative lower neuroinvasiveness than those fromother aeras. Although virulence of all the strains from Culex mosquitoes wereidentical with each other, one strain (HLJ02-144) isolated from Aedes vexanmosquitoes showed very low neuroinvasiveness.Vaccination-challenge protection test was done in mice for the evidence ofprotective effect induced by the live attenuated vaccine and inactivated vaccineagainst the newly isolated virus strains. Mice were immunized by serial dilutedvaccine and challenged ip. by all the virus strains 14 days later. Results showed thatall the isolates could be well protected (over 70%)by vaccination with 10^-3, 10^-4dilution of live attenuated vaccine and 1:5 dilution of inactivated vaccine. Even theamount of antigen reduced to as low as 10^-5 dilution(23pfu) of the live attenuatedvaccine and 1:125 dilution of the inactivated vaccine, the protection effects were stillapparently (30%～80%)against most virus strains.It proved that both of the twovaccines had extensive spectrum and high efficacy. However, the live attenuatedvaccine appeared superior than the inactivated vaccine in respect of the lower antigenand fewer dose of vaccination. In addition when the vaccinated mice were challengedby ic. inoculation, the protection effects of live vaccine showed much better (20%～90%) than that of inactivated vaccine (0%～40%)o PRNT was performed to determinethe neutralizing ablity of pooled sera from mice vaccinated with live or inactivatedvaccine. Results showed that all the new isolates except one (KT) can be wellneutralized by the sera from both vaccines (neutralizing index 1:40～1:80 and 1:20～1:40).Complete genome of SA4 and SC04-17 were sequenced and aligned with 23complete sequences in Genbank.SA4 was most homologous with SA14 in nt and withJarGar01 in aa; SC04-17 was most homologous with Ishikawa in both aa and nt.Comparison of SA4 with SC04-17 found that they had homology of 88.7% in ntand 98.1% in aa. SC04-17 had several insertions and deletions in 3'UTR just like other virus strains of genotype I.E genes of 5 virus strains with different neuroinvasiveness were sequenced andcompared with sequences of other strains in Genbank. Results found that they hadhigh homology in aa. Although there were several aa sites different between thosevirus strains, no evidence was found that they were responsible for the virulencedifference.