Development And Applications Of The Reverse Genetics System For Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is transmitted by mosquitoes and it is the single most important cause of viral encephalitis in Asia, with fatality rates averaging 30%. JEV is widely distributed in Southeast Asia, but JE has recently spread to other areas of the world, and become a worldwide threat to people's health.The live attenuated vaccine strain SA14-14-2 has been inoculated to 300 million people and its safety and efficacy have been confirmed. The SA14-14-2 virus was derived from the wild-type strain SA14 by serial passages in primary hamster kidney cells and mice brains. Compared with SA14, the SA14-14-2 vaccine strain has almost lost the neurovirulence in adult mice and monkeys, but its possible mechanisms have not been well understood.In this study, we first constructed the JEV replicons based on the attenuated vaccine JE strain SA14-14-2, the function characterizations confirmed that the replicon could self-replicate efficiently and express foreign proteins. Futthermore, we have constructed and recovered the chimeric JE virus by inserting the E gene of SA14 into the replicon. The biochemical characteristics and biological functions of chimeric JE illustrated that the E protein decided the neurovirulence and neuroinvasiveness of JE virus. Moreover, E protein of JEV could affect the ability to block the IFN-α/βinduced JAK-STAT signaling pathway, which resulted in the attenuation of SA14-14-2.1. The construction of the JE virus subgenomic replicon.In this part, we firstly constructed the JEV replicon by reverse genetics technology. The JE replicons were transcript and transfect BHK-21 cells and then identified by RT-PCR and IFA assays. Furthermore, the reporter gene GFP was inserted into the structural region of JE replicon to measure abilities of viral replication and foreign proteins expression. Meanwhile, we constructed the reporter-replicons which contained 3, 25, 71 amino acids on the C-terminal of E protein respectively in the purpose of investigating whether the length of the signal peptide of NS1 protein may influence the viral replication. The results showed that the JEV replicons could self-replicate and express the foreign proteins, what's more, the expression of GFP could last for 10 days. Additionally, the activities of GFP in the BHK-21 transfected by different replicons revealed that the numbers of the last codons of envelope protein influence the replication of JEV. 2. The construction of chimeric JE virus and the identification of the biological characteristics.The research of JEV has been hampered by lacking of a full-length infectious cDNA clones, it is necessary to construct a stable full-length infectious cDNA clones. In this part, we constructed and recovered the chimeric JE virus based on the SA14-14-2 replicon, whose prM/E region was replaced by that of SA14, and the biologic characteristics of the chimeric virus were tested. First, we found that the growth characteristic of chimeric virus was similar to the SA14-14-2 and SA14 in BHK21, Vero and HepG2 cell lines. Secondly, we found that the plague size was in between with the SA14 and the SA14-14-2, whereas, the neurovirulence and neuroinvasiveness were similar to the wild-type strain SA14. Finally, we explored the attenuated mechanism of the vaccine strain SA14-14-2 illustrated by comparing the influence to the JAK-STAT signal pathway after infected with SA14, chimeric JE and the SA14-14-2. The results indicated that the chimeric virus as well as wild strain SA14 could block the nuclear translocation of STAT1 by reduced the expression levels of p-STAT1 upon IFN-αtreatment, and block the IFN-αinduced JAK-STAT signaling pathway. In addition, we infected the IFN-α/β-knockout A129 mice and 129 mice with different strains of JEV by i.p. injection. In the 129 mice model, the vaccine strain SA14-14-2 is not pathogenic while the chimeric virus and SA14 were both lethal. In the A129 mice model, the vaccine strain SA14-14-2 caused death within 3 days, similar to SA14 and chimeric virus. In conclusion, in normal 129 mice model, the SA14-14-2 virus can't block the JAK-STAT signaling pathway and was cleared away by the immune system, while the SA14 and chimeric virus block the JAK-STAT signaling pathway and resistant to the immune system clearance. Whereas, after the IFN-α/βgene was knocked out, the replication of SA14-14-2 was not influenced by the IFN-α/βsimilar to the SA14 and chimeric virus. In results, the E protein of the JEV may affact the ability of antagonizing the interferon pathway induced by IFN-α.Above all, in this study, we first constructed the JE replicon which could self-replicate and continuously express the GFP. Additionally, based on the replicon we constructed and rescued the chimeric JE virus and studied the mechanism of attenuated vaccine strain by comparing the biologic characteristic of chimeric virus and its parent viruses, all the results revealed that the E protein of JEV decided its neurovirulence and neuroinvasiveness and the affected its ability of antagonizing the interferon pathway induced by IFN-α. The establishment of the reverse genetics system of JEV will lay a foundation for the research on the genomic structure, function of JEV and the development of new-type vaccines.