Rescue And Identification Of Mutant At Positions 202 And 226 Of Japanese Encephalitis Virus Nonstructural Protein 3

Japanese encephalitis virus(JEV)belongs to the flavivirus genus of the Flaviviridae family and can cause severe central nervous system diseases,with a fatality rate of up to 25%.Nonstructural protein 3(NS3)has serine protease activity,RNA helicase activity and nucleoside triphosphatase activity and plays an important role in the process of virus replication.The results of previous research in this laboratory show that,the Japanese encephalitis virus NS3 protein can down-regulate the expression of host miRNA-466d-3p.In this study,the Nuc Bind online website(https://yanglab.nankai.edu.cn/Nuc Bind/)was used to predict that the 202 and 226amino acids on NS3 can bind to RNA,the two sites 202 and 226 of the NS3 protein in the full-length expression plasmid PMW219-P3 of Japanese encephalitis virus P3strain were mutated by a point mutation kit to obtain recombinant plasmids P3-NS3(R202W)and P3-NS3(R226G).PMW219-P3,P3-NS3(R202W)and P3-NS3(R226G)viruses were rescued by reverse genetics technology,and the recombinant viruses were identified by RT-PCR and western blot.The growth curve of the F₃ generation virus showed that the proliferation rate of the parental virus PMW219-P3 was significantly higher than that of the recombinant viruses P3-NS3(R202W)and P3-NS3(R226G),with the highest titer of 10⁴PFU/m L,10^3.25PFU/m L and 10³PFU/m L,respectively,and compared with the parent virus,the plaque morphology of the recombinant virus has no significant change.The above three viruses were inoculated into one-day-old C57BL/6 mice by brain injection,and their survival rate was 0%;they were inoculated into four-week-old C57BL/6 mice by intramuscular injection,and the survival rate of mice inoculated with the parental virus PMW219-P3 was 0%;the survival rate of mice inoculated with recombinant virus P3-NS3(R202W)and P3-NS3(R226G)were 50%and 40%,respectively.The results showed that mutations at amino acid positions 202 and 226 on NS3 can reduce the virus's pathogenicity to C57BL/6 mice.qRT-PCR detected the expression of miRNA-466d-3p in NA cells after virus infection,and the results showed that the down-regulation effect of recombinant viruses P3-NS3(R202W)and P3-NS3(R226G)on host miRNA was significantly weaker than that of the parental virus PMW219-P3.In summary,mutations at amino acids 202 and 226 on the NS3 protein of Japanese encephalitis virus can cause the virus titer and its pathogenicity to be significantly reduced,and its down-regulation effect on the host miRNA-466d-3p expression is significantly weakened compared with the parent.This provides new ideas for further research on the pathogenic mechanism of Japanese encephalitis virus.