The Rescue Of Japanese Encephalitis Virus Containing NS2A-C60A Point Mutation And Study Of The Biological Characteristics Of The Virus

Japanese encephalitis virus(JEV)belongs to the Flaviviridae family,the genus Flavivirus.It is one of the main pathogens causing viral encephalitis in Asia and the Pacific.Based on previous work in the laboratory,this study aims to construct and rescue NS2A-C60 A single-point mutation virus and study the biological characteristics of the virus.This study will provide a theoretical basis for deeply analyze the relationship between the changes in the expression of non-structural proteins and the attenuation mechanism during the attenuation of JEV,and to further explore the possible roles of non-structural gene mutations in the life cycle of JEV.1.The rescue of rSA14-NS2A-C60A single point mutation virusThis study successfully constructed and rescued single point mutation virus r SA14-NS2A-C60A(named r JEV-C60A).Firstly,we performed genetic sequence comparison and structural prediction of the attenuated JEV SCYA201201-0901 strain and its original strain SCYA201201-01 in the laboratory,and determined the target site NS2A-C60 A that it may affect neurovirulence of JEV.Secondly,the total protein collected from HEK-293 cells inoculated with the attenuated strain and the original strain was compared and verified by Western Blot,It was found that the expression of NS1' protein of the attenuated strain was lower than that of the original strain.Finally,the r SA14-NS2A-C60 A single-point mutation virus was successfully constructed and rescued using reverse genetic technology,and the rescued virus was identified by combining cytopathic observation,plaque test,PCR,and sequence comparison.2.The study on the biological characteristics of r JEV-C60 A point mutation virusThis study confirmed that the NS2A-C60A point mutation can affect the expression of JEV NS1' protein,but it has no obvious effect on the virus growth characteristics,plaque formation and neurovirulence.Firstly,using plaque formation,growth curve to study the effect of the mutation on the growth characteristics of Japanese encephalitis virus in vitro;it was found that the target point mutation has no effect on the plaque morphology and growth characteristics of JEV.Secondly,dual luciferase analysis and Western Blot results show NS2A-C60 A significantly reduced the expression of viral NS1' protein(p<0.5),indicating that this site is crucial for the production of viral NS1'protein.Finally,we used BV-2 cells and mice models to study the effect of NS2A-C60 A mutation on JEV-induced inflammatory factor and neurovirulence.The inflammatory experiment results showed that the r JEV-C60 A can slightly increase the expression levels of inflammatory factors CCL-2,IL-6 and IFN-?,but it is not significant compared to the original strain(p>0.5);65 mice were divided into 13 groups for neurovirulence experiments,and each virus titer was repeated 3 times for verification.The mortality of mice caused by200 PFU non-mutation virus and point mutation virus was 53.33% and 53.33%,respectively,while the mortality of mice caused by 2000 PFU virus was 100%.