Study On The Mechanism Of Superinfection Exclusion Between JEV And ZIKV

Japanese encephalitis virus(JEV)and Zika virus(ZIKV)are both mosquito-borne flaviviruses.They can cause a series of pathological changes after infecting humans.Currently,four different types of vaccines have been developed against JEV,including two inactivated vaccines and two live attenuated vaccines.However,ZIKV has only gradually spread around the world since 2013.No approved vaccine and specific antiviral drugs have been developed to treat ZIKV.Superinfection exclusion(SIE)is the phenomenon that a virus(primary virus)establishes an infection resistant to a subsequent infection with the homologous or heterologous virus(secondary virus)at the cellular and/or organism level,which is considered as an effective strategy to prevent viral infection and usually occurs among viruses with close homology.This study confirmed the existence of superinfection exclusion between JEV and ZIKV for the first time,and the inhibitory effect of high levels of JEV in cells on ZIKV was identified.The inhibition of JEV on ZIKV occurred at the stage of virus replication and translation,and the molecular mechanism of superinfection exclusion was further studied as follows:1.Identification the phenomena of superinfection exclusion between JEV and ZIKVIn this study,after exploring the conditions of infection between JEV and ZIKV,the replication and infection of viruses were detected by quantitative PCR and immunofluorescence in JEV/ZIKV co-inoculated cells.Finally,it was found that there was a phenomena of superinfection exclusion between JEV and ZIKV,and the high replication level of JEV had a significant inhibitory effect on ZIKV.2.The inhibition of ZIKV by JEV was unrecoverable and specificZIKV was first added to the cells,which allowed viral RNA to enter the cells and establish infection.The replication of ZIKV in cells was detected,and the results showed that the level of ZIKV infectivity could not be recovered to the levels with a single ZIKV infection during a coinfection.Further detection showed that there was no superinfection exclusion between JEV and PRV,indicating that the superinfection exclusion was specific to ZIKV.3.The inhibition of ZIKV by JEV occurs at the step of viral RNA replication and translationIn this study,the changes of ZIKV life cycle at different stages were detected.Viral attachment and entry assays showed that ZIKV was able to enter the cells normally and was not affected by JEV.Quantitative PCR and Western blot analysis showed that JEV significantly inhibited the RNA replication and translation of ZIKV,while plaque assay showed that the inhibition of JEV on ZIKV did not occur at the step of virions releasing.It was confirmed that the superinfection exclusion between JEV and ZIKV occurred at the levels of RNA replication and protein synthesis.4.Molecular mechanism of JEV NS2B protein mediated superinfection exclusionWe transfected with plasmids expressing JEV individual proteins in HEK293T cells,and the levels of viral RNA was quantified.The results showed that the RNA replication of ZIKV were significantly inhibited by NS2B protein in dose-dependent manners.After segmented expression of JEV NS2B protein,we found that the NS2B residues Ser46～Ser103,especially Ser46 to Ile60 were the essential region required for superinfection exclusion and related to its protease activity.Besides,the mutation of NS2B(R56A)eliminated the ability of the JEV to exclude ZIKV superinfection.