Establishment And Application Of A Novel HEV Replicon Cell Line And Gerbil Infection Model

Hepatitis E virus(HEV)is one of the major etiological agents responsible for acute and chronic hepatitis E,leading to an annual death toll about 50 thousand worldwide.Hepatitis E is a particularly dangerous to pregnant women,organ transplant recipients and immunodeficient patients,both in undeveloped and developed countries.The major barrier to the development of therapeutics forHEV infection is the unavailability of an efficient in vitro culture system and the small animal model for persistent infection.Here we reported the success to establish of both cellular and rodent systems of HEV,based on replicon cell lines(S10-3-EZ and BHK-EZ)for p6-EGFP/Zeocin(p6-EZ)HEV and the infection of Mongolian gerbils with genotype 3 human HEV(Kernow-C1/p6 strain).HEV does not replicate efficiently in mammalian cell cultures,thus a useful model that mimics persistent HEV replication is needed to dissect the molecular mechanism of pathogenesis.In this study,we report a genotype-3 HEV RNA replicon expressing an EGFPZeocin(EZ)resistant gene(p6-EZ)that persistently self-replicated in cell lines of human(Huh-7-S10-3)or hamster(BHK-21)origin after transfection with in vitro RNA transcripts and subsequent drug screening.Two cell lines,S10-3-EZ and BHK-21-EZ,stably expressed EGFP in the presence of Zeocin during continuous passages.Both genomic and subgenomic HEV RNAs and viral replicase proteins were stably expressed in persistent HEV replicon cells.The values of the cell models in antiviral testing,innate immune RNA sensing and type I IFN in host defense were further demonstrated.We revealed a role of RIG-I like receptor(RLR)-interferon regulatory factor 3(IRF3)in host antiviral innate immune sensing during HEV replication.We further demonstrated that treatment with interferon(IFN-?)or ribavirin significantly reduced expression of replicon RNA in a dose-dependent manner.The availability of the models will greatly facilitate HEV-specific antivir al development,and delineate mechanisms of HEV replication.Furthermore,in this study,we established a reproducible conventional rodent infection model for human genotype 3 HEV with gerbils,and tested the genotype 1 and 4 HEV for retrospective studies.The values of the practical models in antiviral testing,innate immune RNA sensing and type I IFN in host defense were further studied.We showed that gerbils inoculated intrahepatically with capped HEV RNA transcripts,via oral with infectious HEV or intraperitoneally with infectious HEV produced robust infection,as evidenced by presence of HEV in livers,spleens,and feces for up to 7 weeks post-inoculation,seroconversion,and pathological liver lesions.We revealed a role of RIG-I like receptor(RLR)-interferon regulatory factor 3(IRF3)in host antiviral innate immune sensing during HEV replication using two molecule inhibitors(BX795 and MRT67307)in gerbils model.We further demonstrated that the gerbil model was invaluable in investigating antiviral IFN? and ribavirin responses.The availability of the model will greatly facilitate HEV-specific antiviral development and assess the mechanism of host innate immune response during HEV infection in vivo.Finally,we screened a small molecule inhibitors library with the help of the HEV replicon cell lines,founding that all the 17 HSP90 inhibitors could effectively inhibit the replication of HEV.HSP90 targeting markedly suppressed HEV replication with an efficacy exceedingly superior to conventional antivir al medicines.Unexpectedly,i HSP90 compounds released viral replicase(ORF1)from the ORF1-HSP90 complex.Notably,the anti-HEV strategy targeting the ORF1-HSP90 interaction was safe,considerably efficient,and substantially prevented the liver damage in HEV-inflicted rodents,as proofed in a preclinical trial in gerbils with chronic infection of human genotype 3 HEV.Collectively,in this study,HEV replicon cell system and gerbil infection model were established and our findings describe the critical mechanism and key host factors in host-HEV interactions and propose a feasible and promising therapeutic strategy for preventing hepatitis E.