Establishment Of Anti-hepatitis E Virus Drug Screening Platform

Hepatitis E virus(HEV)is a viral hepatitis pathogen that is transmitted through the intestines and can infect humans and a variety of animals across species,seriously endangering human health.Hepatitis E is spreading all over the world.According to WHO statistics,about 20 million people worldwide are infected with hepatitis E every year and 70,000 people die.Especially in some developing countries,the incidence of hepatitis E is high and it shows a trend of continuous growth.At present HEV has been fund 8 genetypes,China’s main popular genetype IV HEV.No effective and specific treatment of HEV has been found.Although the clinical use of over-the-counter,broad-spectrum antiviral drugs to antiviral,such as ribavirin and interferon,but these drugs not only can not completely clear the virus in patients or produce drug-resistant strains,but also lead to anemia,peripheral blood leukopenia and thrombocytopenia and other side effects in patients.Therefore,the screening of anti-HEV drugs has become a hot and difficult point in current research.This paper aims to constructa screening platform for anti-hepatitis E virus drug and study the effects of different drugs on HEV replication at the cellular level,laying the foundation for screening anti-HEV drugs.The main work of this study includes three parts:(1)Establishment of HEV model in vitro culture;(2)Construction of EGFP-HEV recombinant virus;and(3)Screening of anti-HEV drugs.(1)Establishment of HEV model for in vitro cultureVirus culture is the basis for studying the mechanism of viral replication and pathogenesis.The in vitro culture of viruses has always been the focus,hotspot and difficulty of research.To investigate whether HEV can successfully replicate in Huh7.5.1 cells,we inoculated cells with HEV strain and cultured for multiple generations.The results showed that HEV virus copies were able to continue to grow in Huh7.5.1 cells and were able to be cultured for multiple generations.At the same time,we identified the HEV ORF2-specific protein by immunofluorescence.As time passed,the HEV ORF2 fluorescence increased gradually in the cell and the signal gradually increased,indicating that HEV was successfully replicated in vitro.(2)Construction of EGFP-HEV recombinant virusIn order to observe the replication of HEV virus more intuitively,the full-length HEV gene was recombined with EGFP gene by reverse genetics,and the recombinant EGFP-HEV virus was successfully constructed and transfected into Huh7.5.1 cells and BALB/c nude mice.It was confirmed that the recombinant virus was able to replicate normally in the cells,and fluorescence was observed in nude mice by in vivo imaging technology,and HEV was detected in the organs of nude mice,indicating that the EGFP-HEV recombinant virus was successfully constructed.(3)Screening of anti-HEV drugsIn order to further screen anti-HEV drugs,we used the successful in vitro culture model of HEV and the recombinant EGFP-HEV virus to infect Huh7.5.1 cells HEV.We observed the replication of HEV by adding RBV,IFN-α/2β,Sofosbuvir,Glycyrrhizinand,Silymarin,Hugan and Shugan drugs,and collected cells at different time points for quantitative analysis and fluorescence observation.It was found that the results of cell quantitative analysis were consistent with those of fluorescence observation.This indicates that the model can successfully simulate the replication of HEV,which lays a foundation for further screening of anti-HEV drugs.