Analysis Of Infectivity Of Hepatitis C Virus To Human Diploid Cells

Chronic hepatitis C virus(HCV)infection is one of the main causes of end-stage liver disease,which seriously affects people’s health.At present,there is no effective HCV prevention and treatment vaccine,and clinical antiviral treatment strategies are mainly used.However,because HCV infection has a very strong tissue specificity,the current HCV in vitro culture is mainly completed in the liver cancer cell line Huh7.5,so obtaining safe sources of HCV virus particles is an urgent problem in the development of HCV vaccines.KMB17 is a human diploid cell line with proven safety,which has been used in the production of various vaccines.The study takes KMB17 as the research object,hoping to construct a cell matrix that can be used for HCV production.To study the characteristics of HCV infection in KMB17 cells,we prepared high-sensitivity HCV pseudovirus particles(HCVpp)and used HCVpp to infect KMB17 cells.It was found that KMB17 cannot effectively support HCV infection under natural circumstances.Further research shows that HCV infection is closely associated with the expression of CD8 1 receptor,human scavenger receptor class B type 1,SR-BI,blocking protein claudin-1(CLDN1),human tight junction protein occludin(OCLN).In order to find the cytokines lacking in the process of HCV infection of KMB17 cells,we analyzed the expression levels of four receptors related to HCV infection in KMB17 cells from the transcription level and translation level.After the four receptors gene by lentivirus packaging and introduced into KMB17 cells separately or co-expression.HCVpp was infected with KMB17 to detect the activity of luciferase reporter gene,or HCVcc was infected with KMB17 to detect the expression of intracellular core protein.By analyzing the changes of HCV infection after overexpressing the corresponding receptors in KMB17 to determine the types of receptors that support HCV infection lacking in KMB17 cells.The results showed that after KMB17 cells overexpressed both OCLN and CLDNI receptors,the HCV infectivity was significantly enhanced,indicating that the simultaneous expression of OCLN and CLDNI is a necessary condition to support the successful infection of KMB17 cells with HCV.In order to study the replication efficiency of HCV in KMB17 cells,we first used HCV replicon with luciferase reporter gene and blasticidin resistance gene to detect the replication of HCV in KMB17 cells,and found that KMB17 and HCV replication is not supported.In order to enable KMB17 cells to support the replication of HCV,we introduced the HCV replication enhancer miR122 into KMB17 cells through the method of pseudovirus infection,and then detected the replication of HCV replicons in KMB17 cells,and found that KMB17 can support a small amount of HCV replication.However,due to the limited number of passages of KMB17 cells,long-term screening of replicon cell clone formation experiments was not possible.We tried to immortalize KMB17 cells and successfully obtained KMB17 immortalized cell line KMB17TT.After miR122 was introduced into KMB17TT cells,the cells were found it can support the replication of HCV in a small amount,but it failed to obtain clones of replicon cells after resistance screening.It is suggested that there may be other factors in KMB17 cells to restrict the replication of HCV.The limiting factors affecting the replication of HCV in KMB17 cells still need to be further studied.Pseudoviruses are widely used in the research of infectivity of various viruses,and the detection process is simple and fast.The study used the pseudoviruses packaging system used for HCVpp packaging in the infectivity study of the Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV2).The results show that SARS-CoV2 is more adaptable to the production of commonly used Vero cells,and SARS-CoV2 is also observed.Human liver,kidney,and intestinal cells are more susceptible,and in different species,they are more infectious to tree shrews and human hepatocytes,monkeys hepatocytes are relatively less infectious.This result is consistent with clinical data and the safety of operation Higher,so that in vitro infectivity studies of SARS-CoV2 can be carried out in ordinary laboratories.