Study On The Effect Of BCP/PC Mutation On The Phenotype Of Hepatitis B Mouse Model

Background and ObjectiveHepatitis B virus(HBV)infection is a major public health problem.Approximately 248 million people worldwide are infected with HBV,which chronic infection can lead to a series of serious liver diseases,including liver fibrosis,liver cirrhosis and hepatocellular carcinoma.HBV has the characteristics of species and tissue specificity as well as high replication and mutation rate.The common gene mutation sites are 1762(A1762T)and 1764(G1764A)in the basic core promoter(BCP)region and 1896(G1896A)and 1899(G1899A)in the pre-core(PC)region.It has been found that BCP/PC combined mutation can block or reduce the expression of HBeAg in vitro or clinical samples.However,the more effect of BCP/PC combined mutation on HBV phenotype has not been reported.On the basis of BCP/PC(M)mice model,we established the chronic hepatitis B mice model with reverse mutation(BCP/PC(R)).The effects of BCP/PC(M)on the phenotype of HBV major markers and immune phenotype of organism were studied by comparative analysis.It will provide a theoretical basis for clinical diagnosis and discussion of the mechanism of phenotypic change caused by BCP/PC combined mutation.Methods1.BCP/PC(R)covalently closed circular DNA(cccDNA)was prepared by molecular biological method and identified by sequencing.2.HepG2 cells were transfected with BCP/PC(M)cccDNA and BCP/PC(R)cccDNA.The secretion levels of HBsAg and HBeAg were detected by ELISA.3.Two kinds of HBV cccDNA and / or normal saline were injected into CBA/CaJ male mice(8-10 w)by tail vein high pressure injection.Serum and liver samples were taken at the specified time point.4.The levels of HBsAg and HBeAg in serum samples were detected by radioimmunoassay.5.The levels of HBV DNA and cccDNA in serum and / or liver tissue were detected by qPCR.6.The protein levels of HBsAg and HBcAg in liver were detected by immunohistochemistry.7.The mRNA levels of IL-1?,IL-4,IL-6 and IL-10 in liver were detected by RT-qPCR.8.The pathological changes of liver were detected by HE staining.9.The correlation between HBV markers in liver and serum was analyzed by statistical method.Results1.The results of digestion and sequencing showed that BCP/PC(R)cccDNA was successfully constructed.2.BCP/PC(M)cccDNA and BCP/PC(R)cccDNA were transfected into HepG2 cells respectively.After BCP/PC(M)cccDNA was transfected,HBeAg was not detected in the supernatant,and the level of HBsAg was significantly lower than that of BCP/PC(M)group(p<0.05).3.After BCP/PC(M)cccDNA and BCP/PC(R)cccDNA were injected into CBA/CaJ mice,the HBV markers(HBsAg,HBeAg,HBV DNA and cccDNA)in the both group were continuously expressed for more than 26 weeks.4.In the early stage of infection,the HBsAg and HBV DNA levels in serum and HBV DNA and cccDNA levels in liver in BCP/PC(M)group were significantly lower than those in BCP/PC(R)group(P < 0.05).In the whole infection period,the level of serum ALT in BCP/PC(M)group was significantly higher than that in BCP/PC(R)group(P < 0.05).Compared with BCP/PC(R)group,after 4 weeks of infection,the mRNA levels of IL-4 and IL-10 in the liver of the BCP/PC(M)group were significantly decreased;.However,after 26 weeks of infection,the mRNA levels of IL-4 and IL-10 in the liver of the BCP/PC(M)group were significantly increased,and the mRNA levels of IL-1? were significantly decreased.5.After 26 weeks of infection,inflammatory cell infiltration appeared in both groups,and the inflammatory cell infiltration in BCP/PC(M)group was less than that in BCP/PC(R)group.6.In both groups,the level of HBV DNA in serum was positively correlated with the level of HBV DNA and cccDNA in hepatocytes.Conclusions1.BCP/PC(R)cccDNA was successfully reverse mutated,and BCP/PC(M)and BCP/PC(R)chronic hepatitis B mice models were established.The expression of HBeAg in BCP/PC(R)group was positive continuously,and the HBV markers in both groups were expressed stably for more than 26 w.2.The mutation of BCP/PC resulted in the decrease of HBV markers in serum and liver at the early stage of infection,and the increase of ALT in serum during the whole infection cycle.3.The level of HBV DNA in serum could better reflect HBV replication and cccDNA copy number in hepatocytes than HBsAg.4.BCP/PC mutation reduced the inflammatory response in the later stage of infection.The mechanism might be that in the early stage of infection,BCP/PC mutation inhibited the expression of Th2 cytokines in mice liver,while in the later stage of infection,BCP/PC mutation inhibited the expression of Th1 cytokines in mice liver.