Studies Of The Genotypes, Mutations In The Basal Core Promoter And Precore Regions Of Hepatitis B Virus

Objective:Hepatitis B virus(HBV) infection is a serious public health problem globe-wide. It is estimated that 0.35 billion people are chronically infected with HBV, among them about one million die each year of HBV-related liver cirrhosis, liver failure, and hepatocarcinoma(HCC). The development of hepatitis B-related disease may be associated with immune response of hosts. HBV gene mutation may be one of the key factors contributing to abnormal immune response. HBV is a double-standed DNA virus containing four open reading frames, and has high frequency of mutations. Now based on an intergroup divergence of 8% or more of the complete genomes, HBV has been classified into eight genotypes(A~H). The most common hotspot mutations in HBV genome are located in the basal core promoter(BCP) and precore(PC) regions. The genotype can influence the incidence of BCP/PC regions mutations, and bring more complexity to the result analysis. Therefore, deep insight of the genotypes and their polymorphic characteristics of HBV will be imperative for prognosis of HBV carriers and approaches for hepatitis B molecular epidemiology. Herewith, we examined the HBV genotypes, HBV DNA loads and the mutations in HBV BCP/PC regions to analyze the character of HBV infection, and the function of genotypes and mutations of HBV in the progress of the disease for prevention and treatment of HBV infection in Bengbu area.Methods:1. Totally, 268 clinical isolates of HBV serum samples were collected for extraction of HBV DNAs from the First Affiliated Hospital of Bengbu Medical College from Jun 2013 to Jun 2014. Real-Time Genotyping and Quantitative PCR(GQ-PCR) was used to genotype and quantitate the HBV strains, and analyze the relationship of HBV loads and Hbe Ag, genotypes, and the prevalent characteristic and distribution of HBV genotypes. 2. Retrospective analysis of the proportion of genotypes in different liver diseases: Mutation analysis of the sequenced basal core promoter/precore region was performed by nested PCR amplification. The mutants of nt 1762, 1764 in BCP region and nt 1896 in PC region were analyzed by Vector NTI Suite software, and by the diversity of HBV PC/BCP in different liver-disease groups and the relationship between the genotypes and HBe Ag.Results:1. The genotyping showed that among the 268 serum samples of HBV-infected patients, 46 and 202 cases belong to the genotype B and C, and 20 cases to genotype B/C, with positive rates of 17.16%, 75.37%, and 7.46%, respectively. The virus loads of HBV were positively correlated with the rate of HBe Ag, and significant differences were found in HBe Ag-positive patients between genotypes B and C. No difference was noted in HBV DNA levels(log10 copies/ml) of the three genotypes. The patients younger than 35 years were mainly distributed in the genotype C, and those over 35 years in genotype B.2. We detected 134 cases of mutations in 245 individuals, the mutations at nt 1762/1764 in the four liver disease groups(CHB, CSHB, HCC, and LC) were 22.4%,57.4%,46.7%, and 67.6%, respectively, and mutants at nt 1896 were 17.3%,48.5%,31.1%, and 41.2% respectively. All mutations were 8.2%,36.8%,15.6%,and 26.5%, respectively. The mutation rates in CSHB,HCC,and LC groups were significantly higher than those in CHB groups. The mutation rates of genotype C infection were significantly higher thanthose of the B and B+C. Additionally, we detected 107 cases in the HBe Ag-negative groups, and the mutation rates in the HBe Ag-negative were significantly higher in the HBe Ag-positive group. Moreover, the mutation rates of HBV BCP/PC increased with the deterioration of the disease.Conclusions:1. The HBV genotype C is predominant in Bengbu, followed by genotype B. And the genotype is closely associated with HBe Ag and the age of patients with HBV infection.2. The mutation rates of A1762T/G1764 A and G1896 A in CSHB, HCC, and LC groups are significantly higher than those in CHB groups.3. Genotype C of HBV varies more frequently than other genotyes in virus mutation.4. The mutations of A1762T/G1764 A and G1896 A may be associated with the severity of negative HBe Ag, and may aggravat the development of HBe Ag-negative CHB.