Study On Relation Between Genotype Of HBV And Clinic Of Hepatitis B And Its Pathogenesis

Part I: Study on relation between genotype of HBV and clinical situations of the cases with hepatitis B virus infection Objective To determine the genotypes of hepatitis B virus (HBV) in the cases with hepatitis B virus infection in Hunan Province of China by a nested PCR with multiplex pairs of genotype-specific primers, and to study the relation-ship between HBV genotype and clinical situation of the cases with hepatitis B virus infection. Method Ten outer and inner primers were designed on the basis of the conserved nature of nucleotide sequences in regions of the Pre-S1 through S genes,in which 8 inner primers had HBV genotype specificity and were devided into mix A and B to amplify HBV of genotype A,B,C and D,E,F respectively. The PCR products after second-round PCR amplification were electrophoresed on a 3% agarose gel and stained by Ethidium bromide (EB). Genotypes of HBV were determined directly by the size of PCR products. To test its reliability and veracity, the results of typing specific primer nested PCR was compared with PCR-RFLP, followed by repeated experiments. Sera of 220 cases with HBV infection from Hunan Province, China, including chronic asymptomatic carriers(ASC), chronic hepatitis patients with mild, moderate, severe manifestations and chronic severe hepatitis B, were genotyped by PCR with genotype-specific primers. The results were statistically analyzed by SSPS. Result The data showed complete concordance between the two assays of PCR with genotype-specific primers and PCR-RFLP and 100% recurrence in the repeated experiments. Of the 220 HBV infected cases, 190 (86.4%) were genotype B and 30 (13.6%) were genotype C. The percent of severe-symptomatic appearance in the patients with genotype C HBV infection is clearly higher than that in the patients with genotype B HBV infection (P<0.05). Genotype C HBV infected patients had a higher level of alanine aminotransferase (ALT), total bilirubin (TBIL) and HBV-DNA than genotype B HBV infected patients, but there were no statistical difference among them. However, the ALT elevation rate in the genotype C HBV infected patients (96.7%) was significantly higher than that of genotype B HBV infected patients (75.2%) (P<0.05). There was no statistical difference in HBeAg positive between genotype B and C HBV infected patients in general, but in patients with chronic severe hepatitis or aged in 21-30, the HBeAg positive in genotype C HBV infected patients (35.0%, 50.0%) were significantly higher than that of genotype B HBV infected patients (14.4%, 24.5%) (P<0.05). Conclusion Genotype-specific primer nested PCR could help to determine HBV genotypes clearly and directly with reliable and accurate results. The predominant HBV genotypes in Hunan were confirmed to be genotype B and C by genotype-specific primer nested PCR. Genotype of HBV correlated with clinical situations of hepatitis B patients, and genotype C of HBV infection might cause more severe type of hepatitis B than that of genotype B of HBV infection. Part Ⅱ: Pilot study on HBV genotypes related to the pathogenesis of hepatitis B Obiective To explore the pathogenesis of different genotypes of HBV infection and to know whether the hepatocyte apoptosis induced by HBV is dependent on the genotypes of HBV or not. Method Four sera sample named A21, B1, E1 and E61, belonging to different genotypes and clinical situations, were used to amplify HBV-C gene fragment (639bp) by PCR. PCR products of HBV-C gene were cloned into T vector, then subcloned into the vector pEGFP-C1, which could express enhanced green fluorescent protein, to construct recombinant eukaryon expressing plasmids, pEGFP-C1/HBV-C. These plasmids were identified by PCR, dual restriction endonuclease digestion and DNAsequencing. The homogeneity of HBV DNA amplified from four samples in sequences of DNA and amino acid was detected by Blast. Then the plasmids were transfected into hepatocarcinoma cell HepG2. The cell cycle and apoptosis of transfected cells were determinated by MTT and flow cytometer respectively and the data were analyzed statistically by SSPS. Result Four recombined eukaryon expressing plasmids were all constructed successfully. Blast showed that the genotypes of four samples were same as what had determined by nested PCR with genotype-specific primers described in Part I, which further proved the reliability of genotyping with nested PCR with genotype-specific primers. Analysis on the homogeneities of their DNA sequences showed that E1 (genotype C, severe situation) was mostly like B1 (genotype C, mild situation) in 98.7% and variant from A21 (genotype B, asymptotic carrier) in 96.4%. Moreover, analysis on the homogeneities of their amino acid sequences showed that A21 was the same as B1 (100% ) and E1 was diverse from E61 (genotype B, severe situation) in 97.2%. The inframe sequences of HBV-C were correctly followed by the sequences of EGFP gene by DNA sequence analysis and enhanced green fluorescent protein successfully expressed after recombinant plasmid, pEGFP-C1/HBV-C transfection. The proliferation rate of HepG2 cells after pEGFP-C1/HBV-C(A21) transfection was highest of that in four kinds of gene transfected HepG2 cells, and that of pEGFP-C1/HBV-C(E1) gene transfected cells was lowest, but there was no clearly difference among the four kinds gene transfected cells(P>0.05). It was found that the apoptosis of the HepG2 cells transfected by pEGFP-C1/HBV-C(E1) gene (8.8±2.0)was clearly higher than that of the pEGFP-C1/HBV-C(A21) transfected HepG2 cells (6.4±0.8) by Flow cytometer (P<0.05). Conclusion HBV of genotype C could induce cell apoptosis more seriously than HBV of genotype B.Part ⅢStudy on subgenotype and quasispecies of hepatitis B virus Objective To know the relationship between subgenotype and quasispecies of hepatitis B virus and clinical situation of HBV infection. Method Fifty HBV-positive serum samples identified as genotype B by nested PCR with genotype-specific primers were chosen randomly, and their subgenotypes (Bj and Ba) were determinated by nested-PCR-RFLP. HBV of genotype B serum samples, taken from CSA(32 samples) and the chronic hepatitis patients(28 samples), were chosen to detect quasispecies of HBV DNA by melt curve. Then, the relation between quasispecies of HBV belonging to the same genotype and clinical situation of HBV infection would be studied. Result All the subgenotypes of 50 samples were HBV genotype Ba, and no subgenotype of Bj was found. Therefore, further study on the relation between subgenotypes of HBV and clinical situation of HBV infection couldn't be carried on. The data of 60 serum samples of HBV of genotype B detected by melt curve showed that the quasispecies of HBV DNA had more complicate in chronic hepatitis patients than that in CSA (P<0.05). Conclusion The predominant subgenotype of HBV in Hunan Province is HBV genotype Ba. The quasispecies of HBV correlates with clinical situations of HBV infection, and The complicate quasispecies of HBV were infected, the more severe hepatitis would be. It could explain the phenomenon that HBVs with one genotype infection caused many different clinical situations.