| Hepatitis B virus (HBV) infection is a global health problem, and more than 400 million people in the world are chronic carriers of the virus. The clinical manifestations of HBV infection range from acute self-limiting infection, inactive carrier state, fulminant hepatic failure and chronic hepatitis with progress to liver cirrhosis, and hepatocellular carcinoma (HCC). Based on an inter-group divergence of 8% or more in the complete genome nucleotide sequence, HBV has been classified into A～H eight genotypes. HBV genotypes exhibit distinct geographical distributions. It's well known that HBV genotypes might influence the severity of liver disease, mutation patterns in precore and core promoter regions, and response to antiviral treatment.In the first part HBV strains from 238 HbeAg-positive chronic hepatitis B(CHB) patients in five adefovir dipivoxil(ADV) phaseⅢclinical trials who received ADV 10mg per day for one year had been genotyped. 218 out of 238 HBV strains were genotyped by type-specific primer PCR, and the other 20 strains were genotyped with type-specific nucleotide analysis. Only three genotypes (genotype B, C and D) were observed in Chongqing, China. 159 (66.8%) cases were genotype B, 69(29.0%) cases were genotype C, 6(2.5%) cases were mixtures of genotype B and C, and 4(1.7%) cases were mixtures of genotype B and D. Genotype B and C were the main genotypes in Chongqing municipality, genotype A, E, F, G and H were not observed.The whole genome sequence of three samples (No.21, No.55 and No,124) were analyzed. The nucleotides sequence variation between No.21, No.55 and genotype Ba were less than 0.16%, and the sequence variation between No.124 and genotype C were less than 2.0%. Nucleotides at 1838 were A in both No.21 and No.55. Both results suggested that No.21, No.55 belonged to genotype Ba . These results conformed to the type-specific primer PCR methods.We analyzed the nucleotides and the amino acids sequences of retrotranscriptase of 41 HBV strains. Three sites of amino acids of 40 strains were identical with the subgenotype Bj(rt153R, rt207V and rt272C), while subgenotype Ba had different amino acids at these sites(rt153W, rt207M and rt272W, respectively). Retrotranscriptase is relatively conserved in the virus evolution process. This results suggested HBV strains in Chongqing area had the similar genetic background with subgenotype Bj, however, the Chongqing strains also owned its unique characteristics from other subgenotype Ba, although it belonged to Ba. Based on these observations, we think that subgenotype Ba can be further divided into sub-subgenotype Bac, c stands for Chongqing area where Bac is first reported.In the second part, we investigated the role of HBV genotypes on the response to ADV antiviral therapy. Data of 177 HBV samples with known genotypes from HBeAg-positive CHB patients treated with ADV had been retrospectively analyzed. The clinical data include: serum HBV DNA seroclearance, mean HBV DNA reduction, HBeAg loss, anti-HBe seroconversion and serum ALT. The number of genotype B and genotype C were 102 and 65 cases, respectively. Baseline data of both groups were comparable. The mean HBV DNA reduction in patients with genotype B and genotype C at the treatment of 12, 24 and 48 weeks were 2.2log,2.1log (P>0.05), 2.7log,2.4log(P>0.05) and 3.6log,3.1log(P<0.05), respectively. At the end of therapy (48 weeks), 43(42.2%) patients and 22(33.8%) patients with genotype B and genotype C infection had achieved HBV DNA seroclearance (P<0.05), respectively. Our results suggested that HBV genotype B seemed to have a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than genotype C. But longer terms of ADV treatment are needed to verify this conclusion.Third part: Based on the HBV DNA values, 30 HBV ADV-resistant strains (including: 21 primary resistant strains and 9 secondary resistant strains) had been selected out. After PCR amplification of the retrotranscriptase region and the nucleotide, amino acid alignment, we first found that polymorphism sites of rtN118H, rtM271L and rtY141F occurred with frequency of 23.8%, 14.3% and 14.3%, respectively. One patients with chronic hepatitis B had one amino acid variation in the RT C conserved domain,Six patients had nine amino acids variation in the RT none-conserved domain. We further constructed and analyzed the whole HBV genome sequence of three secondary resistant strains, and found that regulatory and structure elements such as X gene, BCP region and core/e gene also had important variations.In short, our results suggested that ADV-resistant HBV strains may harbor several amino acid variances.not only in the conserved regions of RT, but also in the none-conserved and the regulatory regions.
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