Study On HBV Genotype/subgenotype And Their Association With Clinical Implications

Research on HBV genotypes during the last decade has demonstrated significant associations between the HBV genotypes and the severity of liver disease, clinical outcomes and the response to antiviral therapies, and also HBV genotype/subgenotype may vary in geographical distribution. However, genotyping method used by most former researcher was PCR RFLP, which was prone to be influenced by the function status and reaction conditions of restriction endonuclease and proficiency of operator. As golden standard of genotyping, direct sequencing of PCR product can obtain abundant information but has low detection sensitivity (only 104 IU/ml). Besides, we found it inefficient to analyze large sample of sequence artificially, while existing HBV sequence databases on website mainly collect HBV genotype A, D and E sequences predominating in Europe and America and most databases need to be paid.Objective: To analyze hepatitis B virus (HBV) subgenotypes geographical distribution in North China and association of HBV subgenotypes with the occurrence of genotypic nucleos(t)ide analog-resistance mutations, precore/basal core promoter (BCP) mutations, cytotoxic T lymphocytes (CTL) epitope mutations and the influence of HBV subgenotypes on dessase progression.Contents: A total of 2377 patients chronically infected with hepatitis B (1981 men and 396 women, mean age 38±13) from North China, including Beijing, Hebei, Shanxi, Tianjin, Inner Monglia province, that admitted to Beijing 302 Hospital from July 2007 to December 2008, were included in the present study. They were identified as four groups due to disease stage: chronic hepatitis B (1874) and HBV-related liver cirrhosis (354), liver failure (32) and hepatocarcinoma (117). Co-infection with hepatitis C, hepatitis D and human immunodeficiency viruses were excluded. The criteria used for evaluating disease stage and grouping are 2000 Xi'an Viral Hepatitis Management Scheme issued by the Chinese Society of Infectious Diseases and Parasitology, and the Chinese Society of Hepatology, of the Chinese Medical Association. Methods: Serum HBV DNA was extracted from 2377 patients. A nested PCR assay with high sensitivity and specificity developed by our laboratory was employed for the preS/S (overlapping complete RT gene) gene and precore/BCP gene amplification. Direct sequencing of PCR products were performed. HBV genotypes/subgenotypes were classified based on the RT/S sequences. Establish a comprehensive HBV database with various bioinformatics functions to assist analysis of variations/mutations associated with drug resistance (including rtL80I, rtV84M, rtV173L, rtL180M, rtA181T, rtT184A/I/L/P/S, rtS202G, rtM204I/V, rtN236T and rtM250L), precore/BCP (T1753A/C/G, T1758C, A1762T, G1764A, C1766T, T1768A, G1862T, G1896A and G1899A) and CTL specific epitopes.Results:.1. Establishment of highly efficient and sensitive amplification method of preS/S and preC/BCP region: By optimizing experimental condition, we have successfully established a highly efficient and sensitive amplification method of complete preC/BCP and RT region. The detection sensitivity of RT gene was up to 100 IU/ml, which was 2 order of magnitude higher than international standard (≥104 IU/ml). We also conquered the difficulty of high viral load tolerance and can detect serum samples with different levels of viral load ranging from 102 to 109 simultaneously.2. Establishment of the first HBV comprehensive database in mainland of China: Epidemical information and clinical test information of about 6800 patients infected with HBV were collected in the database, as well as more than 11000 HBV fragments.3. Geographical distribution of HBV genotypes/subgenotypes in North China: Three HBV genotypes B, C, D were detected, occupying 83.8% (1992/2377), 15.5% (368/2377) and 0.7%(17/2377)respectively. The case numbers (proportion) for subgenotypes in total patients were 10 (0.4%) for B1, 344(14.5%) for B2, 6 (0.3%) for B3,8 (0.3%) for B4, 28 (1.2%) for C1, 1937 (81.4%)for C2, 21 (0.9%) for C3 , and 6 (0.3%) for C4. B2 (344/368, 93.5%) was predominant subgenotype in genotype B, and C2 (1937/1992, 97.2%) was predominant subgenotype in genotype C. The prevalent feature of HBV subgenotypes among different provinces and municipalities was similar.4. The clinical implications of HBV genotypes/subgenotypes:a. Demographical and clinical background: No obvious difference was observed in sex, HBV DNA level, ALT/AST, TBIL, CHE, PTA and PTT between patients infected with C2 and B2 viruses.b. Relationship between HBV subgenotypes and nucleos(t)ide analogue resistance mutation: C2 virus infection had higher occurrence of lamivudine-resistance mutations (L80I, V173L, L180M, M204I) but no difference in occurrence of adefovir- and entecavir-resistance mutations than B2 virus infection. c. Relationship between HBV subgenotypes and BCP/PC mutation: The subgenotype B2 virus had significantly lower prevalence of BCP mutations T1753A/C/G, T1758C, A1762T/G1764A and C1766T, but significantly higher prevalence of precore mutation G1896A in comparison with subgenotype C2 virus.d. Relationship between HBV subgenotypes and CTL specific variation: Statistical significant difference was found between B2 and C2 virus infected patients in five S gene CTL specific epitopes: preS2 44-53, S 14-22, S 20-28, S 88-96 and S 172-180 and in 1 pol gene CTL specific epitopes: Pol 455-463. The variation rate of S 20-28 was 100% compared with C2 9%; the variation rate of Pol 455-463 was 2% compared with C2 81%. The variation pattern between B2 and C2 also show different characteristics, such as the S 38-47 variation pattern of B2 virus is mainly SLNFLGGTPV while C2 is SLNFLGGAPT; and S207-216 variation pattern of B2 was NILSPFMPLL while C2 was NILSPFLPLL.e. Relationship between HBV subgenotypes and clinical implication: The ratio of HBV subgenotypes B2 versus C2 among five groups of patients diagnosed by patients with acute hepatitis B (data of this group were not included in debate paper but can be used as a meaningful complementation), chronic hepatitis B, HBV-related liver cirrhosis, acute-on-chronic liver failure, and hepatocellular carcinoma were analyzed and showed gradually increase in a stepwise manner.Conclusion: We established a comprehensive HBV database with complete clinical information and powerful bioinformatics tools. C2 was the most predominant subgenotype followed by B2 in North China which together account for 95.9% of HBV infection cases. Between the two subgenotypes difference was shown in drug resistance mutation rate, BCP/PC mutation rate and variation rate and patterns of CTL epitopes. Generally C2 HBV infection patients may have higher probability of chonicity and severity of chronic hepatitis B than B2 HBV infection.