Relationship Between The Gene Heterogeneity Of Hepatitis B Virus And The Potential Clinical Implications

Hepatitis B virus (HBV) is a highly variant virus which result in the heterogeneity of HBV DNA, leading to different genotypes , serotypes and quasispecies in infected individuals. The relationship between HBV DNA heterogeneity and potential clinical implications is still the focus of studies in the field of chronic infection of HBV, currently.Objective: To investigate the prevalence of hepatitis B virus genotype in Fujian Province and the clinical implications of distinct genotypes in HBV-related liver disease; and three different methods were compared in monitoring the lamivudine-resistant HBV mutants to find a more sensitive, special and quick method which can be used method to study the correlation between HBV genotypes and lamivudine therapy in the treatment of chronic hepatitis B.Methods:1. HBV genotype was determined by the restriction fragment length polymorphism analysis in patients with chronic HBV infection in 5 cities of Fujian Province.2. The sensitivities and specialties of melting curve assay and PCR microplate hybridization-ELISA assay were compared with mPCR-RFLP and sequence analysis for the detection of HBV YMDD mutants in 44 serums from patients receiving lamivudine monitherapy with viral breakthrough.Results:1. Of the 443 patients with HBV DNA positive detected by PCR, 275(62. 1 %) were HBV genotype B, 100(22.6%) were genotype C, 51(11.5%) were genotype D and D-mixed genotype. Genotype A 12(2.7%)was only limited in Fuzhou and Putian cities and it was more prevalence in Putian than Fuzhou (10.6% vs. 2.6%, X2 =7.1, P=0.008). Genotype E and F were not found.2. Correspondence analysis showed that genotype B was significantly more common in patients with asymptomatic carriers (ASC), chronic hepatitis B (CHB) and severe hepatitis (SH) compared with liver cirrhosis (LC) and hepatocellular carcinoma (HCC)(P<0.05). Genotype C was most prevalence in patients with LC than in those with other liver diseases (P<0.05). Genotype D and D-mixed genotype were found to correlated with HCC at first time.3. The positive rate of hepatitis B e antigen was lower in patients with genotype D than in those with genotype B, genotype C and mixed genotype. (30.8% vs.59.6%,X2 =8.1, P=0.005, and 30.8% vs.56%,X2 =5.3, P=0.022, and 30.8% vs.60%, X2 =4.8, P=0.03, respectively).4. mPCR-RFLP assay was more sensitive (104copies/ml) than both PCR microplate hybridization-ELISA (105copies/ml) and melting curve assay (106copies/ml).5. Among the 44 serums, 26 YMDD mutants and 18 wild-types were determined by the means of mPCR-RFLP. Out of the 26 mutants detected by mPCR-RFLP, only 16 and 18 mutants were found by melting curve assay and PCR microplate hybridization-ELISA, respectively. Whereas, out of the 1 8 wild-types, 2 and 13 mutants were detected by melting curve assay and PCR microplate hybridization-ELISA assay, respectively. To confirm the different results determined by the three methods in 16 samples, sequence analysis was conducted and showed that the rate of consistency to sequencing was 93.8% by mPCR-RFLP, 43.8% by melting curve and 18.8% by PCR microplate hybridization-ELISA, respectively( X2=18.7, P<0.001).Conclusion:1 . The major genotype of HBV popularizing in Fujian was B; genotype C and genotype D were also popular too.2. The data of our study suggested a geographic distribution of genotype A in Fujian.3. HBV genotype B was associated with SH and genotype C was closed to LC.4. Genotype D appeared a tendency to the development of HCC, which should be needed a further study to confirm.5. The mPCR-RFLP assay was useful for its reliability in monitoring HBV YMDD mutants. Melting curve assay and PCR microplate hybridization-ELISA assay should be further improved to increase their sensitivity and specialty.