Distinct quasispecies characteristics and positive selection within precore/core gene in hepatitis B virus HBV associated acute-on-chronic liver failure.

BACKGROUND AND AIM: The cause of hepatitis B virus associated acute-on-chronic liver failure (ACLF) remains unclear. Quasispecies can contribute to virus persistence and pathogenesis. We used a bioinformatics-based molecular evolution approach to compare quasispecies characteristics and positive selection sites within HBV precore/core gene between ACLF and chronic hepatitis B (CHB) patients. METHODS: HBV precore/core gene were amplified from 11 ACLF and 10 CHB patients harboring HBV genotype B; following DNA cloning and sequencing quasispecies complexity, diversity, and positive selection sites within the precore/core gene were determined by bioinformatics analysis, and compared between the patient groups. RESULTS: Both quasispecies complexity (P=0.022 at nucleotide level and 0.008 at amino acid level) and diversity (P<0.05) were found to be significantly greater in ACLF than in CHB. The frequency of G1896/A mutation in ACLF (175/298 clones, 58.7%) was also significantly higher than in CHB (100/230 clones, 43.5%) (P=0.0005). Moreover, analysis of positive selection revealed that significantly more patients with such sites were present in ACLF than in CHB (8/11 VS 2/10, P=0.03); the majority of these positive selection sites lay within HLA-restricted epitopes. CONCLUSIONS: The ACLF patients showed distinct quasispecies characteristics with higher complexity and diversity within the HBV precore/core gene. The increased HBV quasispecies complexity and diversity, together with a distinct set of positive selection sites, is likely associated with the development of ACLF.

[Polymerase region mutations and hepatitis B virus genotypes in chronic hepatitis B patients with poor response to lamivudine].

OBJECTIVE: To investigate the mutations in Polymerase region and hepatitis B virus (HBV) genotypes in chronic hepatitis B patients with poor response to Lamivudine treatment. METHODS: 631 chronic hepatitis B patients with poor response to Lamivudine were recruited in this study. Real-time PCR and DNA sequencing were used to determine HBV genotypes; direct sequencing was performed to detect mutations, and real-time PCR was used to quantify HBV DNA load. Mutations in polymerase region were investigated in different HBV genotypes. RESULTS: 272 patients were infected with HBV of genotype B, and 359 patients were infected with HBV of genotype C. The mean age of patients infected with HBV of genotype C (39.1+/-11.4 years old) were significant higher than that of patients infected with HBV of genotype B (33.7+/-9.7 years old) (t = -6.55, P less than 0.01). The patients infected with HBV of genotype C had relatively higher HBV DNA load [(5.96+/-1.22) log10 copies/ml] than the patients infected with HBV of genotype B [(5.58+/-1.21) log10 copies/ml] (t = -2.01, P less than 0.05). The overall incidence rate of A181V/T mutation in genotype C (5.3%) was significantly higher than that in genotype B (0.4%) (x2=12.23, P less than 0.01), but the incidence rate of M204I/V, L180M, T184A/G/I/S, S202G/I and V173L mutations was not significantly different between genotype B and C (each P more than 0.05). M204I mutation in genotype B (20.6%) was more frequent than that in genotype C (13.9%) (x2=4.91, P less than 0.05). The Lamivudine resistance mutations were not significantly different between genotype B and genotype C (x2 = 0.00, P more than 0.05). CONCLUSIONS: The incidence rate of lamivudine resistance mutation is not significantly different between genotype B and genotype C, but patients infected with HBV of genotype C have higher HBV DNA load than patients infected with HBV of genotype B.

[Evaluation of different methods in monitoring YMDD motif mutations associated with lamivudine resistance].

OBJECTIVES: To evaluate the three different methods in monitoring the lamivudine-resistant HBV mutants in lamivudine-treated patients with chronic hepatitis B. METHODS: The sensitivity and specialty of melting curve assay and polymerase chain reaction microplate nucleotide hybridization-enzyme linked immunosorbent assay (PCRmnh-ELISA) were compared with those of mismatch polymerase chain reaction-restriction fragment length polymorphism (mPCR-RFLP) and sequence analysis, through detection of HBV YMDD mutants in 44 serums from chronic hepatitis B patients receiving lamivudine monotherapy at the time of viral breakthrough. RESULTS: mPCR-RFLP assay was more sensitive (10(4) copies/ml) than both PCRmnh-ELISA (10(5) copies/ml) and melting curve assay (10(6) copies/ml). 26 YMDD mutants and 18 wild-types were determined by the means of mPCR-RFLP. Among the 26 mutants, only 16 and 18 mutants were found by melting curve assay and PCRmnh-ELISA, respectively. Whereas, out of the 18 wild-types, 2 and 13 mutants were detected by melting curve assay and PCRmnh-ELISA, 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 with sequencing was 93.8% by mPCR-RFLP, 43.8% by melting curve, and 18.8% by PCRmnh-ELISA, respectively (chi2=18.7, P<0.01). CONCLUSIONS: The mPCR-RFLP assay is reliable to monitor HBV YMDD mutations. Melting curve assay and PCRmnh-ELISA should be further improved to increase their sensitivity and specialty.

[Distribution of hepatitis B virus genotype in 5 cities of Fujian province and the clinical implications of HBV genotype].

OBJECTIVE: To study the prevalence of hepatitis B virus (HBV) genotype in 5 cities of Fujian province and the clinical implications of distinct genotypes in HBV-related liver diseases. METHODS: HBV genotype was determined by the restriction fragment length polymorphism analysis in patients with chronic HBV infection in 5 cities of Fujian province. The relationship between HBV genotype and its clinical implications was studied by multinomal logistic regression and correspondence analysis. RESULTS: Of the 431 HBV DNA positive patients detected by PCR, 275 (63.8%) belonged to HBV genotype B, 100 (23.2%) to genotype C, 51 (11.8%) to genotype D and D-mixed genotype. Genotype A, E and F were not found. Multinomal logistic regression showed that genotype B was more prevalent in Quanzhou and Sanming cities than in Fuzhou (P = 0.002, P = 0.006), and genotype B appeared significantly more common in asymptomatic carriers (ASC), chronic hepatitis B (CHB) and severe hepatitis (SH). Genotype C was most prevalent in patients with liver cirrhosis (LC) (47.0%) than in those with ASC (14.5%) and SH (14.7%) (P = 0.009, P < 0.001). The positive rate of hepatitis B e antigen was higher in patients with genotype C than in those with genotype B and genotype D (56.0% vs. 52.4%, P = 0.008, and 56.0% vs. 30.8%, P = 0.051, respectively). By correspondence analysis, genotype D and D-mixed genotype seemed to be correlated with hepatocellular carcinoma (HCC). CONCLUSIONS: (1) The major popular genotypes of HBV were B, C and D in Fujian. (2) Data of our study suggested that the geographic distribution of genotype B and C might be different in some cities of Fujian. (3) Genotype B might have a tendency to lead to SH in younger patients with chronic hepatitis B and the development of LC might be associated with genotype C among the elder patients. (4) Genotype D appeared to associate with development of HCC, which called for further study to confirm.