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Li X.-D.,Institute of Infectious Diseases and Liver Failure Research Center | Wang L.,Institute of Infectious Diseases and Liver Failure Research Center | Liu Y.,Institute of Infectious Diseases and Liver Failure Research Center | Xu Z.-H.,Institute of Infectious Diseases and Liver Failure Research Center | And 6 more authors.
Chinese Medical Journal

Background There is still a paucity of data on hepatitis B virus (HBV) subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen (HBeAg)-loss-associated mutations in patients with chronic hepatitis B (CHB) is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence. Methods Sera were collected from 1301 nucleoside analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter (BCP) regions. Results HBV/B, HBV/C, and HBV/D were detected in 190 (14.6%), 1096 (84.2%), and 15 (1.2%) patients, respectively. HBV/B2 (182/190), HBV/C2 (1069/1096), and HBV/D1 (12/15) were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area (77.2%) than in other north areas (84.9%-87.4%). HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase (ALT) levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM204I/V (44.9% vs. 30.2%, P <0.01) and BCP mutations A1762T+G1764A (65.8% vs. 40.0%, P <0.01) compared with HBV/B infection. Conclusions C2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection. Source

Wang X.,Guilin Medical College | Liu Y.,Institute of Infectious Diseases and Liver Failure Research Center | Si L.-L.,Institute of Infectious Diseases and Liver Failure Research Center | Chen L.,Institute of Infectious Diseases and Liver Failure Research Center | And 7 more authors.
Medical Journal of Chinese People's Liberation Army

Objective To identify a novel mutation rtN236V in the hepatitis B virus (HBV) reverse-transcriptase (RT) region of an adefovir dipivoxil (ADV)-refractory patient with chronic hepatitis B, and analyze its phenotypic resistant characteristics. Methods HBV DNA was extracted from an ADV-refractory patient with chronic hepatitis B, and the full-length RT region was amplified by nested PCR. The PCR product was cloned into the pGEM-Teasy vector, and then transfected into JM109 cells. Thirtyfour clones were randomly selected for DNA sequencing, and drug-resistance-associated mutations were analyzed. After restriction double enzyme digestion and ligation procedure, the 1.1-ploid genome length HBV recombinant plasmids harboring wild type and three different mutants in RT region were constructed. The replication-competent constructs were then transiently transfected into the HepG2 cells. Four hours post-transfection, new medium containing different concentrations of lamivudine, ADV, entecavir and tenofovir were supplemented every other day for 4 days. The phenotypic characteristics of the HBV mutants were analyzed under the drug pressure, the supernatant was collected and HBV DNA production was quantitatively detected using real-time PCR. Results Virological and biochemical breakthrough appeared in this patient after ADV treatment for 15 months. Sequence analysis of 34 clones showed that there were 20 strains (58.8%) for rtN236V mutant, 11 (32.4%) for rtN236T, 2 (5.9%) for wild type and 1 (2.9%) for rtA181V+N236V. The relative viral replication capacity of rtN236T, rtN236V and rtA181V+N236V mutants was 89.43%, 83.60% and 75.44% of the wild-type strain, respectively. Phenotypic resistance analysis showed that the susceptibility of mutant harboring rtN236T, rtN236V and rtA181V+N236V was 1/4.75, 1/3.10 and 1/5.10 of the wild-type virus. The three mutants were still susceptible to lamivudine, entecavir and tenofovir. Conclusion A novel mutation rtN236V concomitant with rtN236T and rtA181V mutations has been first identified in viral pool of an ADV-refractory patient. rtN236V may decrease the viral replication capacity and the susceptibility to ADV, which might be a novel ADV resistant mutation. Source

Ling J.,Peking University | Xu Z.-H.,Institute of Infectious Diseases and Liver Failure Research Center | Liu Y.,Institute of Infectious Diseases and Liver Failure Research Center | Li W.-D.,Institute of Infectious Diseases and Liver Failure Research Center | And 5 more authors.
Medical Journal of Chinese People's Liberation Army

Objective:To evaluate the influence of hepatitis B virus (HBV) genome nucleotide A1846T mutation on the viral replication capacity and the transcription activity of HBV core promoter (CP) in vitro. Method A total of 385 patients with hepatitis B admitted to the 302 Hospital of PLA were enrolled in the study, including 116 with moderate chronic hepatitis B (CHB-M), 123 with severe chronic hepatitis B (CHB-S), and 146 with acute-on-chronic liver failure (ACLF). Serum HBV DNA was isolated and full-length HBV genome was amplified. The incidence of A1846T was analyzed. Full-length HBV genomes containing 1846T mutation were cloned into pGEM-T easy vector, and the counterpart wild-type 1846A plasmids were obtained by sitedirected mutagenesis. The full-length HBV genome was released from recombinant plasmid by BspQ /Sca digestion, and then transfected into HepG2 cells. Secreted HBsAg level and intracellular HBV core particles were measured 72 hours post-transfection to analyze the replication capacity (a 1.0-fold HBV genome model). 1846 mutant and wild-type full-length HBV genomes were extracted to amplify the fragment of HBV CP region, and the dual luciferase reporter of the pGL3-CP was constructed. The luciferase activity was detected 48 hours post-transfection. Result The incidence of A1846T mutation gradually increased with the severity of hepatitis B, reaching 31.03%, 42.27%, and 55.48% in CHB-M, CHB-S and ACLF patients respectively (P0.01). The replication capacity of 1846T mutants, level of secreted HBsAg, and transcriptional activity of CP promoter were increased by 320%, 28% and 85% respectively, compared with 1846A wild-type strains. While the more common double mutation A1762T/ G1764A in CP region was increased by 67%, 9% and 72% respectively, compared with its counterpart wild-type strains. A1846T had a greater influence on viral replication capacity in vitro. Conclusions A1846T mutation could significantly increase the replication capacity of hepatitis B virus, secretion of HBsAg and transcription activity of CP promoter, and cis-activate the downstream gene transcription. The finding indicates that HBV genome A1846T mutation might play a role in liver disease progression. Source

Ji D.,Institute of Infectious Diseases and Liver Failure Research Center | Ji D.,Liver Fibrosis Diagnosis and Treatment Center | Liu Y.,Institute of Infectious Diseases and Liver Failure Research Center | Li L.,Institute of Infectious Diseases and Liver Failure Research Center | And 9 more authors.
Journal of Clinical Virology

Background: It remains unclear whether hepatitis B virus (HBV) reverse-transcriptase (RT) rtL229 substitutions influence HBV drug resistance. Objective: The study was to investigate the association of HBV rtL229 substitutions with viral resistance to lamivudine (LAM). Study design: Entire HBV RT genes were amplified by nested PCR and sequenced from sera of 6000 nucleos(t)ide analog-experienced patients with chronic HBV infection. The incidence and clinic relevance of rtL229 substitutions were analyzed. Replication-competent viral amplicons which harbored HBV genomes of wild-type, rtM204I, or rtM204I in conjunction with various rtL229 substitutions (rtL229F/W/M/V) were constructed. The amplicons were transfected into HepG2 cells for phenotyping of replication capacity and susceptibility to nucleos(t)ide analogs. Results: The rtL229 substitutions were detected in 6.57% (394/6000) of patients. Individual substitution incidences were 2.77%, 0.97%, 0.83% and 0.55% for rtL229V, rtL229F, rtL229M and rtL229W, respectively. The incidence of rtL229 substitutions was significantly higher in LAM-experienced patients (341/4220, 8.1%) than in LAM-naïve patients (53/1780, 3.0%), and were independently associated with genotypic LAM resistance (77.9% vs. 21.2%, OR 8.806, 95%CI 6.345-12.223) and low viral replication (HBV DNA <1000. IU/mL) (4.60% vs. 24.2%, OR 0.478, 95%CI 0.254-0.898). Representative cases follow-up showed that rtL229F developed subsequent to rtM204I emergence during LAM treatment and regressed with rtM204I after LAM withdrawal. Functionally, rtL229F did not confer reduced susceptibility to LAM, but could restore replication capacity of rtM204I strain. Conclusion: The rtL229 substitutions were potentially associated with LAM resistance in Chinese patients and rtL229F had characteristics of a compensatory mutation of rtM204I mutant. © 2012 Elsevier B.V. Source

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