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Ye X.-L.,Guilin Medical University | Liu Y.,Institute of Infectious Diseases Liver Failure Research Center | Chen R.-J.,Institute of Infectious Diseases Liver Failure Research Center | Xu Z.-H.,Institute of Infectious Diseases Liver Failure Research Center | And 5 more authors.
Medical Journal of Chinese People's Liberation Army | Year: 2015

Objective To analyze the evolution of rtI233V mutation in the reverse transcriptase domain of hepatitis B virus (HBV) and its association with adefovir dipivoxil (ADV) resistance. Methods The rate of detection of rtI233V mutation in 9830 patients with chronic HBV infection was analyzed. HBV reverse transcriptase genes isolated from serial serum samples of two patients were amplified by nested PCR, and clonal sequencing (>20 clones/sample) was performed to analyze the evolution of rtI233V mutations. The replica of pTriEx-HBV1.1 vectors harboring wild-type and mutant strains (rtI233V, rtN236T, rtI233V+rtN236T) were respectively constructed and transiently transfected into HepG2 cells. Media containing serial concentrations of lamivudine (LAM), ADV, entecavir (ETV), or tenofovir (TDF) were used to treat the cells. Then HBV DNA in the supernatants was quantitatively determined by real-time PCR in order to analyze HBV mutants' replication competence and phenotypic characteristics under the drug pressures. Results The detection rate of rtI233V mutation in 9830 nucleos(t)ide analogues-treated patients was 0.28% (28/9830), including 0.19% (19 patients) with rtI233V individual mutation and 0.09% (9 patients) with rtI233V mutation combining with rtN236T or other mutations. All of the patients had rtI233V had ADV exposure history: 16 (57.1%) of them received ADV monotherapy for over six months, and 12(42.9%) of them received ADV combined sequential therapy for over 12 months. Replication competence and phenotypic resistance analysis showed rtI233V and wild-type strains had similar viral replication competence, while rtN236T exhibited significantly lower replication competence compared with wild-type strains. rtI233V strains remained sensitive to LAM, ADV, ETV, and TDF and showed little influence on drug resistance when combined with rtN236T, but it showed ability to restore the defected replication capacity of rtN236T strains. Conclusions The rtI233V mutation is associated with sub-optimal response to ADV. Though it does not directly reduce virus sensitivity to ADV, rtI233V mutation enhances replication competence of ADV resistant strains, and it is a complementary mutation. © 2015, People's Military Medical Press. All rights reserved. Source


Su H.,Guilin Medical University | Liu Y.,Institute of Infectious Diseases Liver Failure Medical Center | Xu Z.,Institute of Infectious Diseases Liver Failure Medical Center | Cheng S.,Third Peoples Hospital of Guilin | And 6 more authors.
PLoS ONE | Year: 2014

Hepatitis B virus (HBV) genotypes and subgenotypes may vary in geographical distribution and virological features. Previous investigations, including ours, showed that HBV genotypes B and C were respectively predominant in South and North China, while genotypes A and D were infrequently detected and genotype G was not found. In this study, a novel A/C/G intergenotype was identified in patients with chronic HBV infection in Guilin, a city in southern China. Initial phylogenetic analysis based on the S gene suggested the HBV recombinant to be genotype G. However, extended genotyping based on the entire HBV genome indicated it to be an A/C/G intergenotype with a closer relation to genotype C. Breakpoint analysis using the SIMPLOT program revealed that the recombinant had a recombination with a arrangement of genotypes A, G, A and C fragments. Compared with the HBV recombinants harboring one or two genotype G fragments found in Asian countries, this Guilin recombinant was highly similar to the Vietnam (98-99%) and Long An recombinants (96-99%), but had a relatively low similarity to the Thailand one (89%). Unlike those with the typical genotype G of HBV, the patients with the Guilin recombinant were seropositive for HBeAg. Moreover, a relatively high HBV DNA viral load (> 2x106 IU/ml) was detected in the patients, and the analysis of viral replication capacity showed that the Guilin recombinant strains had a competent replication capacity similar to genotypes B and C strains. These findings can aid in not only the clarification of the phylogenetic origin of the HBV recombinants with the genotype G fragment found in Asian countries, but also the understanding of the virological properties of these complicated HBV recombinants. © 2014 Su et al. Source


Liu Y.,Institute of Infectious Diseases | Xin S.,Institute of Infectious Diseases | Ye X.,Third Peoples Hospital of Guilin | Chen R.,Institute of Infectious Diseases | And 5 more authors.
Antiviral Therapy | Year: 2016

Background: The study aimed to clarify whether the rtI233V substitution affects adefovir (ADV) resistance. Methods: A total of 18,419 patients from Beijing 302 Hospital were investigated. HBV complete reverse transcriptase region of the polymerase was screened by direct sequencing and veriied by clonal sequencing if necessary. Replication-competent wild-type and mutant HBV genomic amplicons were transfected into HepG2 cells for phenotypic analysis of viral replication capacity and drug susceptibility. Results: The rtI233V substitution was detected in 38/5,344 (0.71%) ADV-treated patients and in 8/13,075 patients without receiving ADV (P<0.001). Eight patients with rtI233V ± rtA181V/rtN236T had virological breakthrough in the clinical course of ADV treatment. Phenotypic analysis showed that rtI233V mutants from patient 1 and patient 2 exhibited 1.57-fold and 1.51-fold decreased susceptibility to ADV, respectively, compared to wild-type virus; by contrast, rtN236T and rtI233V+N236T mutants from patient 1 had 6.82-fold and 5.28-fold decreased susceptibility to ADV. rtI233V, rtN236T and rtI233V+N236T mutants had 97.5%, 30.2% and 69.7% of replication capacity compared to wild-type virus in the absence of antivirals and all remained susceptible to lamivudine, entecavir and tenofovir. Viral replication capacity correspondingly decreased after eliminating rtI233V from rtI233V+N236T mutant and was restored after introducing rtI233V into rtN236T mutant. In clinical practice, switching to entecavir rescue therapy suppressed HBV DNA to an undetectable level for both patients. Conclusions: rtI233V usually emerged in ADV-treated patients with little impact on ADV susceptibility but it effectively restored replication capacity of the rtN236T mutant, suggesting that rtI233V may partly serve as a compensatory mutation associated with ADV resistance. © 2016 International Medical Press. Source

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