Analysis for phenotypic resistant characteristics of a novel mutation rtN236V in the reverse- transcriptase domain of hepatitis B virus isolated from an adefovir dipivoxil-refractory patient with chronic hepatitis B
- Author:
Xiao WANG
1
Author Information
1. School of Graduated, Guilin Medical College
- Publication Type:Journal Article
- Keywords:
Drug resistance;
Hepatitis B virus;
Variation;
Viral
- From:
Medical Journal of Chinese People's Liberation Army
2013;38(4):256-259
- CountryChina
- Language:Chinese
-
Abstract:
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.
