| Background In China, hepatitis B virus (HBV)chronically infected 93 million people and HBV-related hepatic diseases leads to 300 throusand deaths annually. Currently, five kinds of oral nucleos(t)ide analogues (NA)agents, including lamivudine (LAM), adeforvir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) have been approved to be used for anti-HBV therapy. Although these oral antiviral agents can effectively suppress viral replication and they are welltolerated with minimal side effects, prolonged viral suppressionruns the risk of selecting for antiviral drugresistance, resulting in virologic and biochemical breakthrough. Antiviraldrug resistance may also cause hepatitis reoccurrcnccand eventually lead to fulminant liver failureand death.The current study aimed at the difficulties and hot spots in HBV drug-resistance mutation investigation, such as HBV rtA181T, rtN238T mutations and HBV genotypes and their impact on drug resistance. In addition, preexisting drug resistance in NA-untreated patiens, which remained controversial in many reports, was analyzed.Multiple investigative tools including large-scale sequencing of clinical samples, dynamic follow-up, clononal sequencing and assessment of replication capacity and phenotypic characteristics by transient tranfection were employed in this study. The results will broaden the knowledge about HBV NA-resistant profiles in real clinical practices and may provide the basis for optimizing HBV drug resistance management in China.Objective (1) To investigatethe mutational profile and clinical implications of rtA181T/sW172through population-based analysis of clinical samples.(2)To identify clinical prevalence of untypical ADV-resistant mutations in hepatitis B virus (HBV) reverse transcriptase (RT) region, and to analyze their phenotypic resistance characteristics.(3)To clarify whether HBV drug-resistant mutations were affected by different genotypes.(4)To investigate drug-resistant HBV in NA-untreated patients with chronic HBV infection in real clinical practice.Methods (1) Serum samples from 18419 patients were collected andHBV NA-resistant mutations were determined by direct sequencing of RT gene. The clinical prevalence of differentrtA181T/sW172mutational pattern and their impact on HBV DNA and HBsAg levels were investigated.(2)A total of 11143 patients were selected from patient cohort of part one, who were either treated with single NA or untreated. Seven untypical adefovir-resistant mutations were analyzed in those patients. Serial serum samples were obtained from one patient and viral mutation changes were dynamically analyzed by clonal sequencing. Replication-competent wild-type and mutant HBV genomic amplicons (rtN238T, rtA181V+rtN238T and rtA181V) were constructed into pTriEx-1.1-HBVvector, transfected into HepG2 cells and cultured in the presence or absence of serially-diluted nucleos(t)ide analogs. Intracellular HBV replicative intermediates were quantitated for calculating the replication capacity and phenotypic resistance to NAs.(3)A total of 13847 NA-experienced patients were selectedfrom patient cohort of part one, all of whom were non-HCC patients. HBV genotypes were determined throughphylogenetic trees constructed by Mega 6 software. The association of HBV genotypes and drug resistance was analyzed.(4)A total of 845 NA-naive hoptialized patients were selected from patient cohort of part one. HBV drug-resistant mutations were examined by direct sequencing of viral RT gene. Replication-competent wild-type and mutant HBV genomic amplicons (rtL80I+M204Iã€rtL180M+M204I and rtA181V+N236T) were constructed into pTriEx-1.1-HBV vector. Phenotypic analysis of viral replication capacity and drug susceptibility were performed by measuring viral replicative intermediate level in the mutant or wild-type HBV amplicon-transfected HepG2 cells in the absence or presence of serially-diluted NA, respectively. Follow-up and clonal sequencing were perfomed to clarify the dynamic evolution of one NA-untreated patient with preexisting mutation.Results(1)rtA181T incidence was 4.1%(750/18419) across the study population. Nine kinds of rtA181T-deduced-sW172 mutational patterns were detected.22.1% (166/750) of rtA181T mutation led to sW172stop alone, while 69.9%(525/750) of rtA181T-positive samples were detected with wild-type nucleotide coexistence at the site. No significant difference in average serum HBsAg and HBV DNA levels was observed between patients with and without the mutant.(2)Patients treated with ADV alone were more likely to develop rtN238T mutation than those treated with other NAs, while other six untypical ADV-resistant mutations did not show the same tendency. Clonal sequencing of the patient with rtN238T+A181V mutation showed that switching-to entecavir rescue therapy suppressed HBV DNA to an undetectable level and normalized alanine aminotransferase and converted all virus strains into wild type strains. The viral replication capacity showed that rtN238T+A181V strain was higher than rtA181V strain. Compared to the wild type virus, rtN238T+A181V and rtA181V variants were relatively less susceptible to adefovir, while rtN238T variant was susceptible to adefovir.(3) LAM-resistant mutations was more frequently detected in HBV/C-infected patients compared to HBV/B-infected ones (31.67% vs.25.26%). Adefovir- and entecavir-resistant mutation incidence was similarly in overall but biased with mutational pattern between the two genotypes. For adefovir, HBV/C-infected patients had a higher incidence of rtA181V (5.29% vs.1.36%) but a lower incidence of rtN236T (2.70% vs.6.54%). For entecavir, HBV/C-infected patients had a higher incidence of rtM204+rtT184/S202 (H 3.66% vs.2.16%) but a lower incidence of rtM204+rtM250 (0.67% vs.1.46%). In addition, HBV/C-infected patients had a higher incidence of multidrug-resistant mutation (HBV/C 0.83% vs. HBV/B 0.35%, P<0.05) that was mainly presented as rtM204V/I+rtA181V (HBV/C 0.72% vs. HBV/B 0.30%, P<0.05).(4)Drug-resistant mutations were detected in 2.01%(17/845) of the patients by direct sequencing. Clonal sequencing verified 13 drug-resistant HBV strains: rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T, and rtN236T. Phenotypic analysis showed that two preexisting lamivudine-resistant strains (rtL80I+M204I, rtL180M+M204V) had> 1,000-fold resistance to lamivudine, and one preexisting adefovir-resistant strain (rtA181V+N236T) had 15.4-fold resistance to adefovir compared to the wild-type strain. rtM204I population outgrew from 20% at baseline to 85% after entecavir treatment with corresponding recession of wild-type population in the viral pool in one of the follow-up patients.Conclusions (1) Coexistence of the rtA181T mutant with the wild-type was common in patients, which may had littleimpact on serum HBsAg and HBV DNA levels.(2)rtN238T mutation may serve as a novel compensatory drug-resistant mutation for ADV.(3) HBV/C-infected patients took more risk to develop LAM-resistant and multidrug-resistant mutations compared to HBV/B-infected patients.(4)Drug-resistant HBV mutations were infrequently detected in NA-naive Chinese patients with chronic HBV infection. Resistance testing is not definitely required to start NA treatment in clinics given the fact of its very low incidence. |
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