scholarly journals Use of Massively Parallel Ultradeep Pyrosequencing To Characterize the Genetic Diversity of Hepatitis B Virus in Drug-Resistant and Drug-Naive Patients and To Detect Minor Variants in Reverse Transcriptase and Hepatitis B S Antigen

2008 ◽  
Vol 83 (4) ◽  
pp. 1718-1726 ◽  
Author(s):  
Mariacarmela Solmone ◽  
Donatella Vincenti ◽  
Mattia Carlo Felice Prosperi ◽  
Alessandro Bruselles ◽  
Giuseppe Ippolito ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Direct Sequencing ◽  
Relative Sensitivity ◽  
Massively Parallel ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
B Virus ◽  
Drug Naïve

ABSTRACT Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of ≥20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.

scholarly journals Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis

2016 ◽  
Vol 54 (11) ◽  
pp. 2661-2668 ◽  
Author(s):  
Yi Mou ◽  
Muhammad Ammar Athar ◽  
Yuzhen Wu ◽  
Ye Xu ◽  
Jianhua Wu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Sanger Sequencing ◽  
Melting Curve ◽  
Melting Curve Analysis ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
B Virus

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.

scholarly journals Hepatitis B virus pre-existing drug resistant mutation is related to the genotype and disease progression

2017 ◽  
Vol 11 (09) ◽  
pp. 727-732 ◽  
Author(s):  
Liping Wang ◽  
Fangzheng Han ◽  
Hualing Duan ◽  
Fang Ji ◽  
Xuebing Yan ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Infection ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Hbv Genotype ◽  
B Virus ◽  
Drug Resistant Mutation ◽  
Genotype C ◽  
Resistant Mutation

Introduction: Previous studies have indicated that the drug-resistant mutations of hepatitis B virus (HBV) are a major obstacle to antiviral therapy. However, it is still unclear whether there are pre-existent resistance mutations in patients with HBV infection and the relationship between drug-resistant mutation, genotypes, and progression of hepatitis B disease. Methodology: A total of 357 treatment-naïve patients with HBV infection were involved in this retrospective study. The drug-resistant mutations of HBV reverse transcriptase domain were screened by direct gene sequencing. Results: Lamivudine (LAM) resistance was detected in 8 patients (3.7%) with chronic hepatitis B (CHB), 13 (11.7%) patients with liver cirrhosis (LC), and 6 (21.4%) patients with hepatocellular carcinoma (HCC). Adefovir(ADV)-resistant mutations were detected in 10 (4.6%) patients with CHB, 15 (13.5%) patients with  LC and 4 (14.5%) patients with HCC. Both LAM and ADV resistant mutations were detected in 2 patients (0.9%) with CHB, 1 patient (0.9%) with LC and 1 patient (3.6%) with HCC. Significant differences (p <0.01) were observed in the drug-resistance rates among patients with CHB, LC and HCC. Meanwhile, all the drug-resistant mutations were found in patients with HBV genotype C. Conclusions: This study demonstrated higher risk of pre-existing drug-resistant mutations in patients with HBV genotype C comparing to patients with HBV genotype B. Likewise, increasing prevalence of pre-existing drug-resistant mutations was shown, alongside with the progression of the disease.

Characterization of mutations in the reverse transcriptase region of hepatitis B virus in treated and untreated chronic hepatitis B patients

2020 ◽  
Author(s):  
Weihua Zou ◽  
Fuchu Qian ◽  
Fang Jin ◽  
Dongli Li ◽  
Jing Chen
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Chronic Hepatitis B ◽  
Direct Sequencing ◽  
Antiviral Treatment ◽  
Functional Domain ◽  
Novel Mutations ◽  
B Virus

Abstract Background The reverse transcriptase (RT) region of the hepatitis B virus (HBV) is the target of antiviral treatment. However, the discrepancy in RT mutations between nucleos(t)ide analogue (NA)-treated and -untreated chronic hepatitis B (CHB) patients is un clear. Methods Serum samples were collected from 119 NA-treated and 135 NA-untreated patients. The sampling time was decided by the clinician. Full-length HBV RT regions were amplified using nest polymerase chain reaction. The mutations within the RT region were analysed by direct sequencing. Results The incidence of RT mutations in treated patients was higher than that in untreated patients (p&lt;0.05). The classic drug-resistant mutations were detected in 44.5% (53/119) of treated patients, which was significantly higher than in untreated patients (6.7% [9/135]) (p&lt;0.05). The non-classical mutations showed their complexity and diversity in both patient groups. Multiple mutations (three or more) were more frequent in treated patients than in untreated patients (p&lt;0.05). Several novel mutations might be related to NA resistance. Conclusions The selection pressures of NAs accelerated the development of RT mutations, especially within the functional domain. Mutations in the RT region occurred not only at classical sites, but also at other non-classical sites, which might be related to drug resistance and/or viral replication. The biological function and fitness of HBV isolates harbouring these novel mutations need further in vitro and in vivo verification experiments.

scholarly journals Retrospective Analysis of HBV Pre-Existing Mutations and Drug-induced Resistance Mutations in Patients with Hepatitis B Virus-Related Cirrhosis

2019 ◽  
pp. 185-192
Author(s):  
Wang Liping ◽  
Dai Mingjia ◽  
Li Chunyang ◽  
Hao Jungui ◽  
Ji Fang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Resistance Mutation ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Drug Induced ◽  
B Virus ◽  
Treatment Naïve ◽  
Mutation Pattern

Background: The reported prevalence and necessity of detection of HBV reverse transcriptase (RT) mutation prior to treatment is varied and remains controversial. This study aimed to identify the prevalence of HBV pre-existing gene resistance mutations and compare the difference between pre-existing mutations and drug-induced resistance mutations in patients with hepatitis B virus-related cirrhosis.Methods: 180 patients with hepatitis B virus-related cirrhosis which included 68 patients with virological breakthrough and 112 treatment-naive cirrhosis patients were retrospectively enrolled. The drug-resistant mutations of HBV reverse transcriptase domain were screened by direct gene sequencing. One-way ANOVA analysis was performed in the comparison among different groups. Ratios difference was compared with the chi-square test.Results: There were 48 patients (48/112, 42.86%) with drug resistance mutations in nucleoside/nucleotide analogues (NAs) treatment-naive group, 59 patients (59/68, 86.76%) showed drug-resistant mutations in the NAs treatment group. The gene resistance mutation patterns in treatment-naive group were mainly rtS213T, rtV214A, 191V/I and rtN/H238T/D, and the types of resistance mutations in the treated group were different. The adefovir (ADV) group: mainly rtA181T/V and rtS213T; lamivudine/ telbivudine (LAM/LDT) group: rtL180M+ rtM204I/V/S and rtM204I/V/S or a complex mutation pattern containing 204 site; entecavir (ETV) group: The drug resistance pattern is the simultaneous presence of multiple site mutations. LAM/LDT sequential ADV group: The variant type was multi-site and resistant to both ADV and LAM.Conclusion: There was a prevalence of pre-existing mutations in RT region of HBV polymerase in patients with hepatitis B virus-related cirrhosis, The mutation pattern is mainly related to LAM and ADV-related compensatory mutations, while the drug-induced mutation pattern is more complicated, mainly related to the antiviral drugs used and there are mainly primary mutations. Patients with cirrhosis should be tested genetic resistance mutation before using antiviral drugs.

scholarly journals The L80I Substitution in the Reverse Transcriptase Domain of the Hepatitis B Virus Polymerase Is Associated with Lamivudine Resistance and Enhanced Viral Replication In Vitro

2007 ◽  
Vol 51 (7) ◽  
pp. 2285-2292 ◽  
Author(s):  
Nadia Warner ◽  
Stephen Locarnini ◽  
Michael Kuiper ◽  
Angeline Bartholomeusz ◽  
Anna Ayres ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Resistance Mutations ◽  
Hbv Genotype ◽  
Lamivudine Resistance ◽  
Replication Efficiency ◽  
B Virus ◽  
High Level

ABSTRACT Long-term lamivudine (LMV) treatment of chronic hepatitis B almost inevitably engenders viral resistance. Mutations that result in the replacement of the methionine at position 204 of the deoxynucleoside triphosphate-binding site of the hepatitis B virus (HBV) reverse transcriptase (rt) by isoleucine, valine, or (rarely) serine (rtM204I/V/S) confer high-level resistance to LMV but reduce replication efficiency. The subsequent selection or coselection of secondary mutations that partially restore replication efficiency is common and may influence drug resistance. Genotyping has shown that LMV treatment can select for HBV rtL80V/I mutants, but their prevalence and phenotype have not been documented. Analysis of a large sequence database revealed that rtL80V/I occurred almost exclusively in association with LMV resistance, and 85% of these isolates encoded rtL80I. Coselection of rtL80V/I occurred in 46% of isolates in which LMV resistance was attributable to rtM204I but only 9% of those in which resistance was attributable to rtM204V. Moreover, rtL80V/I did not occur in HBV genotype A isolates but occurred at similar frequencies in genotype B, C, and D isolates. In vitro phenotyping showed that although the rtL80I mutant by itself replicated less efficiently and was hypersensitive to LMV compared to the replication efficiency and sensitivity of its wild-type parent, the presence of rtL80I enhanced the replication efficiency of rt204I/V mutants without significantly affecting LMV resistance. Molecular modeling revealed that rt80 does not interact directly with the enzyme's substrates. Collectively, these results suggest that coselection of rtL80V/I and rtM204I/V occurs because the former compensates for the loss of replication efficiency associated with the acquisition of LMV resistance, particularly in the case of rtM204I.

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