Lamivudine-resistant Mutants Research Articles

Phylogenetic Analysis and Emerging Drug Resistance against Different Nucleoside Analogues in Hepatitis B Virus Positive Patients.

Several nucleotide analogues have been approved for use in treating hepatitis B virus (HBV) infection. Long-term exposure to therapy leads to the emergence of mutations within the HBV DNA polymerase gene, resulting in drug resistance, a major factor contributing to therapy failure. Chronic HBV patients from the Khyber Pakhtunkhwa province, Pakistan, who had completed 6 months of therapy participated in this study. Samples were collected from 60 patients. In this study, the entire reverse transcriptase domain of the HBV polymerase gene was amplified using nested polymerase chain reaction and sequenced. Drug-resistant mutations were detected in nine (22.5%) patients. All of these patients had lamivudine-resistant mutations (rtM204V + L180M), while seven individuals (17.5%) had both lamivudine- plus entecavir-resistant mutations (L180M + M204V + S202G). N236T, a mutation that gives rise to tenofovir and adefovir resistance, was observed in two (5%) patients. T184A, a partial drug-resistant mutation to entecavir, was found in five (12.5%) patients. Furthermore, other genotypic variants (100%) and vaccine escape mutations (5%) were additionally observed. Moreover, pN459Y (35%), pN131D (20%), pL231S (20%), pP130Q (17.5%), pS189Q (12.5%), pP161S (5%), pH160P (2.5%), pT322S (2.5%), and pA223S (2.5%) mutations in the polymerase gene, as well as sA166V (17.5%), sQ181K (12.5%), sV184R (7.5%), sA17E (5%), sP153S/K (5%), sW156C (5%), sC76Y (2.5%), and S132F (2.5%) mutations in the small surface gene, were identified for the first time in this study. Phylogenetic analysis showed that genotype D was predominant amongst the HBV carriers. Subtype D1 was found in most patients, while two patients were subtype D9. These novel findings may contribute to the body of knowledge and have clinical significance for treating and curing HBV infections in Pakistan.

High frequency of Lamivudine and Telbivudine resistance mutations in hepatitis B virus isolates from human immunodeficiency virus co-infected patients on highly active antiretroviral therapy in Bucaramanga, Colombia.

Hepatitis B virus (HBV) antiviral Resistance-Associated Mutations (RAMs) in human immunodeficiency virus (HIV) coinfected patients undergoing highly active antiretroviral therapy (HAART) are complex and incompletely understood. We aimed to determine the prevalence of HBV coinfection, HBV genotypes, and RAMs in a cohort of people living with HIV (PLWH) in the northeastern region of Colombia. This cross-sectional study was carried out between February 2013 and February 2014. Virological, immunological and HAART data were collected from clinical records. In-house nested PCR and Sanger sequencing of the HBV pol gene were used to identify coinfections, genotypes, RAMs and HBV s antigen (HBsAg) escape mutants. Among 275 PLWH, HBV coinfection was confirmed in 32 patients (11.6%), of whom nine (28.2%) were HBsAg positive (active hepatitis B), and 23 (71.8%) were occult hepatitis B infections (OBI). All HBV sequences (n = 23) belonged to the genotype F3. Among HIV/HBV coinfections, 71.9% had CD4+ T cell counts above 200 cells/mm3 and 37.5% had undetectable HIV viral loads. The RAMs rtL80I, rtL180M, and rtM204V, which confer resistance to Lamivudine/Telbivudine and partially resistant to Entecavir, were found in all HBV isolates. An unknown rt236Y mutation to Tenofovir was also identified. Most patients under HAART received first-generation HBV antiviral therapy with a low genetic barrier to resistance. Antiviral Drug-associated Potential Vaccine-escape Mutations (ADAPVEMs) in the S gene were observed in all isolates ranging from 1-20 amino acid substitutions. However, no vaccine escape mutants were detected. In Conclusion, these findings highlight the importance of HBV molecular screening, antiviral resistance monitoring and new guidelines for PLWH to overcome RAMs and prevent HBV-related liver disease.

HIV-1 resistance against dolutegravir fluctuates rapidly alongside erratic treatment adherence: a case report

We report a case of incomplete HIV-1 suppression on a dolutegravir, lamivudine, and abacavir single-tablet regimen with the emergence of the H51Y and G118R integrase resistance mutations. Integrase sequencing was performed retrospectively by Sanger and next-generation sequencing. Rates of emergence and decline of resistance mutations were calculated using next-generation sequencing data. Dolutegravir plasma concentrations were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of H51Y and G118R on infectivity, fitness, and susceptibility to dolutegravir were quantified using cell-based assays. During periods of non-adherence to treatment, mutations were retrospectively documented only by next-generation sequencing. Misdiagnosis by Sanger sequencing was caused by the rapid decline of mutant strains within the retroviral population. This observation was also true for a M184V lamivudine-resistant reverse transcriptase mutation found in association with integrase mutations on single HIV genomes. Resistance rebound upon treatment re-initiation was swift (>8000 copies per day). Next-generation sequencing indicated cumulative adherence to treatment. Compared to WT HIV-1, relative infectivity was 73%, 38%, and 43%; relative fitness was 100%, 35%, and 10% for H51Y, G118R, and H51Y+G118R viruses, respectively. H51Y did not change the susceptibility to dolutegravir, but G188R and H51Y+G118R conferred 7- and 28-fold resistance, respectively. This case illustrates how poorly-fit drug-resistant viruses wax and wane alongside erratic treatment adherence and are easily misdiagnosed by Sanger sequencing. We recommend next-generation sequencing to improve the clinical management of incomplete virological suppression with dolutegravir.

Hepatitis B virus genetic multiplicity and the associated HBV lamivudine resistance mutations in HBV/HIV co-infection in Western Kenya: A review article.

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co-infections are common as the two viruses use same routes of transmission. Studies show that HIV infection modifies the natural course of chronic HBV infection, leading to more severe and progressive liver disease, and a higher incidence of cirrhosis, liver cancer and mortality. Therefore, determining HBV status and genotypes among HIV co-infected patients would improve their therapeutic management. This article reviewed the HBV genetic multiplicity and the associated HBV Lamivudine resistance mutations in HBV/HIV co-infection in western Kenya. Comprehensive literature searches and analysis were performed in peer-reviewed journals in the National council for biotechnology information (NCBI), PubMed, and Web of science using key words of HIV, Hepatitis B genotypes, HBV/HIV co-infection and Lamivudine resistance. HBV genotype A is predominant. D and E are also present in Kenya and neighboring countries in the region. HBV polymerase rtV173L, rtL180M, and rtM204V major substitutional mutations were identified. Currently, TDF+3TC+DTG are recommended for treatment of HBV/HIV co-infection. Evidence shows that HBV/HIV co-infection places a heavy burden to the society. Along with ART regimen, HBV genotype is a major factor determining the course of disease and treatment outcome. Treating HIV in HBV/HIV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistance mutations. Therefore, improved screening for HBV and extended follow-up of HBV/HIV co-infected individuals is needed to better understand the impact of different ART regimens on clinical outcomes.

Identification and Characterization of Besifovir-Resistant Hepatitis B Virus Isolated from a Chronic Hepatitis B Patient.

Hepatitis B virus (HBV) is known to cause severe liver diseases such as acute or chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis B (CHB) infection is a major health problem with nearly 300 million individuals infected worldwide. Currently, nucleos(t)ide analogs (NAs) and interferon alpha are clinically approved treatments for HBV infection. NAs are potent antiviral agents that bind to HBV polymerase and block viral reverse transcription and replication. Besifovir dipivoxil maleate (BSV) is a newly developed NA against HBV in the form of acyclic nucleotide phosphonate that is available for oral administration similar to adefovir and tenofovir. Until now, resistance to BSV treatment has not been reported. In this study, we found a CHB patient who showed viral breakthrough after long-term treatment with BSV. The isolated HBV DNA from patient’s serum were cloned into the replication-competent HBV 1.2 mer and the sequence of reverse transcriptase (RT) domain of HBV polymerase were analyzed. We also examined the drug susceptibility of generated clones in vitro. Several mutations were identified in HBV RT domain. A particular mutant harboring ten RT mutations showed resistance to BSV treatment in vitro. The ten mutations include rtV23I (I), rtH55R (R), rtY124H (H), rtD134E (E), rtN139K (K), rtL180M (M), rtM204V (V), rtQ267L (L), rtL269I (I) and rtL336M (M). To further identify the responsible mutations for BSV resistance, we performed in vitro drug susceptibility assay on several artificial clones. As a result, our study revealed that rtL180M (M) and rtM204V (V) mutations, already known as lamivudine-resistant mutations, confer resistance to BSV in the CHB patient.

Evaluation of Lamivudine Resistance Mutations in HBV/HIV Co-infected Patients

Evaluation of Lamivudine Resistance Mutations in HBV/HIV Co-infected Patients

Molecular and computational analysis of natural drug resistance mutations among Moroccan chronic hepatitis B carriers

Despite the effectiveness of nucleotide analogs (NAs), drug resistance and cccDNA inaccessibility are still significant obstacles to long-term efficacy of antiviral therapy in chronic hepatitis B infection (CHB). Mutations pre-exist spontaneously in Hepatitis B Virus (HBV) reverse transcriptase (RT) gene in minor viral populations and can emerge to confer drug resistance and treatment failure. This study aimed to analyze mutations within the HBV polymerase gene in treatment-naive Moroccan patients with CHB.The HBV polymerase gene was analyzed by nested-PCR and direct sequencing in 123 subjects. A computational analysis was performed to confirm the effect of detected mutations on targeted NAs, using homology and docking tools.Sixty samples were successfully amplified by PCR. Analyses revealed an intermediate rate (13%) of pre-treatment NA resistance mutations and a high frequency of putative resistance mutations (65%). Among these, 56% were lamivudine resistance mutations and 44% adefovir resistance mutations. Two pretreatment resistance mutations, “Y124N” and “N139D/K/H/Q” were detected in 8/60 patients, and 11 putative resistance mutations (N53S/T/D, L91I, H126Y, T128I, V207I, V214A, Q215P/S, L217R, F221Y, L229V, N238H/T) in 39/60 patients, where two have a combination of F221Y and A194T “a classical NA drug resistance mutation.” The computational analysis demonstrated that the combination of A194T + F221Y mutations on HBV RT sequence does not affect its susceptibility to ADV.These results confirm that HBV mutations conferring resistance against currently available anti-HBV NA predate treatment in naïve Moroccan patients. The high rate of non-classical NA resistance mutations needs further in vitro phenotype analysis.

Comprehensive Analysis of Clinically Significant Hepatitis B Virus Mutations in Relation to Genotype, Subgenotype and Geographic Region

Hepatitis B virus (HBV) is a highly variable DNA virus due to its unique life cycle, which involves an error-prone reverse transcriptase. The high substitution rate drives the evolution of HBV by generating genetic variants upon which selection operates. HBV mutants with clinical implications have been documented worldwide, indicating the potential for spreading and developing their own epidemiology. However, the prevalence of such mutants among the different HBV genotypes and subgenotypes has not been systematically analyzed. In the current study, we performed large-scale analysis of 6,479 full-length HBV genome sequences from genotypes A-H, with the aim of gaining comprehensive insights into the relationships of relevant mutations associated with immune escape, antiviral resistance and hepatocellular carcinoma (HCC) development with HBV (sub)genotypes and geographic regions. Immune escape mutations were detected in 10.7% of the sequences, the most common being I/T126S (1.8%), G145R (1.2%), M133T (1.2%), and Q129R (1.0%). HBV genotype B showed the highest rate of escape mutations (14.7%) while genotype H had no mutations (P < 0.001). HCC-associated mutations were detected in 33.7% of the sequences, with significantly higher frequency of C1653T, T1753V and A1762T/G1764A in genotype G than C (P < 0.001). The overall frequencies of lamivudine-, telbivudine-, adefovir-, and entecavir-resistant mutants were 7.3, 7.2, 0.5, and 0.2%, respectively, while only 0.05% showed reduced susceptibility to tenofovir. In particular, the highest frequency of lamivudine-resistant mutations was observed in genotype G and the lowest frequency in genotype E (32.5 and 0.3%; P < 0.001). The prevalence of HBV mutants was also biased by geographic location, with North America identified as one of the regions with the highest rates of immune escape, antiviral resistance, and HCC-associated mutants. The collective findings were discussed in light of natural selection and the known characteristics of HBV (sub)genotypes. Our data provide relevant information on the prevalence of clinically relevant HBV mutations, which may contribute to further improvement of diagnostic procedures, immunization programs, therapeutic protocols, and disease prognosis.

Dolutegravir plus lamivudine for maintenance of HIV viral suppression in adults with and without historical resistance to lamivudine: 48-week results of a non-randomized, pilot clinical trial (ART-PRO)

BackgroundWe investigated the efficacy of a switch to dolutegravir plus lamivudine in aviremic individuals without evidence of persistent lamivudine resistance-associated mutations in baseline proviral DNA population sequencing.MethodsOpen-label, single-arm, 48-week pilot trial. HIV-1 infected adults, naïve to integrase inhibitors, with CD4+ above 350 cell/μL and fewer than 50 HIV-1 RNA copies per mL the year prior to study entry switched to dolutegravir plus lamivudine. Participants were excluded if baseline proviral DNA population genotyping detected lamivudine resistance-associated mutations. To detect resistance minority variants, proviral DNA next-generation sequencing was retrospectively performed from baseline samples. Primary efficacy endpoint was proportion of participants with fewer than 50 HIV-1 RNA copies per mL at week 48. Safety and tolerability outcomes were incidence of adverse events and treatment discontinuations. ART-PRO is registered with ClinicalTrials.gov, NCT03539224.Findings41 participants switched to dolutegravir plus lamivudine, 21 with lamivudine resistance mutations in historical plasma genotypes. Baseline next-generation sequencing detected lamivudine resistance mutations (M184V/I and/or K65R/E/N) over a 5% threshold in 15/21 (71·4%) and 3/20 (15%) of participants with and without history of lamivudine resistance, respectively. At week 48, 92·7% of participants (38/41) had fewer than 50 HIV-1 RNA copies per mL. There were no cases of virologic failure. Three participants with historical lamivudine resistance were prematurely discontinued from the study (2 protocol violations, one adverse event). Ten participants (4 in the group with historical lamivudine resistance) had a transient viral rebound, all resuppressed on dolutegravir plus lamivudine. There were 28 drug-related adverse events, only one leading to discontinuation.InterpretationIn this pilot trial, dolutegravir plus lamivudine was effective in maintaining virologic control despite past historical lamivudine resistance and presence of archived lamivudine resistance-associated mutations detected by next generation sequencing. Further studies are needed to confirm our results.FundingFondo de Investigaciones Sanitarias, Instituto de Salud Carlos III PI16/00837-PI16/00678.

Prevalence and molecular characterization of occult hepatitis B virus in pregnant women from Gondar, Ethiopia

BACKGROUND: The greatest risk of chronic hepatitis B (CHB) is from mother-to-child transmission. Approximately 20% of individuals in sub-Saharan Africa are hepatitis B virus (HBV) surface antigen–positive (HBsAg+), but the prevalence of occult hepatitis B (OHB) is unknown. Aim: This study investigated CHB and OHB prevalence and viral variants in a cohort of pregnant women in Gondor, Ethiopia. METHODS: Patients were prospectively recruited from the University of Gondar Hospital ( N = 200; median age 27 [inter-quartile range] 8.3y) from March through June 2016. Data were collected using an investigator-administered questionnaire. Plasma was tested for HBsAg and HBV core antibody (anti-HBc), and HBV genotype and presence of HBV variants (ie, vaccine escape mutants [VEMs]) were determined by polymerase chain reaction, Sanger sequencing, and phylogenetic analysis. RESULTS: Of women tested, 1% (2/200) were HBsAg+; 26.8% (47/182) of HBsAg-negative patients were anti-HBc+, of whom 37/47 (78.7%) had detectable HBV DNA. The overall rate of OHB was 20.3%. Both HBsAg+ cases were HBV genotype D, and 36/37 (97.3%) of OHB individuals were genotype D. None carried VEM, but both HBsAg+ cases and 32/37 (86.5%) of the OHB cases showed lamivudine-resistant mutations. CONCLUSIONS: Twenty-seven percent of pregnant women in this cohort showed evidence of CHB or prior HBV exposure (ie, HBsAg+ or anti-HBc+) and clinically relevant HBV variants. Data from this single-centre study suggests high HBV prevalence, reinforcing the World Health Organization’s recommendation for universal prenatal HBV screening and infant vaccination.

The Prevalence of Hepatitis B and D Viruses and Evaluating YMDD Mutation in HBV-Suspected Patients in Qom Province, Iran

Background: Hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are known as major health concerns world-wide. Nucleoside analogs (NAs) are widely used in anti-HBV therapy. However, HBV resistance to Nas may result in disease recurrence. Objectives: The present study aimed to evaluate the prevalence of HBV and HDV infections, and to investigate the presence of YMDD motif in HBV-infected patients. Methods: In this cross-sectional study, a total of 1600 blood specimens were collected from HBV-suspected patients, who referred to diagnostic centers in Qom Province, Iran from July 2018 to March 2019 before and after lamivudine therapy. The presence of HBV in the specimens was investigated using ELISA and nested PCR assays. Also, HDV and YMDD motif were detected in HBV-infected patients using the PCR technique. In order to confirm the presence of HDAg gene of HDV, the PCR products of some HBV/HDV-positive specimens were subjected to direct sequencing. Results: The number of HBsAg positive patients was 180 (11.25%) (P < 0.05) according to ELISA and nested PCR assays; these patients were in the age range of 32 - 38 years. Also, 11 (6.1%) patients were infected with HBV/HDV. The frequency of lamivudine resistance mutation in the YMDD region was 28 (15.5%) among chronic HBV patients. The constructed phylogenetic trees for HDAg gene of HDV showed that most HBV/HDV isolates had similar sequences to the Iranian isolates. Conclusions: The present results showed that it is important to increase our knowledge about the frequency of chronic HBV and HDV infections. Also, development of new antiviral medications is important for prevention and prompt and appropriate treatment.

361. Residual Lamivudine-Resistant Hepatitis B Virus Detected on Next-Generation Sequencing of Treatment-Experienced HIV Patients Failing Antiretrovirals

BackgroundHepatitis B is highly prevalent in the Philippines, with 17% of the population infected. With the fastest-growing HIV epidemic in the Asia-Pacific, 12% of HIV patients are HBsAg reactive. With the use of lamivudine and tenofovir-based antiretrovirals (ARVs), hepatitis B virus (HBV) treatment in co-infected HIV patients is not usually an issue. However, there is a potential to develop HBV resistance when patients are switched off tenofovir when antiretroviral resistance develops. With high rates of acquired K65R tenofovir resistance, the potential for inadvertently causing re-emergence of lamivudine-resistant HBV is present. We report two HIV patients with residual whole-genome HBV with lamivudine and telbivudine resistance mutations.MethodsAs part of a surveillance study on acquired drug resistance in the Philippines, samples with an HIV viral load >1,000 copies underwent Sanger sequencing of RT and PR for genotyping and HIV drug-resistance testing. Near-whole-genome next-generation sequencing (NGS) for HIV using Illumina HiSeq was also performed on these samples.ResultsTwo patients had coincidental whole-genome amplification of HBV on NGS (Table 1). HBV serology for both showed reactive anti-HBsAg and non-reactive HBsAg and Anti-HBc. The two HBV samples were genotype A and were resistant to lamivudine and telbivudine, with intermediate resistance to entecavir.ConclusionResidual HBV may be present in patients on ARVs. Antibody responses for HBV serology may not be very reliable in highly immunosuppressed patients. The potential of lamivudine-resistant HBV to emerge when HIV patients are shifted off tenofovir due to resistance in patients should be considered when deciding on second-line ARVs. Disclosures All authors: No reported disclosures.

Design, synthesis, and biological evaluation of novel 2′-deoxy-2′-fluoro-2′-C-methyl 8-azanebularine derivatives as potent anti-HBV agents

Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent. In this paper, a series of novel 2′-deoxy-2′-fluoro-2′-C-methyl-β-d-arabinofuranosyl 8-azanebularine analogues (1 and 2a) and N4-substituted 8-azaadenosine derivatives (2b-g) were designed, synthesized and screened for in vitro anti-HBV activity. Two concise and practical synthetic routes were developed toward the structural motif construction of 2′-deoxy-2′-fluoro-2′-C-methyl-β-d-arabinofuranosyl 8-azainosine from the ribonolactone 3 under mild conditions. The in vitro anti-HBV screening results showed that these 8-azanebularine analogues had a significant inhibitory effect on the expression of HBV antigens and HBV DNA at a concentration of 20 μM. Among them, halogen-substituted 8-azaadenosine derivative 2g displayed activities comparable to that of 3TC. In particular, 2g retained excellent activity against lamivudine-resistant HBV mutants.

Hepatitis B virus mutation pattern rtL180M+A181C+M204V may contribute to entecavir resistance in clinical practice

ABSTRACT Background and Aims: Entecavir (ETV) resistance of hepatitis B virus (HBV) conventionally requires rt184, 202, or 250 mutations plus lamivudine-resistance mutation (rtM204V/I ± L180M). This study aimed to clarify whether rtL180M+A181C+M204V mutations may contribute to HBV ETV resistance. Methods: Serum samples were collected from 22,009 patients who underwent resistance testing in Beijing 302 Hospital from 2007 to 2016. HBV reverse transcriptase (RT) gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility. Results: Classical ETV-resistance mutations of HBV were detected in 1252 patients who were receiving ETV therapy. The rtA181C mutation was detected with rtL180M+M204V mutations in 18 lamivudine-experienced ETV-treated patients, and the emergence of the mutations was associated with virological breakthrough or inadequate virological response to ETV. Patient-derived representative rtA181C-containing mutants, rtL180M+A181C+M204V, rtL180M+A181C+M204V+M250V, and rtL180M+A181C+S202G+M204V, exhibited 45.7%, 25.9%, and 25.0% replication capacity and 85.6-, 356.1-, and 307.1-fold decreased susceptibility to ETV respectively compared to the wild-type strain, while the three mutants remained sensitive to tenofovir (TDF). Artificial elimination of rtA181C largely restored the rtL180M+A181C+M204V mutant’s sensitivity to ETV. Molecular modelling of viral RT binding to ETV showed that the rtL180M+A181C+M204V mutant had a less stable conformation compared to rtL180M+M204V mutant. In clinical practice, undetectable serum HBV DNA was achieved in two of five longitudinally followed rtA181C-positive patients who received switching-to TDF therapy, but not in the other three who received add-on adefovir therapy during observation. Conclusions: Both clinical and experimental data support rtL180M+A181C+M204V as a novel non-classical ETV-resistance mutation pattern.

Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it “HepG2.RL1”. Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

Clinical observation of continuous and intermittent application of lamivudine or entecavir resistance mutations in patients with chronic hepatitis B

Objective: To observe continuous and intermittent application of lamivudine or entecavir resistance mutations in patients with chronic hepatitis B. Methods: Data of patients with active stage of chronic hepatitis B over the past 6 years were collected and analyzed retrospectively. The incidence of drug resistance mutation and related factors between patients taking LAM or ETV continuously and intermittently were compared with those taking LAM or ETV. Data comparison was performed using χ(2) test. Results: Patients with HBV DNA≥10(5) copies / ml at the time of initial treatment had higher resistance mutation rates than those with HBV DNA < 10(5) copies / ml at either continuous or intermittent treatment, and patients with intermittent treatment had higher resistance mutation rates than those with continuous treatment. Simultaneously, the incidence of drug resistance mutation in LAM and ETV in the first, second and third years were significantly higher in intermittent treatment than that of continuous treatment (P < 0.05). There was a positive correlation between the frequency of drug withdrawal and the rate of drug resistance mutation. There were no individual difference and drug difference between LAM and ETV. Conclusion: In the treatment of chronic hepatitis B with oral nucleoside analogues, drug resistance may occur in either continuous or intermittent treatment. When comparing continuous with intermittent treatment, it suggests that intermittent is more likely to cause viral resistance mutation.

Comparison of replication competence of wild-type and lamivudine-resistant hepatitis B virus isolates from a chronic hepatitis B patient

In lamivudine-refractory chronic hepatitis B (CHB) patients, discontinuation of lamivudine therapy may lead to loss of lamivudine-resistant hepatitis B virus (HBV) and reappearance of wide-type HBV as dominant strains, yet the underlying mechanism remains unclear. In this study, we cloned wide-type and lamivudine-resistant HBV genomes from the sera of a CHB patient who stopped lamivudine therapy after occurrence of resistant virus and determined the biologic properties of the two isolates in hepatoma cell lines. Sequencing reverse transcriptase region of HBV revealed that the patient developed lamivudine-resistant mutations (rtV173 L, rtL180 M, and rtM204 V) 36 months after the start of lamivudine therapy, and lamivudine-resistant mutants reversed to wild-type after the treatment was stopped for 8 months. Our data showed that the wild-type and mutant isolates had similar transcriptional and translational activity. However, comparison of intracellular and released HBV DNA levels showed that replication efficiency of the mutant virus was approximately 50% of wild-type HBV, while the infectivity of released virus was not affected by the lamivudine-resistant mutations. In conclusion, the reversion of lamivudine-resistant mutants to wild-type HBV after discontinuation of lamivudine in hepatitis B patients may be attributed to better replication fitness of wild-type HBV.

Hepatitis B virus rtA181T/sW172non-stop mutation may increase resistance fold to adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation

The study aimed to characterize rtA181T/sW172stop (*) and rtA181T/sW172non-stop mutations of hepatitis B virus (HBV). Total of 22,009 patients who visited Beijing 302 Hospital from 2007 to 2016 were enrolled. These patients all received nucleos(t)ide analogues (NAs) treatment and their serum samples were collected for sequence analysis of HBV reverse-transcriptase (RT) and S regions. The rtA181T mutation was detected in 5.37% (1182/22,009) of the patients' samples. The rtA181T-causative sW172*, sW172non-stop (sW172 L/S), and mixed sW172*/non-stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively. The patients with rtA181T/sW172non-stop mutants had a higher HBV DNA level compared to those with rtA181T/sW172* mutants. 44.33% (524/1182) rtA181T-positive samples were detected with signature drug-resistant mutations, including 325 with adefovir-resistant mutation rtA181V/N236T, 57 with lamivudine-resistant mutation rtM204V/I, 99 with entecavir-resistant mutation rtM204V/I plus rt184/202/250 substitution(s), and 43 with multidrug-resistant mutation rtA181V/N236T + rtM204V/I ± rt184/202/250 substitution(s). The rtA181T/sW172non-stop mutation had a higher ratio of coexistence with adefovir-resistant mutation compared to rtA181T/sW172* mutation (42.86% vs. 24.59%, P < 0.05). rtA181T/sW172S + rtN236T and rtA181T/sW172L + rtN236T mutants exhibited higher HBV DNA production and adefovir resistance fold than that of rtA181T/sW172* + rtN236T mutant (98.02% and 85.5% vs. 42.1% in HBV DNA production, and 7.38-fold and 5.49-fold vs. 3.69-fold in half maximal effective concentration of wild-type strain); rtA181T/sW172L + rtS202G + rtM204V strain exhibited higher HBV DNA production and entecavir resistance fold than that of rtA181T/sW172* + rtS202G + rtM204V strain (50.98% vs. 34.49%, 524.00-fold vs. 69.33-fold). In conclusion, rtA181T/sW172non-stop mutation may increase resistance fold of adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation and might influence clinical presentation of NAs-treated patients.

High Prevalence of HBV Lamivudine-Resistant Mutations in HBV/HIV Co-Infected Patients on Antiretroviral Therapy in the Area with the Highest Prevalence of HIV/HBV Co-Infection in China

Objectives: We aimed to determine the prevalence of hepatitis B virus (HBV) drug-resistant mutations in patients co- infected with HBV/human immunodeficiency virus (HIV), including both drug-naïve subjects and those who received antiretroviral therapy (ART) in Guangxi, where the prevalence of HIV/HBV co-infection is highest in China. Methods: Two hundred and three subjects co-infected with HBV/HIV were recruited, including 123 drug-naïve patients (group 1) and 80 who received ART (group 2). The polymerase gene of HBV in the serum of all study subjects was analysed. Results: The results showed that the prevalence of HBV drug-resistant mutations in group 2 (76.5%, 95% CI 56.3–96.7) was significantly higher than that in group 1 (1.4%, 95% CI –1.4 to 4.2; χ² = 50.955, p < 0.05). The major pattern of lamivudine (3TC)-resistant mutations is L180M+M204I+L80I (35.7%). In total, 95% of subjects with resistant mutations had cross-resistance to telbivudine and entecavir. No putative tenofovir disoproxil fumarate (TDF) resistance change was found. Five subjects (6.5%) in group 2 had HBV viral loads over 10 × 10⁶ copies/mL. Four of them had 3TC-resistant mutations. Multivariate analysis showed that ART was the only factor associated with the development of drug-resistant mutations. Conclusion: Treating HIV in HIV/HBV co-infection with antiretroviral agents may result in a very high prevalence of HBV 3TC-resistant mutations. TDF could not completely suppress HBV replication.

Design, synthesis, and biological evaluation of new 1,2,3-triazolo-2′-deoxy-2′-fluoro- 4′-azido nucleoside derivatives as potent anti-HBV agents

Novel drugs are urgently needed to combat hepatitis B virus (HBV) infection due to drug-resistant virus. In this paper, a series of novel 4-monosubstituted 2′-deoxy-2′-β-fluoro-4′-azido-β-d-arabinofuranosyl 1,2,3-triazole nucleoside analogues (1a-g) were designed, synthesized and screened for in vitro anti-HBV activity. At 5.0 μM in the cellular model, all the synthetic compounds display activities comparable to that of the positive control, lamivudine at 20 μM. Of the compounds tested, the amide-substituted analogue (1a) shows the most promising anti-HBV activity and low cytotoxicity in the cell model. In particular, it retains excellent activity against lamivudine-resistant HBV mutants. In duck HBV (DHBV)-infected duck models, both the serum and liver DHBV DNA levels (67.4% and 53.3%, respectively) were reduced markedly by the treatment with 1a. Analysis of the structure of HBV polymer/1a-triphosphate (1a-TP) complex shows that 1a-TP is stabilized by specific van der Waals interactions with the enzyme residues arising from 4-amino-1,2,3-triazole and the 4′-azido group.