rtM204V Mutation Research Articles

Susceptibility of Drug Resistant Hepatitis B Virus Mutants to Besifovir.

Currently, interferon alpha and nucleos(t)ide analogues (NAs) are clinically available to treat hepatitis B virus (HBV) infection. Several NAs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV) and tenofovir (TDF or TAF) have been approved and administered to chronic hepatitis B (CHB) patients. NAs inhibit HBV DNA synthesis by targeting the reverse transcriptase (RT) domain of HBV polymerase. Several mutations in the RT domain which lead to drug resistance against NAs have been reported, even for TDF and TAF which are highly potent with very low resistance rate. Besifovir (BFV) is a new antiviral dGMP analogue able to be used as a new NA drug for the control of CHB infection. Drug resistance to BFV is not well known due to its shorter duration of clinical use. Recently, we reported that rtL180M (M) and rtM204V (V) mutations, already resistant to LMV, are associated with BFV resistance. However, the susceptibility to BFV of previously known HBV mutants resistant to various drugs has not been studied. To investigate this, we performed in vitro drug susceptibility assays using natural and artificial mutants that are associated with resistance to LMV, ADV, ETV or TDF. As a result, LMV-resistant mutants were not susceptible to BFV and ETV-resistant clones showed partial resistance against BFV as well. However, ADV-resistant mutants were highly sensitive to BFV. In case of tenofovir-resistant mutations, the HBV mutants harboring primary mutations to tenofovir resistance were susceptible to BFV. Therefore, our study revealed that BSV may serve as an alternative drug for patients with ADV-, ETV-, TDF- or TAF-resistance.

A small molecule targeting hepatitis B surface antigen inhibits clinically relevant drug-resistant hepatitis B virus.

Currently approved oral antivirals for chronic HBV infection target the reverse transcriptase (RT) domain of the HBV polymerase. Emergence of drug resistance has been reported in a small proportion of chronic HBV patients on prolonged treatment with antivirals. We recently reported ZINC20451377, a small molecule targeting hepatitis B surface antigen (HBsAg) that effectively inhibits both WT HBV and tenofovir-resistant HBV. Due to the partial overlap between the RT domain and HBsAg, drug-resistant mutants are associated with corresponding mutations in HBsAg. To evaluate the efficacy of ZINC20451377 against nine clinically relevant drug-resistant HBV mutants that lead to simultaneous mutations in the overlapping HBsAg gene. Huh7 cells were transfected with 1.2× HBV replicons corresponding to WT HBV or drug-resistant HBV mutants and treated with different concentrations of ZINC20451377. We assessed the IC50 values of ZINC20451377 for HBsAg levels in the culture supernatants using ELISAs. HBV secretion was measured by immunocapture of secreted virions followed by real-time PCR quantitation of virion-associated DNA. ZINC20451377 led to a dose-dependent inhibition of secreted HBsAg encoded by WT HBV and all nine drug-resistant mutants tested and the IC50 values were in the low micromolar range. ZINC20451377 inhibited HBV secretion from drug-resistant mutants except for mutants harbouring the rtL180M + rtM204V (MV) mutation. The small molecule ZINC20451377 inhibits HBsAg and virion secretion in some of the clinically relevant drug-resistant HBV mutants. ZINC20451377 has a modest overall effect, and it was not effective against the MV mutants (lamivudine- and entecavir-resistant mutants).

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.

Entecavir-resistant hepatitis B virus decreases surface antigenicity: A full genome and functional characterization.

Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. The rtL180M+rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.

HBV genotypes and drug resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected patients.

The presence of HBV resistance mutations upon initiation or during antiretroviral therapy (ART) in HIV-coinfected patients is an important determinant of treatment response. The main objective of the study was to determine the prevalence of HBV resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected Ghanaian patients with detectable HBV viraemia. HBV-HIV-coinfected patients who were ART-naive or had received at least 9 months of lamivudine (3TC)-containing ART were enrolled in a cross-sectional study. Demographic and clinical data were collected and HBV DNA quantified. Partial HBV sequences were amplified by PCR and sequenced bi-directionally to obtain a 2.1-2.2 kb fragment for phylogenetic analysis of HBV genotypes and evaluation of drug resistance mutations. Of the 100 HBV-HIV-coinfected study patients, 75 were successfully PCR-amplified, and 63 were successfully sequenced. Of these 63 patients, 27 (42.9%) were ART-experienced and 58 (92.1%) had HBV genotype E. No resistance mutations were observed in the 36 ART-naive patients, while 21 (77.8%) of 27 treatment-experienced patients had resistance mutations. All patients with resistance mutations had no tenofovir in their regimens, and 80% of them had HIV RNA <40 copies/ml. The 3TC resistance mutations rtL180M and rtM204V were observed in 10 (47.6%) of the 21 patients, while 5 patients (23.8%) had rtV173L, rtL180M and rtM204V mutations. A high proportion of HBV-HIV-coinfected patients with detectable viraemia on 3TC-containing ART had resistance mutations despite good ART adherence as determined by HIV RNA suppression. This study emphasizes the need for dual therapy as part of a fully suppressive ART in all HBV-HIV-coinfected patients in Ghana.

Association between clinical features and YMDD mutations in patients with chronic hepatitis B following lamivudine therapy.

The aim of the present study was to investigate the correlation between feature and genotype with regard to the tyrosine-methionine-aspartate-aspartate (YMDD) mutation in chronic hepatitis B patients after lamivudine (LAM) therapy. A total of 30 patients with chronic hepatitis B were recruited, who underwent one year of LAM therapy. The patients' alanine aminotransferase (ALT) level and hepatitis B envelope antigen (HBeAg) seroconversion were evaluated, hepatitis B virus (HBV) DNA was genotyped using a new genotyping method and YMDD mutations were analyzed prior to treatment and at 6 and 12 months after LAM treatment. Furthermore, the secondary protein structure of the HBV DNA polymerase gene (P gene) was analyzed. Following treatment, the results suggested that LAM therapy improved ALT normalization. There was no correlation between clinical effects and ALT level before treatment. After 12 months treatment, the rate of HBeAg loss increased and the rate of HBeAg seroconversion decreased linearly with the rise of baseline ALT level. While ALT normalization and HBeAg seroconversion were highest in patients with HBV genotype B, HBeAg loss and HBVDNA loss were highest in those with genotype C. The effect was predominant in genotype D. No YMDD mutations were identified prior to 6 months of LAM therapy. The rate of YMDD mutations after 12 months LAM therapy was 12.12%. Two patients with rtM204V + rtL180M belonged to genotype C and another patient with rtL180M alone belonged to genotype D. The turn of secondary protein structure of P gene changed to β sheet when a rtM204V mutation occurred, and no change of secondary protein structure was associated with the rtL180M mutation. Thus, the present results indicate that one year of LAM therapy is able to improve ALT normalization. Long-term LAM therapy may induce YMDD mutation and drug resistance.

Investigation by the Method of INNO-LiPA of Primary Resistance to Lamivudine in Patients with Chronic Hepatitis B Who Have Not Used Antiviral Therapy.

Two billion people around the world are exposed to the hepatitis B virus (HBV) and about 350 million are infected with chronic HBV. The infection can be acquired early (neonatal) and becomes chronic in 90%; this rate reduces to 30% between ages one and five years. There is a 25% risk of chronicity in adults. Nowadays, immunomodulatory and antiviral pegylated-interferons or oral antiviral agents are used in the treatment of chronic hepatitis B. Lamivudine is an effective oral antiviral agent which inhibits the replication of HVB by blocking reverse transcriptase enzyme. The study aims to detect the resistance of HBV to lamivudine in the community and evaluate the effectiveness and suitability of early treatment with lamivudine. One hundred patients who presented to our Faculty of Medicine Hospital Infectious Diseases and Clinical Microbiology Department and had not received any antiviral treatment were recruited. The INNO-LiPA method was applied to investigate primary lamivudine resistance in patients. Seventy-eight patients were HBeAg-negative and 22 patients were HBeAg-positive. A statistically significant correlation was found between HBeAg positivity, alanine aminotransferase (ALT) elevation and HBV DNA (p < 0.05). The rtM204V and L180M mutation motif was found in one patient with HBeAg positivity. Hepatitis B virus in our region is not a lamivudine-resistant strain and early treatment with lamivudine is an effective and convenient method.

High incidence of lamivudine-resistance-associated vaccine-escape HBV mutants among HIV-coinfected patients on prolonged antiretroviral therapy.

Worldwide, frequent emergence of lamivudine (3TC)-resistant HBV mutants has been reported in HIV-HBV-coinfected patients during long-term antiretroviral therapy (ART) that contains 3TC as the sole anti-HBV drug. Three major patterns of mutations in HBV polymerase gene, namely single (rtM204V), double (rtL180M+rtM204V) and triple (rtV173L+rtL180M+rtM204V) mutations, are associated with 3TC-resistance; additionally, the triple mutation has vaccine-escape potential due to a corresponding change in overlapping surface gene. Data from India, a major reservoir for HIV and HBV infection, is lacking. Here we investigated the effect of long-term 3TC treatment on virological response for HBV and characterized the 3TC-resistant HBV mutations in a cohort of HIV-HBV-coinfected patients from eastern India. A cross-sectional study was performed in HIV-infected patients (n=563) receiving 3TC-containing ART for ≥6 months from the major ART centre of eastern India during 2011-2012. The hepatitis B surface antigen-positive HIV-infected patients (n=62) were categorized into four groups with comparable sample size according to the 3TC exposure for ≥6-<12 months (group I; n=15), ≥12-<24 months (group II; n=20), ≥24-<48 months (group III; n=13) and ≥48 months (group IV; n=14). Patients' plasma samples were examined for hepatitis B e antigen (HBeAg), HBV DNA, viral load and covalently closed circular DNA (cccDNA). HBV reverse transcriptase region was sequenced. With a longer period of 3TC exposure, the frequency of HIV-HBV-coinfected patients having HBV DNA suppression decreased. The prevalence of HBeAg-positivity, serum HBV DNA load >2,000 IU/ml and 3TC-resistant mutations simultaneously increased. Remarkably, the 3TC-resistant triple mutation predominated over the double mutation in this cohort (32.26% versus 19.34%) and prevailed in significantly higher frequency among HBV viraemic patients experiencing 3TC for ≥48 months (60% versus 10%; P=0.03). Patients with 3TC-resistant triple mutants had HBV genotype-D, high serum HBV DNA load and elevated alanine aminotransferase level, and presence of cccDNA in their serum. Considering this alarmingly high incidence of 3TC-resistant triple mutation and its possible clinical/public health implications, proper management of 3TC-resistance among HIV-HBV-coinfected patients is an urgent necessity in India.

Generation of a human hepatoma cell line supporting efficient replication of a lamivudine resistant hepatitis B virus

Emergence of lamivudine (LAM) resistance causes treatment failure in patients with chronic hepatitis B and compromise the efficacy of subsequent salvage therapies with other nucleot(s)ide analogs (NAs). Establishment of cell-based assays supporting LAM-resistant hepatitis B virus (HBV) replication will not only provide tools for investigating the replication property, but also screening for antiviral agents efficiently inhibiting the replication of LAM-resistant HBV variants. Accordingly, a human hepatoma (HepG2)-derived cell line was established by stable transfection of a plasmid containing a 1.2 unit length of HBV genome harboring rtL180M and rtM204V mutations that confer LAM resistance. In addition to support efficient viral genome replication, the cell line also produces high levels of HBV virions and subviral particles. As expected, HBV DNA replication in this cell line is completely resistant to lamivudine, but sensitive to adefovir (ADV), entecavir (ETV) and tenofovir (TDF). The cell line is suitable for screening for antiviral agents that inhibit LAM-resistant HBV replication and inhibitors of HBsAg biosynthesis and secretion, which may reduce HBsAg antigenemia and ultimately help to restore host antiviral immune response against HBV and cure chronic HBV infection.

Prevalence and mutational patterns of lamivudine-resistant HBV strains in chronically infected Serbian patients

This study aimed to determine the prevalence and mutational patterns of lamivudine-resistant hepatitis B virus (HBV) strains in chronically infected Serbian patients. The study included 154 patients on long-term lamivudine monotherapy. Resistance-associated mutations were identified by direct sequencing of the S/P gene. The genotypic resistance to lamivudine was confirmed in 54.5% of patients. Three primary resistance-associated mutations were found: rtM204V (55.9%), rtM204I (40.5%), rtA181T (3.6%) and two compensatory mutations rtV173L (17.8%) and rtL180M (67.8%). Seven mutational patterns were discovered with rtL180M+rtM204V being dominant (41.6%). The presence of resistance associated mutations was correlated to the older age of patients, the presence of clinically relevant HBsAg mutations and higher values of viral load. No correlation with HBV genotypes, subgenotypes or HBsAg subtypes was observed. High prevalence of resistance supports the use of genotypic testing in monitoring patients on lamivudine therapy and selecting those who would benefit from therapy with newly developed nucleos(t)ide analogs.

Profile of hepatitis B virus resistance mutations against nucleoside/nucleotide analogue treatment in Chinese patients with chronic hepatitis B

AimAntiviral drug-resistant HBV mutants are complex and currently partly understood. This study was performed to analyze the profile of hepatitis B virus (HBV) resistance mutations against nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB).MethodsThis was a population-based cross-sectional study. Serum samples of 179 patients with virological breakthrough undergoing different NAs treatment were obtained between January 2008 and December 2012. The HBV reverse transcriptase region was sequenced and the following NAs-resistant changes including rtL80, rtI169, rtV173, rtL180, rtA181, rtT184, rtA194, rtS202, rtM204, rtN236 and rtM250 were analyzed.ResultsIn this cohort, 21.2% (38/179) were genotypes B and 78.8% (141/179) were genotypes C; and 89.4% (160/179) of them detected NAs-resistant mutations. The prevalence of HBV mutations at rtM204 was 93.0% (106/114) in patients with lamivudine (LAM) or telbivudine (LdT)-based therapies, and that of rtN236 mutations was 76.1% (35/46) in patients with adefovir dipivoxil (ADV)-based therapies. Among LAM/LdT based therapies, HBV rtM204I was significantly associated with HBV rtL80I/V mutations [rtM204I+rtL80I/V (50.0%, 32/64) vs. rtM204V+rtL80I/V (27.3%,9/33), P=0.032]; while the HBV rtM204V mutations was significantly associated with HBV rtL180M mutations [rtM204V+rtL180M (100%, 33/33) vs. rtM204I+rtL180M (60.9%, 39/64), P<0.001]. Additionally, HBV rtA181 mutations were observed in 19.3% (22/114) of patients with LAM/LdT-based therapy and 23.9% (11/46) of patients with ADV-based therapy.ConclusionsMajority of virological breakthrough is associated with NAs-resistant HBV, and the mutation patterns of NAs-resistant HBV are complicated in real clinical practice.

The incidence of Drug- Resistant Mutants in Patients with Chronic HBV Infection After Lamivudine Treatment

Background: Hepatitis B virus (HBV) is replicated through reverse transcription of polymerase gene. Lamivudine can postpone the clinical progression in Hepatitis B infected patients, but the long-term monotherapy causes the emerge of YMDD (tyrosine-methionine-aspartate-aspartate), LLAQ (leucine–leucine–alanine–glutamine) motifs and increases the Alanine aminotransferase (ALT) and HBV DNA levels. Objectives: The aim of this study was to investigate rtL180M and rtM204V mutations in polymerase gene of HBV in infected patients after Lamivudine therapy. Patients and Methods: Fifty sera samples collected from patients who referred to Blood Transfusion Center in Tehran were studied. The samples were divided into two groups; treated and untreated based on antiviral treatment status. From 50 patients, 34% were males and 66% were females, aged between 18 and 80 years.rnAll of samples were tested for anti hepatitis B e antibody (anti-HBe), hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg) and hepatitis B core antibody (anti-HBc) by enzyme-linked immunosorbent assay (ELISA) method. The ALT levels were measured using a commercial kit. Then HBV-DNA was extracted from serum samples using a commercial kit and a fragment of the P gene was amplified by nested PCR. Also, HBV viral load was detected by Real-Time PCR. HBV genotypes and polymerase gene mutations were determined by direct sequencing of the polymerase gene of HBV. Phylogenetic tree was constructed using neighbor-joining (NJ) method. Results: About 6% (3 of 50) of samples were HBeAg positive and 94% (47 of 50) of patients were HBeAg negative by ELISA method. The patients' ALT level was between 16 and 95 IU/L with the mean of 37.58 IU/L. Also, 48% (24 of 50) of samples had 104 IU/mL viral load. About 10% (5 of 50) of samples was treated with Lamivudine with specific substitution of amino acid located at codons 80, 180 and 204. In addition, phylogenetic tree showed that genotype D of HBV was dominant among Iranian HBV infected patients Conclusions: This study showed that the presence of mutation at codons rtL80V, rtL180M and rtM204V in A, B and C domains is associated with higher viral load and resistance to Lamivudine (3TC) respectively.

Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants

BackgroundSelective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored.MethodsTo determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China.ResultsWestern-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg.ConclusionsDrug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg warrants further consideration.

Multidrug‐resistant hepatitis B virus resulting from sequential monotherapy with lamivudine, adefovir, and entecavir: Clonal evolution during lamivudine plus adefovir therapy

Whether multidrug-resistant (MDR) hepatitis B virus (HBV) harbors mutations co-located in the same HBV clones that confer reduced sensitivity to antiviral therapy remains uncertain. This study investigated the evolution of MDR HBV strains developed from sequential monotherapy with lamivudine (LAM), adefovir (ADV), and entecavir (ETV) during LAM plus ADV salvage therapy. Sera were obtained from six patients who had developed sequential resistance to LAM, ADV, and ETV before and during LAM plus ADV therapy. The HBV genomes from each patient were amplified, cloned, and sequenced. Among 6 sets of 20 clones obtained before salvage therapy, all clones harbored the rtM204V mutation, and ETV-resistant mutations were detected with the rtM204V in 108 clones. The rtA181 mutation was not detected at baseline, but emerged in five patients during therapy. Among 9 sets of 20 clones obtained during salvage therapy, 39 clones harbored rtA181T/V ± rtN236T mutations, which were detected in the absence of rtM204 and ETV-resistant mutations in 37 clones (94.9%). Only two clones (5.1%) harbored both rtA181T/V and ETV-resistant mutations. The rtA181T/V mutation emerged after reversion from ETV-resistant mutants to wild-type HBV. Five patients achieved a partial virologic response to LAM plus ADV therapy. In conclusion, the majority of MDR mutations existed in different genomes. Suboptimal response to LAM plus ADV therapy may not result from the co-localization of MDR HBV mutations in the same genome, but instead the low antiviral potency of these drugs. Thus, more potent antiviral drug combinations may be an effective salvage therapy for patients infected with MDR HBV.

Do different lamivudine‐resistant hepatitis B genotypes carry the same risk of entecavir resistance?

Entecavir switch is one of the treatment options for lamivudine-resistant hepatitis B (HBV) patients in Asia. This study examined the outcome of patients with different baseline resistance genotypes in a cohort study. In this study, 14 patients with chronic HBV were treated with entecavir 1 mg/day for 5 years. Enrolment criteria include: documented lamivudine resistant mutations, treatment with adefovir 10 mg/day for at least 24 weeks, and Child-Pugh score <7. Most had previous failed adefovir therapy and compensated cirrhosis of the liver. Clinical outcomes, liver biochemistries, and HBV DNA were monitored regularly. Patients with virologic breakthrough were rescued with add-on adefovir. At the end of the treatment period, the mean HBV DNA fell from 5.92 × 10(6) (baseline) to 3.67 × 10(1) IU/ml. The presence of a HBV polymerase rtM204V mutation at the baseline was found to be the major risk factor for adverse outcomes. Compared to the patients with the rtM204I mutant, patients with the rtM204V mutant had increased risk of virologic breakthrough (80% vs. 0%, P = 0.010) requiring add-on adefovir, slower virologic responses (log rank test, P = 0.0011), failure to reach undetectable HBV DNA levels (60% vs. 0%, P = 0.045), and higher risk of entecavir-resistance (60% vs. 0%, P = 0.045). All the patients with rtM204I and rtA181 mutants had undetectable HBV DNA from 18th month. In summary, lamivudine-resistant HBV patients with the rtM204V mutation have the highest risk of developing entecavir resistance, and entecavir monotherapy should be avoided. Those with the rtM204I and rtA181V mutations may have lower risks, but regular surveillance for viral breakthrough is required.

Clinical characteristics and chronicity of acute hepatitis B induced by lamivudine‐resistant strains

Whether resistant hepatitis B virus (HBV) strains are transmissible and can lead to chronic infection remains to be studied. The aim of this study was to investigate the clinical characteristics of patients with acute hepatitis B caused by lamivudine (LAM)-resistant strains. Sera were collected from 234 Chinese patients with acute hepatitis B. LAM-resistance mutations were identified by direct polymerase chain reaction (PCR) sequencing. LAM-resistant HBV variants were detected in 11 of the 234 (4.7%) patients. Among these patients, six harbored the rtM204I mutation, two harbored the rtL180M + rtM204I mutations, one harbored the rtM204I + rtM204V mutations, one harbored the rtL80I + rtM204I mutations, and one harbored the rtV173L + rtL180M + rtM204V mutations. Three patients were infected with genotype B HBV and eight patients were infected with genotype C HBV. Two patients infected with viruses with LAM-resistance mutations developed severe acute hepatitis. One patient developed chronic hepatitis B. This patient was infected with genotype C HBV that had LAM-resistance mutations (rtL180M + rtM204I). The patient was diagnosed with an occult hepatitis B virus infection based on the presence of HBV DNA in the liver and the absence of detectable hepatitis B surface antigen (HBsAg) in the serum. LAM-resistant HBV strains in China are transmissible, can cause acute hepatitis B, and can even progress to chronic infection in China.

Impact of Drug-Resistance Polymerase Mutations on the Replication of HBeAg-Positive and HBeAg-Negative Hepatitis B Virus Strains in Vitro

Impact of Drug-Resistance Polymerase Mutations on the Replication of HBeAg-Positive and HBeAg-Negative Hepatitis B Virus Strains in Vitro

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).

Risk Factors of Gene-Resistant Mutations in Different Nucleosides

To explore the risk factors of gene-resistant mutations during nucleotide treatment. A total of 320 patients with CHB were randomly divided into lamivudine (LAM, 107 cases), adefovir (ADV, 106 cases) and entecavir (ETV, 107 cases) groups, and P gene mutations of HBV were regularly detected. Kaplan-Meier and Cox regression analysis were performed. There was no statistical significance in baseline data between the three groups. The gene-resistant mutation rates were 20.0%, 0.0% and 0.0% one year later, 37.1%, 3.8% and 0.0% two years later, 42.8%, 9.5% and 0.9% three years later, and 64.7%, 25.7% and 0.9% four years later in LAM, ADV and ETV groups, respectively. In LAM group, rtL180M combined with rtM204V mutations accounted for 32.7% and in ADV group, rtN236T mutation accounted for 12.3%. The gene-resistant mutation rate was the most strong in LAM group. With an extension of time, gene-resistant mutation rates are increased, but it is the highest in LAM group and the lowest in ETV group. Family history, the negative conversion time of HBV DNA and different nucleosides are independent risk factors of gene-resistant mutations.

Identification of hepatitis B virus rtS117F substitution as a compensatory mutation for rtM204I during lamivudine therapy

The replication defect of hepatitis B virus (HBV) lamivudine-resistant mutants can be restored by the development of compensatory mutations. Such mutations have long been recognized for the rtM204V mutant, while little is known about any compensatory mutation specific to the rtM204I mutant. The aim of this study was to search for previously unrecognized compensatory mutations following development of lamivudine-resistant mutants. Of 83 lamivudine-resistant patients, 49 and 34 patients harboured the rtM204I and rtM204V mutations, respectively. Serial serum samples obtained during the therapeutic course were submitted to sequence analysis. Site-directed mutagenesis experiments were performed to examine the functions of the identified associated mutations. Of the 49 patients carrying the rtM204I mutation, 5 subsequently developed an rtS117F substitution during the follow-up, whereas 4 harboured an rtN124D substitution prior to the development of the rtM204I mutation. Emergence of the rtS117F mutation was associated with an increase in hepatitis activity, whereas prior existence of the rtN124D mutation was associated with decompensated liver function upon development of the rtM204I mutation. Site-directed mutagenesis experiments showed that the rtS117F mutation by itself did not confer lamivudine resistance but it compensated for replication deficiency of the rtM204I mutant in HepG2 and Mahlavu cells. Additionally, virion and hepatitis B surface antigen secretion of the rtS117F mutant was significantly impaired. The rtS117F substitution served as a compensatory mutation for rtM204I. Emergence of the rtS117F mutation in lamivudine-resistant patients carrying rtM204I was associated with increased hepatitis activities. Prior existence of the rtN124D substitution was associated with liver decompensation upon development of the rtM204I mutation.