Hepatitis B Virus Reverse Transcriptase Research Articles

Possible biological mechanisms of entecavir versus tenofovir disoproxil fumarate on reducing the risk of hepatocellular carcinoma.

Hepatitis B virus (HBV) is a life-threatening infectious virus associated with the risk of liver failure and hepatocellular carcinoma (HCC). Regarding HBV treatment, the recent development of nucleoside/nucleotide analogs (NUC), HBV reverse transcriptase inhibitors, enabled favorable viral control as well as improved prognosis in patients with chronic hepatitis B. However, NUC fails to clear HBV because the formation of covalently closed circular DNA or HBV surface antigen occurs upstream of the point of action of NUC. Recently, we found that acyclic nucleoside phosphonates (ANP) such as adefovir or tenofovir, but not lamivudine or entecavir, induced IFN-λ3 productions in the gastrointestinal tract and modulated lipopolysaccharide (LPS)-mediated cytokine profiles in peripheral blood mononuclear cells, such as interleukin (IL)-12p70 induction and IL-10 inhibition, which are immunologically favorable cytokine profiles for HBV elimination. Furthermore, IFN-α, in combination with ANP, showed additional and synergistic effects on IFN-λ3 and IL-12p70 production, respectively, while not affecting IL-10 levels. Mechanistic analyses of the cytokine modulation by ANP revealed that ANP blocked the mammalian target of the rapamycin (mTOR) pathway by inhibiting Akt translocation to the plasma membrane, thereby inhibiting Akt phosphorylation. As it has been reported that IFN-λ inhibits tumor growth directly or indirectly and the mTOR pathway is generally activated in most cancer cells, ANP might have potential anti-HCC effects. Our in vitro and ex vivo findings might stir the debate on whether types of NUC affect the risk of HBV-related HCC incidence.

Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2.

Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1-6 suppressed the production of cccDNA. These results suggest that KPNA1-6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.

Phylogenetic and drug- and vaccine-resistance profiles of Hepatitis B Virus among children with HIV co-infection in Pakistan.

HIV-1 and hepatitis B virus (HBV) share common routes of transmission and therefore co-infection is common. In 2019, an HIV-1 outbreak that resulted in >1000 children being infected, predominantly through nosocomial transmission, occurred in Sindh, Pakistan. We conducted a phylogenetic and drug resistance analysis of the HBV Reverse Transcriptase (RT) gene in children with HIV-1 and HBV co-infection. Blood samples were collected from 321 children with HIV who were recruited as part of a study to investigate the HIV-1 outbreak. All samples were tested for HBV surface antigen (HBsAg) using an ELISA assay, and positive samples were used to amplify and sequence the HBV RT gene. The phylogenetic relationship between sequences was analyzed, and drug- and vaccine- resistance mutations in the RT gene were explored. Of 321 samples, 23% (n=75) were positive for HBsAg on ELISA. Phylogenetic analysis of the sequences revealed that 63.5% of HBV sequences were sub-genotype D1, while the rest were sub-genotype D2. Cluster analysis revealed grouping of sub-genotype D1 sequences exclusively with Pakistani sequences, while clustering of sub-genotypes D2 predominantly with global sequences. The 236Y mutation associated with resistance to tenofovir was observed in 2.8% of HBV sequences. Additionally, seven vaccine escape mutations were observed, the most common being 128V. Our study suggests ongoing transmission of HBV D1 and D2 sub-genotypes in the HIV-1 co-infected population, likely nosocomially, given common routes of HVB and HIV-1 transmission. The prevalence of major HBV drug- and vaccine-resistant mutations remains low. Surveillance for further transmissions and the possible emergence of major drug- or vaccine-resistant variants is required.

Putative and pretreatment drug resistance mutations in reverse transcriptase gene among untreated chronic hepatitis B patients at Arifin Achmad Regional District Hospital, Riau, Indonesia

Background: Mutations within the hepatitis B virus (HBV) reverse transcriptase (RT) gene have been associated with drug resistance against nucleos(t)ide analogs (NAs).
 Objective: This study aimed to identify mutations in the RT gene among patients with chronic hepatitis B (CHB) before receiving antiviral therapy and its relationship with the HBV genotypes.
 Methods: A total of 26 HBV DNA was extracted from the blood plasma of CHB patients. HBV RT gene was amplified and sequenced using the Sanger dideoxy sequencing method. The HBV genotype was determined through phylogenetic analysis using the Maximum Likelihood method.
 Results: The study subjects comprised 14 CHB patients without complications and 12 CHB patients with cirrhosis/hepatoma. CHB patients with cirrhosis/hepatoma were older than those without complications. The HBV genotypes comprised 15 (57.7%) genotype C and 11 (42.3%) genotype B. All treatment-naïve CHB patients did not demonstrate any classical NA resistance mutations within the RT gene. However, several putative and pretreatment resistance mutations, including F221Y, N238H, and V224I, were high frequency in more than 40% of study subjects. In addition, F221Y and N238H/Q mutations were frequently observed in genotype B, while V224 I was only found in patients infected with genotype C (p=0.000).
 Conclusions: There was no evidence of classical RT gene mutations associated with NA resistance in treatment-naïve patients with CHB. However, several putative and pretreatment mutations were identified as genotype-specific mutations and may contribute to antiviral resistance against NAs.

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.

Early dynamic changes of quasispecies in the reverse transcriptase region of hepatitis B virus in telbivudine treatment

BackgroundTelbivudine (LdT) - a synthetic thymidine β-L-nucleoside analogue (NA) - is an effective inhibitor for hepatitis B virus (HBV) replication. The quasispecies spectra in the reverse transcriptase (RT) region of the HBV genome and their dynamic changes associated with LdT treatment remains largely unknown. MethodsWe prospectively recruited a total of 21 treatment-naive patients with chronic HBV infection and collected sequential serum samples at five time points (baseline, weeks 1, 3, 12, and 24 after LdT treatment). The HBV RT region was amplified and shotgun-sequenced by the Ion Torrent Personal Genome Machine (PGM)® system. We reconstructed full-length haplotypes of the RT region using an integrated bioinformatics framework, including de novo contig assembly and full-length haplotype reconstruction. In addition, we investigated the quasispecies' dynamic changes and evolution history and characterized potential NAs resistant mutations over the treatment course. ResultsViral quasispecies differed obviously between patients with complete (n = 8) and incomplete/no response (n = 13) at 12 weeks after LdT treatment. A reduced dN/dS ratio in quasispecies demonstrated a selective constraint resulting from antiviral therapy. The temporal clustering of sequential quasispecies showed different patterns along with a 24-week observation, although its statistic did not differ significantly. Several patients harboring pre-existing resistant mutations showed different clinical responses, while NAs resistant mutations were rare within a short-term treatment. ConclusionA complete profile of quasispecies reconstructed from in-depth shotgun sequencing may has important implications for enhancing clinical decision in adjusting antiviral therapy timely.

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.

Natural variability in surface antigen and reverse transcriptase domain of hepatitis B virus in treatment-naïve chronic HBV-infected Egyptian patients

Hepatitis B virus (HBV) infection is a serious health problem not only in Egypt, but also worldwide. We collected 57 serum samples from treatment-naïve chronic HBV-infected Egyptians. The DNA segment encoding HBV surface antigen (HBsAg) and reverse transcriptase (RT) domain was partially sequenced. Our data revealed that all viral isolates belonged to genotype D with ayw2 as the predominant serotype (89 %).Regarding HBsAg, 45 substitutions were detected in the collected isolates. Eleven substitutions were found in the major hydrophilic region, including two novel ones (M103T and G130E) that were not correlated before with genotype D. Additionally, 11 occult samples (19 %) were detected, in which the predominant mutations of HBsAg were S143L (7 samples) followed by D144A and T125M (4 samples each). Concerning the RT domain, 26 isolates (45 %) harbored 19 natural mutations that were reported to be associated with antiviral resistance. Eleven different mutations were not correlated previously with genotype D. The most predominant mutation was Y124H (47 samples, 82 %). Interestingly, such mutation was detected in 91 % of the previous reported sequences of HBV isolates collected in Egypt (157 sequences). Furthermore, our study illustrated the presence of viral quasispecies in the HBsAg (10 samples, 17.5 %) and RT domain (9 samples, 15.7 %).In conclusion, we elucidated the presence of natural substitutions in HBsAg and RT domain of HBV isolates obtained from treatment-naïve chronic HBV-infected Egyptian patients. Additionally, we detected viral quasispecies and revealed Y124H as a characteristic substitution in the RT domain for HBV isolates in Egypt. Moreover, novel substitutions in HBsAg and RT domain were reported with genotype D.

Multiple drug-resistant HBV mutation may contribute to poor response of adefovir + entecavir in entecavir-resistant patients.

Adefovir plus entecavir (ADV+ETV) rescue therapy in ETV-resistant patients with chronic hepatitis B virus (HBV) infection is suboptimal in some patients. This study aims to elucidate the evolutionary characteristics of drug-resistant HBV mutants and their association with clinical responses in such patients. Thirty-seven ETV-resistant patients were enrolled, among whom twelve had an inadequate virological response to ADV+ETV rescue therapy. The clonal sequence (³ 20 clones/sample) of HBV reverse transcriptase gene was performed to identify the resistance mutations. Phenotypic analysis was performed to evaluate the replication capacity and drug susceptibility of the mutants. ETV-resistant mutants were continuously detected in 10 of the 12 patients, and multidrug-resistant (MDR) mutants, including a novel strain (rtL180M+A181V+T184A+S202G+M204V), were detected in two patients. Seven of the 12 patients who subsequently received tenofovir (TDF)-based therapy for 38 (23-60) months all achieved undetectable HBV DNA after treatment, and ETV-resistant mutants converted to wild-type in the four patients' samples. In contrast, the other five patients who did not achieve an adequate virological response had remaining of ETV-resistant mutants. The novel MDR strain exhibited multiple resistances to LAM, ADV, and ETV, and 11.2-fold lower susceptibility to TDF. This study is the first to demonstrate that MDR HBV mutations may contribute to the poor efficacy of ADV+ETV combination therapy in ETV-resistant patients. Moreover, a novel MDR HBV strain was identified. Our results indicate that a TDF-based rescue therapy would be effective for the treatment of the refractory cases.

The association of the heterogeneity of HBV reverse transcriptase quasispecies with antiviral efficacy after treatment with nucleos(t)ide analogues for 10years.

To assess the heterogeneity of HBV reverse transcriptase (RT) quasispecies during 10years of antiviral therapy and their association with antiviral efficacy. Nineteen patients with chronic hepatitis B (CHB) infection receiving nucleos(t)ide analogues (NAs) were enrolled. Based on the antiviral efficacy after 1year of treatment, 5 patients were grouped into an early virologic response (EVR) group, while 8 patients were grouped into a late virologic response (LVR) group. Furthermore, 6 CHB patients that had undergone antiviral treatment for 10years were grouped into a virologic breakthrough (VBT) group. The HBV RT from each patient were amplified, cloned, and sequenced. The complexity of the RT gene in the EVR group was significantly higher than that in the LVR (P=0.0393) and VBT groups (P=0.0141). Phylogenetic tree analysis showed that the average branch length of the EVR and LVR groups were significantly greater than that of VBT group (P<0.001). The complexity (at the nucleotide level) of the RT quasispecies was negatively correlated with the corresponding HBV DNA load (P=0.0163) at one year post-antiviral treatment. Moreover, both the LVR and VBT groups accumulated more deleterious mutations than the EVR group. After 1year of NAs treatment, the increased HBV quasispecies complexity and evolutionary topologies, coupled with less deleterious mutations, are likely associated with a favorable efficacy during long-term antiviral treatment.

Identification of a novel long-acting 4’-modified nucleoside reverse transcriptase inhibitor against HBV

While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. Herein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity invitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined. E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate's interactions and anti-HBV potency. E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV. Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.

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

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. 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. The incidence of RT mutations in treated patients was higher than that in untreated patients (p<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<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<0.05). Several novel mutations might be related to NA resistance. 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.

CccDNA Maintenance in Chronic Hepatitis B - Targeting the Matrix of Viral Replication.

Chronic hepatitis B is a numerically important cause of cirrhosis and hepatocellular carcinoma, despite an effective prophylactic vaccine and well-tolerated and effective oral antivirals. Both the incapacity of the immune system to clear hepatitis B virus (HBV) infection and the unique replication strategies adopted by HBV are considered key determinants of HBV chronicity. In this regard, the formation of the HBV DNA minichromosome, the covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes, is essential not only for the production of all viral proteins but also for HBV persistence even after long-term antiviral therapy. Licensed polymerase inhibitors target the HBV reverse transcriptase activity, control the disease with long-term therapy but fail to eliminate the cccDNA. Consequently, the production of viral RNAs and proteins, including the hepatitis B surface antigen (HBsAg), is not abolished. Novel therapeutic efforts that are in the pipeline for early clinical trials explore novel targets and molecules. Such therapeutic efforts focus on achieving a functional cure, which is defined by the loss of HBsAg and undetectable HBV DNA levels in serum. Since a true cure of HBV infection requires the elimination of the cccDNA from infected cells, comprehension of the mechanisms implicated in cccDNA biogenesis, regulation and stability appears necessary to achieve HBV eradication. In this review, we will summarize the state of knowledge on cccDNA metabolism, focusing on insights suggesting potential weak points of the cccDNA that may be key for the development of therapeutic approaches and design of clinical trials aiming at lowering cccDNA loads and activity.

3,7-Dihydroxytropolones Inhibit Initiation of Hepatitis B Virus Minus-Strand DNA Synthesis.

Initiation of protein-primed (-) strand DNA synthesis in hepatitis B virus (HBV) requires interaction of the viral reverse transcriptase with epsilon (ε), a cis-acting regulatory signal located at the 5’ terminus of pre-genomic RNA (pgRNA), and several host-encoded chaperone proteins. Binding of the viral polymerase (P protein) to ε is necessary for pgRNA encapsidation and synthesis of a short primer covalently attached to its terminal domain. Although we identified small molecules that recognize HBV ε RNA, these failed to inhibit protein-primed DNA synthesis. However, since initiation of HBV (-) strand DNA synthesis occurs within a complex of viral and host components (e.g., Hsp90, DDX3 and APOBEC3G), we considered an alternative therapeutic strategy of allosteric inhibition by disrupting the initiation complex or modifying its topology. To this end, we show here that 3,7-dihydroxytropolones (3,7-dHTs) can inhibit HBV protein-primed DNA synthesis. Since DNA polymerase activity of a ribonuclease (RNase H)-deficient HBV reverse transcriptase that otherwise retains DNA polymerase function is also abrogated, this eliminates direct involvement of RNase (ribonuclease) H activity of HBV reverse transcriptase and supports the notion that the HBV initiation complex might be therapeutically targeted. Modeling studies also provide a rationale for preferential activity of 3,7-dHTs over structurally related α-hydroxytropolones (α-HTs).

Prevalence and Phylogenetic Analysis of Hepatitis B in Captive and Wild-Living Pileated Gibbons (Hylobates pileatus) in Cambodia

The risk of transmitting novel pathogens from released animals to wild conspecifics is an important consideration for reintroduction initiatives. Apart from infecting humans, hepatitis B virus (HBV) has been detected in nonhuman primates, including captive gibbons. Gibbons that test positive for HBV are removed from reintroduction programs as the presence of infection in wild populations is uncertain. The aim of this study was to determine the prevalence of HBV in wild-living gibbon populations in the southwest and northeast of Cambodia and compare HBV DNA strains collected from these animals with samples from wild-born captive gibbons at the Phnom Tamao Wildlife Rescue Center, Cambodia. We screened HBV infection in blood and stool samples from captive gibbons and stool samples from wild-living gibbons. We sequenced samples with detectable HBV DNA viral load in the HBV reverse transcriptase coding region overlapping with the S-gene. Eleven of twenty-four (46 %) wild-living pileated gibbons (Hylobates pileatus) from the southwest of Cambodia were positive for HBV. Phylogenetic analysis of HBV DNA sequenced from wild and captive gibbons revealed that viral strains were related and different from human strains. The presence of an HBV variant circulating in the wild similar to that found in captive pileated gibbons suggests 1) the strain identified is associated with the species, 2) wild-born captive gibbons positive for this strain became infected prior to captivity, and 3) it might be appropriate to release captive gibbons that test positive for specific serological markers of HBV, provided they are otherwise suitable candidates for reintroduction.

Evidence of tenofovir resistance in chronic hepatitis B virus (HBV) infection: An observational case series of South African adults.

IntroductionTenofovir disoproxil fumarate (TDF) is widely recommended for treatment of chronic hepatitis B virus (HBV) infection because it is safe, affordable and has a high genetic barrier to resistance. TDF resistance associated mutations (RAMs) have been reported, but data are limited, particularly for Africa. We set out to identify potential RAMs in individuals with detectable HBV viraemia on TDF treatment.MethodsWe recruited adults with chronic HBV infection from Cape Town, South Africa, identifying individuals with a TDF resistance phenotype, defined as persistent HBV vireamia despite >12 months of TDF treatment. We sequenced HBV DNA using MiSeq Illumina with whole genome target enrichment, and sought potential TDF RAMs, based on a pre-defined list of polymorphisms.ResultsAmong 66 individuals with chronic HBV (genotypes A and D), three met our clinical definition for TDF resistance, of whom two were coinfected with HIV. In one participant, the consensus HBV sequence contained nine polymorphisms that have been described in association with TDF resistance. Significant treatment non-adherence in this individual was unlikely, as HIV RNA was suppressed. TDF RAMs were also present in HBV sequences from the other two participants, but other factors including treatment non-adherence may also have had a role in failure of HBV DNA suppression in these cases.DiscussionOur findings add to the evidence that RAMs in HBV reverse transcriptase may underpin a TDF resistant phenotype. This is the first time these RAMs have been reported from Africa in association with clinical evidence of TDF resistance.

Prevalence of Potential Resistance Related Variants Among Chinese Chronic Hepatitis B Patients Not Receiving Nucleos(T)ide Analogues.

Background and AimsPotential drug resistance (DR) related variants in the hepatitis B virus (HBV) reverse transcriptase (RT) region may be associated with the effectiveness of antiviral drugs and disease progression. The aim of this study was to investigate the prevalence and clinical characteristics of potential DR-related variants in Chinese CHB patients not receiving nucleos(t)ide analogues (NAs).Patients and MethodsTwo hundred and six untreated CHB patients from Huzhou Central Hospital in eastern China were recruited for this study. The serum DNA was extracted and the HBV RT region was amplified using nest polymerase chain reaction (nest-PCR). The 42 potential DR-related variants were analyzed by direct sequencing.ResultsAmong these CHB patients, HBV genotype B and genotype C were identified in 121 (58.7%) and 85 (41.3%) patients, respectively. Potential DR-related variants were detected in 42.7% (88/206) of patients. Primary and secondary DR variants were found in 7.3% (15/206) of patients, including rtL80I/V, rtI169T, rtV173L rtL180M, rtA181T/V, rtM204I/V, and rtN236T. The variants at rt53, rt82, rt221, rt233, rt237, and rt256 were specific for genotype B, and those at rt38, rt84, rt126, rt139, rt153, rt191, rt214, rt238, and rt242 were specific for genotype C. Moreover, the variation frequency in the A-B interdomain (3.96%) was significantly higher than that in the functional domains (1.17%) and non-A-B interdomains (1.11%). Multivariate logistic regression analysis showed that lower HBV-DNA load (<106 IU/mL) was an independent factor associated with potential DR-related variants in untreated CHB patients (P <0.05).ConclusionPotential DR-related variants were frequent and complex in untreated Chinese CHB patients. Furthermore, the variants may contribute to decreased serum HBV-DNA loads. However, the effects of potential DR-related variants on the antiviral therapy and liver disease progression require further study.

Hepatitis B virus mutation pattern rtA181S+T184I+M204I may contribute to multidrug resistance in clinical practice: Analysis of a large cohort of Chinese patients

The study aimed to characterize the prevalence and virological features of the rtA181S + T184I + M204I mutant in a large cohort of patients with chronic HBV infection. In total, 22,009 nucleoside/nucleotide analog-treated patients who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between 2007 and 2016 were enrolled. Serum samples were collected for HBV reverse-transcriptase gene sequencing. Phenotypic analysis of the viral replication capacity and drug susceptibility was performed. The rtA181S mutation was detected in 0.82% (180/22,009) of samples. rtA181S-positive patients had significantly higher lamivudine (LAM), adefovir (ADV), and entecavir (ETV) exposure than rtA181S-negative patients. Of 180 rtA181S-positive patients, 42 had no coexistent resistance mutations, 34 had coexisting LAM-resistance mutation (LAMr), 17 had coexisting ADV-resistance mutation (ADVr), and 86 had coexisting ETV-resistance mutation (ETVr), and one had ADVr + ETVr. rtA181S + T184I + M204I occurred in 79.1% (68/86) of patients with rtA181S + ETVr and 37.8% (68/180) of all rtA181S-positive patients. Longitudinal analysis of the clinical course of resistant mutant evolution for four representative cases showed that rtA181S + T184I + M204I developed in all patients who had received LAM/telbivudine ± ADV and was receiving ETV or ADV + ETV. Compared with wild-type, the rtA181S + T184I + M204I mutant had 53.7% lower replication capacity and >1000-, 3.9-, and 383.3-fold greater LAM, ADV, and ETV resistance, respectively, but remained sensitive to tenofovir. Artificial elimination of rtA181S from the rtA181S + T184I + M204I mutant restored viral susceptibility to ADV but decreased viral replication capacity. Our study presented the first evidence that HBV rtA181S + T184I + M204I mutation had features of multidrug-resistance that contributed to resistance to both nucleoside and nucleotide analogs.

HBV variants are common in the 'immune-tolerant' phase of chronic hepatitis B.

Nucleos(t)ide analogues (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk of hepatocellular carcinoma (HCC) persists despite viral suppression. Specific HBV variants have been associated with adverse outcomes, including HCC; however, the frequency of these variants during the seemingly benign immunotolerant (IT) phase is unknown. Next-generation sequencing and detailed virological characterization on a cohort of treatment-naïve IT subjects were performed to determine the frequency of clinically relevant viral variants. Samples from 97 subjects (genotype B/C 55%/45%, median HBV-DNA 8.5log10 IU/mL, median HBsAg 4.8 log10 IU/mL, median HBeAg 3.6 log10 PEIU/mL) were analysed. Despite subjects being in the IT phase, clinically relevant HBV variants were common at baseline, particularly in the basal core promoter (BCP, overlaps the hepatitis B X (HBx) gene), precore and PreS regions. BCP/HBx variants were independently associated with lower baseline HBeAg, HBsAg and HBV-DNA titres. Precore variants were independently associated with higher baseline ALT. Increased viral diversity was associated with increased age and lower HBV-DNA, HBsAg and HBeAg levels. Low-level (<5%) drug resistance-associated amino acid substitutions in the HBV reverse transcriptase were detected in 9 (9%) subjects at pre-treatment but were not associated with reduced antiviral activity. Future studies should evaluate whether the detection of HBV variant during IT CHB is predictive of progression to immune clearance and poor prognosis, and whether early initiation of antiviral therapy during IT CHB to prevent the selection of HBV variants is clinically beneficial.

Structural dynamics and susceptibility of anti-HIV drugs against HBV reverse transcriptase

Hepatitis B virus (HBV), a small enveloped DNA virus, attacks the human liver causing both acute and chronic diseases. Current therapeutic drugs use the nucleos(t)ide analogues (NAs) as a competitive inhibitor against HBV reverse transcriptase (HBV-RT), an essential enzyme pivotally involved in viral replication. Unfortunately, this treatment still causes the development of resistant variants of HBV against NAs. As HBV-RT is homologous to the human immunodeficiency virus reverse transcriptase (HIV-RT), it is reasonable to treat HBV-RT with anti-HIV drugs. In the present study, we aimed to investigate the structural dynamics and susceptibility of the known anti-HIV drugs (stavudine [d4T], didanosine [DDI], and zidovudine [ZDV]) against HBV-RT enzyme in comparison to the anti-HBV drug lamivudine (3TC) and deoxythymidine triphosphate (dTTP) substrate using several computational approaches. The calculations revealed that seven polar residues (K32, R41, D83, S85, D205, N236, and K239) and three hydrophobic residues (A86, A87, and F88) of HBV-RT as well as the adjacent DNA strands play an important role in the ligand binding. In addition, the H-bond pattern of d4T is similar to that of 3TC, especially at the residues A86 and A87. Such interactions promote the favorable conformation of ligand in the HBV-RT binding pocket, while the several different conformations of ligand are found in the unbound state. The predicted binding free energy results based on QM/MM-GBSA and MM/GB(PB)SA methods suggested that the susceptibility towards HBV-RT of d4T and ZDV is higher than that of 3TC and dTTP. Altogether, this work sheds light on the potentiality of d4T and ZDV as a promising drug for HBV-infected patients harboring 3TC resistance. Communicated by Ramaswamy H. Sarma