Adefovir Resistance Mutations Research Articles

Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients

Background: HBV/HIV co-infection impact on high HBV replication, progression to liver cancer and high mortality. Co-infection may lead to cross-resistance of HBV and HIV drugs due to immune therapy pressure or hepatotoxicity. These challenges necessitate continuous monitoring of HBV variants to aid better diagnosis and treatment strategies. We conducted this study to characterise HBV among HIV infected individuals. Methods: Serum was screened for HBsAg using ELISA, followed by DNA extraction, PCR amplification, Sanger sequencing and phylogenetic analysis. Results: Of the 50 samples in this study 100% (N=50/50) were HBsAg positive; 78% (N=41/50) HBV/HIV co-infection and 92% (N=38/41) of the amplicons were successfully sequenced. Samples nucleotide sequences were identified as genotype A. Mutations prevalence in the HBsAg region was 47% (N=18/38); including mutations associated with diagnostic failure (K122R and T143S) and 7 vaccines escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). Mutations showed resistance to lamivudine 71% (n=5/7), telbivudine 57% (n=4/7), 14% (n=1/7) for entecavir and 43% (n=3/7) for adefovir. Mutations causing resistance to lamivudine and telbivudine were M204V, L180M, V163I, and S202K; with S202K also causing resistance to entecavir and adefovir resistance mutation were I253Y, I223V and M250I. Multiple drug resistance mutations within a single sample contained L180M, M204V, S202K and M250I mutations. There was no statistical significance between the RT mutations associated with drug resistance at P>0.005. The correlation test exhibited a weak statistical association between SHB and RT mutations (0.877**). Conclusions: This study shows the predominance of HBV genotype A in HIV-infected patients. We discovered HBV mutations linked to immune evasion and drug resistance. Although there is no statistical significance amongst the mutations associated with drug resistance and vaccine escape. These mutations could have clinical implications that could have therapeutic repercussions by influencing the correct clinical diagnosis and treatment in HBV/ HIV co-infected individuals.

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.

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

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

Three-year efficacy and safety of tenofovir in nucleos(t)ide analog-naïve and nucleos(t)ide analog-experienced chronic hepatitis B patients.

This study compared the efficacy and safety of tenofovir disoproxil fumarate (TDF) up to 3 years of innucleos(t)ide analog (NA)-naïve and NA-experienced chronic hepatitis B (CHB) patients. Tenofovir disoproxil fumarate-treated NA-naïve and NA-experienced CHB patients were retrospectively analyzed. After 3 years of TDF therapy, 97.7%, 71%, and 45.5% NA-naïve patients achieved a virological response, alanine aminotransferase normalization, and hepatitis B e antigen seroconversion, respectively. Compared with NA-naïve patients, NA-experienced patients without drug resistance and infected with lamivudine/telbivudine-resistant mutants showed similar results. In contrast, patients previously infected with adefovir-resistant mutants and with a suboptimal entecavir response showed significantly lower rates of virological response and hepatitis B e antigen loss/seroconverion than NA-naïve patients. Mean estimated glomerular filtration rate markedly reduced within 12 months of TDF therapy; however, it did not decrease significantly during 12-36 months of treatment. Diabetes mellitus was an independent predictor of a ≥ 0.5 mg/dL increase above baseline in serum creatinine level, and age, hypertension, diabetes mellitus, and baseline creatinine level were independent factors for > 20% decline in estimated glomerular filtration rate from baseline. Liver stiffness measurements improved significantly, but bone mineral density did not change significantly during treatment. Hepatocellular carcinoma incidence was low at 36 months. Age of > 60 years, cirrhosis, a low baseline platelet count and a high α-fetoprotein level at 12 months were significant predictors of hepatocellular carcinoma development. Tenofovir disoproxil fumarate is effective and safe for NA-naïve and NA-experienced CHB patients and should be used cautiously in patients with comorbidities because of a renal dysfunction risk.

Tenofovir monotherapy versus tenofovir and entecavir combination therapy in adefovir-resistant chronic hepatitis B patients with multiple drug failure: results of a randomised trial

ObjectiveLittle clinical data are available regarding the optimal treatment of patients who harbour adefovir-resistant HBV.DesignIn this multicentre trial, patients who had adefovir-resistant HBV with serum HBV DNA levels >60 IU/mL...

Investigation into drug-resistant mutations of HBV from 845 nucleoside/nucleotide analogue-naive Chinese patients with chronic HBV infection.

This study aimed to clarify the clinical significance of drug-resistant HBV in nucleoside/nucleotide analogue (NA)-naive Chinese patients with chronic HBV infection in real clinical practice. A total of 845 NA-naive patients who were admitted to Beijing 302 Hospital between July 2007 and March 2012 were included in the study. HBV drug-resistant mutations were examined by direct sequencing of the viral reverse transcriptase gene and verified by clonal sequencing. Phenotypic analysis of viral replication capacity and drug susceptibility were performed by measuring viral replicative intermediate level in 1.1-mer mutant or wild-type HBV amplicon-transfected HepG2 cells in absence or presence of serially diluted drugs. Drug-resistant mutations were detected in 2.01% (17/845) of the patients by direct sequencing, including 15 with lamivudine-resistant mutations (rtM204V, rtM204I), one with adefovir-resistant mutation (rtA181V), and one with both lamivudine- and adefovir-resistant mutations (rtA181V, rtM204I). Clonal sequencing identified 13 drug-resistant HBV strains: rtL80I+M204I, rtL80I+M204V, rtL180M+M204I, rtL180M+M204V, rtM204I, rtM204V, rtL80I+L180M+M204I, rtL80I+L180M+M204V, rtA181V, rtA181V+M204I, rtA181T+N236T, rtA181V+N236T and rtN236T. Phenotypic analysis showed that two pre-existing lamivudine-resistant strains (rtL80I+M204I, rtL180M+M204V) had >1,000-fold resistance to lamivudine, and one pre-existing adefovir-resistant strain (rtA181V+N236T) had 15.4-fold resistance to adefovir compared with the wild-type strain. A follow-up study showed that the presence of pre-existing rtM204I strain in one patient increased from 20% at baseline to 85% after 13 months of entecavir treatment with corresponding recession of wild-type strain in the viral pool. The incidence of drug-resistant HBV mutations was low in NA-naive Chinese HBV-infected patients. Pre-existing mutants had similar resistance characteristics to those from NA refractory patients.

Antiviral efficacy of adefovir dipivoxil in the treatment of chronic hepatitis B subjects from Indian subcontinent

Adefovir is one of the therapeutic options for the treatment of chronic hepatitis B. A total of 30 adefovir-experienced subjects with the median treatment duration of 12 (interquartile range (IQR) 6-18) months were studied. Virological response was measured by hepatitis B virus deoxyribonucleic acid (HBV DNA) levels. HBV reverse transcriptase (rt) domains were sequenced for the identification of resistance mutations. Among the 30 subjects, two (7%) showed virological response and 19 (63%) were non-responders. The virological response for the remaining nine (30%) subjects was not determined. On sequence analysis, two subjects were identified with rtI169L and rtA181V mutation after 9 months and 18 months of adefovir treatment, respectively. Though the frequencies of adefovir resistance mutations are low, a large majority of subjects showed non-response. Therefore, adefovir in the management of HBV should be used judiciously.

HBV clinical isolates expressing adefovir resistance mutations show similar tenofovir susceptibilities across genotypes B, C and D

Hepatitis B virus (HBV) genotypes can influence clinical outcomes as well as response to antiviral therapy. This study evaluated the tenofovir (TFV) susceptibility of HBV genotype B, C and D clinical isolates with adefovir resistance-associated mutations (ADV-R). Full-length HBV isolates from patients infected with genotype B, C and D virus had rtA181T, rtA181V, rtN236T, rtA181T+rtN236T and rtA181V+rtN236T mutations introduced by site-directed mutagenesis. Phenotypic analyses were performed in HepG2 cells and susceptibility to TFV and ADV were assessed. Clinical HBV isolates containing rtA181T, rtA181V or rtN236T as single mutants remained sensitive to TFV across genotypes B, C and D. Clinical isolates containing the rtA181T+rtN236T double mutant remained sensitive to TFV in genotype D but exhibited reduced susceptibility to TFV in genotypes B and C. Viruses containing the double mutant rtA181V+rtN236T in genotypes B and D exhibited reduced susceptibility to TFV with EC50 fold changes (FC) of 3.8 and 2.5, respectively, while genotype C viruses containing rtA181V+rtN236T either remained sensitive (FC=1.3) or exhibited reduced susceptibility to TFV (FC=2.9) depending on the isolate. All rtA181V+rtN236T isolates conferred reduced susceptibility to ADV (FC values 2.3-4.2). Genotype B, C and D isolates with single ADV resistance mutations remained fully sensitive to TFV, while the double mutants rtA181T+rtN236T and rtA181V+rtN236T exhibited either no change or low-level reduced susceptibility to TFV across genotypes. These results are consistent with the clinical efficacy observed with tenofovir disoproxil fumarate (TDF) treatment across all genotypes in vivo and the limited impact of ADV-R mutations on TDF therapy.

Anti-HBV treatment induces novel reverse transcriptase mutations with reflective effect on HBV S antigen

The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification. 356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay. Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181 T/V (WT:-9.63 kcal/mol, rtA181T:-9.30 kcal/mol, rtA181V:-7.96 kcal/mol, rtS78T:-7.37 kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate. Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.

Similar evolution of hepatitis B virus quasispecies in patients with incomplete adefovir response receiving tenofovir/emtricitabine combination or tenofovir monotherapy

Adefovir (ADV) resistance mutations induce low-level cross-resistance to tenofovir in vitro. Our aim was to compare viral kinetics, nucleos(t)ide analog resistance mutations, and quasispecies (QS) evolution during therapy with tenofovir disoproxil fumarate (TDF) or emtricitabine + TDF (FTC/TDF) in selected patients with incomplete ADV responses. Patients with chronic hepatitis B and incomplete response to ADV were randomized in a double-blind trial of TDF vs. FTC/TDF. Extensive analysis of QS evolution was performed in 17 patients through 48 weeks of treatment. At week 24, 48% of patients (9/17) achieved HBV DNA undetectability (<69 IU/ml) with no difference between treatment groups. ADV and/or LAM resistance mutations were detected in all 17 patients at baseline and in 5/6 analyzable patients at week 48. A total of 1224 reverse transcriptase clones were analyzed. Clonal analysis revealed no significant difference at baseline in QS complexity or diversity between treatment groups. There was a trend in both treatment groups for an increase in QS complexity at week 12, followed by a decrease in complexity and diversity by week 48. Analysis of individual patients showed no consistent selection/accumulation of specific viral resistance patterns during treatment, but at week 48, mutations at rtA181 persisted in 4 patients. TDF or FTC/TDF demonstrated strong viral suppression in patients with an incomplete response to ADV and no significant selective pressure on pre-existing ADV or LAM resistant strains. TDF monotherapy and FTC/TDF combination therapy had a comparable impact on QS evolution.

Virological response to entecavir reduces the risk of liver disease progression in nucleos(t)ide analogue-experienced HBV-infected patients with prior resistant mutants

This study investigated the influence of virological response (VR) to entecavir on clinical liver disease progression in nucleos(t)ide analogue (NA)-naive and -experienced patients. We investigated 487 chronic hepatitis B patients (323 NA-naive, 164 NA-experienced) treated with entecavir monotherapy for at least 12 months. VR was defined as hepatitis B virus DNA level <300 copies/mL during entecavir therapy. Clinical events were defined as hepatic decompensation, hepatocellular carcinoma (HCC), death and liver transplantation. Of the 487 patients, 49 developed clinical events during entecavir treatment. Of those, 36 developed HCC. For all patients, Cox regression analysis showed that age, baseline cirrhosis, alanine aminotransferase level ≤200 U/L, albumin level and no VR during entecavir treatment were independent predictors for clinical events and HCC development. However, the benefit of VR to entecavir was significant for clinical events and HCC only in NA-experienced patients, but not in NA-naive patients. For the further analysis of the different subgroups of NA-experienced patients, the benefit of VR to entecavir was significant for clinical events or HCC only in patients with prior lamivudine- or adefovir-resistant mutants, but not in NA-experienced patients who had never developed lamivudine- or adefovir-resistant mutants. VR at month 12 (but not early VR at month 6) remained a significant predictor associated with the development of clinical events and HCC in NA-experienced patients. VR to entecavir was associated with a reduced risk of clinical events and HCC in NA-experienced patients, particularly in those who had prior lamivudine- or adefovir-resistant mutants.

Randomized trial of lamivudine, adefovir, and the combination in HBeAg-positive chronic hepatitis B

The aim of this study was to compare the efficacy of lamivudine or adefovir alone for 96 weeks versus initial treatment with the combination of lamivudine and adefovir for 12 to 24 weeks followed by adefovir alone. One hundred and fifty patients with HBeAg-positive chronic hepatitis B were randomized equally to lamivudine and adefovir diprivoxil combination therapy (LA), lamivudine alone (L), or adefovir dipivoxil alone (A) in a multicenter randomized clinical trial. In the LA group, the earliest time for lamivudine discontinuation was 12 weeks and adefovir monotherapy was continued until 96 weeks. Groups L and A received monotherapies for 96 weeks. At 12 weeks, the decrease in HBV DNA, percentage of patients with negative HBV DNA, and ALT normalization rate for the LA group were comparable to those of group L, but superior to those of group A. At 24 weeks, the rates of negative HBV DNA and HBeAg seroconversion of group LA were significantly higher than the monotherapy groups. This superiority was subsequently preserved during the maintenance phase with adefovir monotherapy. Starting at 48 weeks, the mean HBV DNA level of group L increased over the 24-week level. In contrast, the A group's rates of virological response, biochemical response, and HBeAg seroconversion continued to improve. At week 96, the percentage of patients with undetectable DNA and HBe seroconversion of LA group (100%, 51%) was higher than that of L (66%, 21%) and A group (49%, 33%), while no significant difference was observed between the L and A groups. During the course of therapy, no lamivudine- or adefovir-resistance mutations were discovered in the LA group. Rates of adverse reactions were comparable between the three groups. Combination therapy with lamivudine and adefovir for 12 to 24 weeks followed by adefovir monotherapy significantly improved antiviral efficacy and reduced drug resistance without compromising safety and tolerability compared to either drug alone in HBeAg-positive chronic hepatitis B.

Hepatitis B e antigen‐suppressing mutations enhance the replication efficiency of adefovir‐resistant hepatitis B virus strains

Hepatitis B e antigen (HBeAg) negative hepatitis B virus (HBV) infections caused by precore (PC) or basal core promoter (BCP) mutations are associated with disease progression and complications. PC or BCP mutations may enhance the replication capacity of distinct drug-resistance-associated polymerase mutations, but their effect on adefovir-resistant HBV mutants is unclear. Importantly, BCP mutations were an independent risk factor for virological breakthrough in lamivudine-resistant patients treated with adefovir. We aimed at addressing the functional consequences of PC and BCP mutations on the replication and drug susceptibility of adefovir-resistant HBV mutants. Therefore, HBV constructs with wild type (WT) or adefovir-resistant rtN236T, rtA181V and rtA181T mutations, with or without concomitant PC or BCP mutations, were analysed in vitro using molecular assays. The adefovir-resistant polymerase mutations rtN236T, rtA181V and rtA181T showed a drastically reduced viral replication compared with WT. Interestingly, additional PC or BCP mutations enhanced the reduced replication efficacy of adefovir-resistant constructs and restored HBV replication to WT level. HBV rtA181T mutants displayed abolished hepatitis B surface antigen (HBsAg) secretion, owing to a sW172* stop codon in the overlapping envelope gene. All rtN236T- or rtA181V/T-containing constructs, regardless of concomitant PC or BCP mutations, were resistant to adefovir, but remained susceptible to telbivudine, entecavir and tenofovir. In conclusion, adefovir drug resistance mutations reduced viral replication, which can be significantly increased by additional HBeAg-suppressing PC or BCP mutations. Because increased HBV replication in HBeAg-negative patients has been associated with an unfavourable clinical course, close monitoring appears indispensable during adefovir treatment in HBeAg-negative patients.

Detection of lamivudine and adefovir resistance mutations among Filipino patients with chronic hepatitis B using line probe assay

Detection of lamivudine and adefovir resistance mutations among Filipino patients with chronic hepatitis B using line probe assay

Treatment Response and Evolution of HBV Resistance during Lamivudine plus Adefovir or Entecavir Therapy in Patients with Adefovir-Resistant Mutants

Here, we investigated the treatment response and evolution of HBV resistance during lamivudine (LAM) plus adefovir (ADV) and entecavir (ETV) monotherapy in patients with ADV-resistant mutants. Of the 53 patients with ADV-resistant mutants, 25 received combined LAM plus ADV therapy (LAM+ADV group) and 28 received ETV monotherapy (ETV group) for at least 12 months (median 24 months and range 12-67 months). During 24 months therapy, no significant difference was noted in HBV DNA reduction from baseline, HBV DNA<200 copies/ml, hepatitis B e antigen loss and ALT normalization between the two groups. In the LAM+ADV group, patients with single rtN236T resistant mutation had higher rates of undetectable HBV DNA than those with the double mutant rtA181T/V+rtN236T at months 3-18 of therapy. No virological breakthrough occurred except for one patient with rtN236T resistant mutation who experienced virological and biochemical breakthrough after the emergence of an additional rtA181T mutant under LAM+ADV therapy. Of the 28 patients receiving ETV monotherapy, ETV-resistant mutants developed in 8. The cumulative rates of ETV-resistant mutations and virological breakthrough at months 12, 24 and 36 were 3.6%, 25.7% and 46.8%, respectively. ADV-resistant mutations were rapidly replaced by LAM-resistant mutations (median 12 months) followed by ETV-resistant mutations. There was no significant difference in virological response between the LAM+ADV and ETV groups in patients with ADV-resistant mutants. LAM+ADV were less effective in patients with the double mutant rtA181T/V+rtN236T than the single rtN236T mutation. The incidence of ETV-resistant mutation was high in patients with LAM/ADV-resistant mutants treated with ETV monotherapy.

Evolution of hepatitis B virus during long-term therapy in patients with chronic hepatitis B

Background. Long-term lamivudine (LAM), adefovir (ADV) and entecavir (ETV) treatment induce the emergence of drug-resistant hepatitis B virus (HBV) in patients with chronic hepatitis B infection.Aim. To evaluate the LAM, ADV and ETV resistance mutations detected in our patient group.Materials and methods. Twenty patients who had received at least two years of treatment with nucleoside/tide analogues were enrolled in this study. Patients with detectable HBV DNA were analyzed in order to detect resistance mu-tations and in this group of patients treatment was change.Results. Three patients developed LAM resistance mutations (2 presented rtM204I and one rtL180M+rtM204V/I) and one patient showed rtN236T ADV resistance mutation. During ADV and LAM treatment, one patient developed ADV plus LAM resistance mutations (rtI163V+rtL180M+rtA181V+rtN236T), in this case, HBV strains harbouring polymerase mutations did not develop LAM associated rtM204V/I primary mutation. In addition, ETV resistance mutations (rtL180M+rtT184A+rtS202G+rtM204V) were detected in one patient.Conclusions. These findings suggest that monotherapy resulted in a limited virological response and combination strategies including potent antiviral agents should be recommended for patients with resistant mutations.

Virologic and clinical characteristics of HBV genotypes/subgenotypes in 487 Chinese pediatric patients with CHB

BackgroundThe association of hepatitis B virus (HBV) genotypes/subgenotypes with clinical characteristics is increasingly recognized. However, the virologic and clinical features of HBV genotypes/subgenotypes in pediatric patients remain largely unknown.MethodsFour hundred and eighty-seven pediatric inpatients with CHB were investigated, including 217 nucleos(t)ide analog-experienced patients. HBV genotypes/subgenotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. The stage of fibrosis and degree of inflammatory activity were evaluated by the Metavir score system.ResultsAmong 487 enrolled pediatric patients, HBV genotype C2 and B2 were the most two prevalent (73.7% and 21.1%). Comparing with HBV/B2 infected patients, no significant difference was observed in the incidence rate and mutant patterns of lamivudine- or adefovir-resistant mutations in HBV/C2 infected patients (P > 0.05). Importantly, we found that the degree of hepatic inflammation degree, fibrosis stage and ALT level were significantly higher in HBV/C2-infected HBeAg positive patients than it was in HBV/B2-infected ones.ConclusionsThe pediatric patients with HBV/C2 infection might be more susceptible to develop severe liver pathogenesis.

The main Hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M + rtM204V, rtV173L + rtL180M + rtM204V, rtM204I, rtL180M + rtM204I, rtN236T, rtA181V, rtA181V + rtN236T, rtA181T, rtA181T + rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M + M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.

HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A–H and its use in antiviral phenotyping assays

The aim of this study is to establish a phenotyping assay to analyze patient HBV polymerase/reverse transcriptase (RT) sequences for potential drug resistance against RT inhibitors. HBV RT (pol aa 304–715, including the entire RT) from clinical isolates were amplified and ligated into a plasmid vector (pRTAN) expressing a genotype A HBV genome lacking the RT region. HBV DNA replication of the recombinants and their drug susceptibilities were assessed by transient transfection into HepG2 cells and intracellular core DNA was analyzed either by Southern blot or using a 96-well format and quantification by qPCR. Cloning of the HBV RT gene from clinical isolates representing genotypes A–H was successful and led to virus DNA replication. Recombinants expressing patient derived RT genes containing the rtL180M+M204V lamivudine resistance (LAM-R) mutations demonstrated a LAM-R phenotype. Similarly, patient derived RT genes containing the adefovir resistance (ADV-R) mutations rtA181V or rtN236T demonstrated an ADV-R phenotype. Recombinants containing HBV RT from paired patient samples without genotypic changes had similar EC50 values. In conclusion, a phenotyping assay for HBV RT gene was developed, allowing evaluation of patient-derived HBV RT from genotypes A–H, and confirmed the drug resistance phenotype in samples containing LAM-R or ADV-R mutations.

Virological response to adefovir monotherapy and the risk of adefovir resistance

To evaluate virological response to adefovir (ADV) monotherapy and emergence of ADV-resistant mutations in lamivudine (LAM)-resistant chronic hepatitis B patients. Seventy-seven patients with documented LAM resistance who were treated with 10 mg/d ADV for > 96 wk were analyzed for ADV resistance. At week 48 and 96, eight (10%) and 14 (18%) of 77 LAM-resistant patients developed the ADV-resistant strain (rtA181V/T and/or rtN236T mutations), respectively. Hepatitis B virus (HBV) DNA levels during therapy were significantly higher in patients who developed ADV resistance than in those who did not. Incidence of ADV resistance at week 96 was 11%, 8% and 6% among patients with complete virological response (HBV DNA level < 60 IU/mL); 0%, 5% and 19% among patients with partial virological response (HBV DNA level ≥ 60 to 2000 IU/mL); and 32%, 34% and 33% among patients with inadequate virological response (HBV DNA levels > 2000 IU/mL) at week 12, week 24 and week 48, respectively. HBV DNA levels > 2000 IU/mL at week 24 showed best performance characteristics in predicting ADV resistance. Development of ADV resistance mutations was associated with HBV DNA levels, which could identify patients with LAM resistance who are likely to respond to ADV monotherapy.