S. Amini-Bavil-Olyaee

The rtA194T polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis B e antigen–positive and hepatitis B e antigen–negative hepatitis B virus strains

Tenofovir is a new effective treatment option for patients with chronic hepatitis B, but could be potentially hampered by mutations in the hepatitis B virus (HBV) polymerase conferring drug resistance. Drug resistance may occur preferentially if long‐term administration is required, for example, in patients with hepatitis B e antigen (HBeAg)‐negative HBV infection bearing precore (PC) and basal core promoter (BCP) mutations. The rtA194T polymerase mutation has been found in HBV/HIV coinfected patients during tenofovir treatment and may be associated with tenofovir resistance. We generated replication‐competent HBV constructs harboring rtA194T alone or in addition to lamivudine (LAM) resistance (rt180M + rtM204V), PC mutations, and BCP mutations and assessed their replicative capacity after transient transfection in human hepatoma cells. The rtA194T polymerase mutation alone or in conjunction with LAM resistance reduced the replication efficiency as compared with wild‐type (WT) HBV. In contrast, combination of rtA194T (± LAM resistance) with HBeAg‐negative PC or BCP mutants increased the replication capacity of the drug‐resistant polymerase mutants, thereby restoring the viral replication to similar levels as WT clones. Clones harboring rtA194T showed partial resistance to tenofovir in vitro and also to LAM but remained susceptible to telbivudine and entecavir. Conclusion: The rtA194T polymerase mutation is associated with partial tenofovir drug resistance and negatively impacts replication competence of HBV constructs. Viral replication, however, can be restored to WT levels, if these polymerase mutations occur together with precore or basic core promoter substitutions as found in HBeAg‐negative hepatitis B. Patients with HBeAg‐negative chronic HBV infection may therefore be at particular risk when developing drug resistance to tenofovir. Telbivudine or entecavir should be considered as effective alternative treatment options for these patients. (HEPATOLOGY 2009.)

Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome.

BACKGROUND/AIMS The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. METHODS The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/75 females, 194 treatment-nai ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. RESULTS In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-nai ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. CONCLUSIONS Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications.

Single‐step real‐time PCR to quantify hepatitis B virus and distinguish genotype D from non‐D genotypes

Summary.  Hepatitis B virus (HBV) viral load and its genotype play important roles in clinical outcome, management of disease and response to antiviral therapy. In many parts of the world such as Europe or the Middle East, distinguishing HBV genotype D from non‐D is most relevant for treatment decisions, because genotype D‐infected patients respond poorly to interferon‐based therapeutic regimens. Here, we developed an in‐house real‐time PCR to concordantly assess HBV genotype (D vs non‐D) based on melt curve analysis and quantify the viral load. Genotype distinction was established with control plasmids of all HBV genotypes and validated with 57 clinical samples from patients infected with six different HBV genotypes. Our in‐house real‐time PCR assay could discriminate HBV genotype D from non‐D using single‐step melt curve analysis with a 2 °C difference in the melt curve temperature in all samples tested. Viral load quantification was calibrated with the WHO HBV international standard, demonstrating an excellent correlation with a commercial kit (r = 0.852; P < 0.0001) in a linear range from 3.2 × 102 to 3.2 × 1010 IU/mL. In conclusion, we developed a rapid, simple and cost‐effective method to simultaneously quantify and distinguish HBV genotypes D from non‐D with a single‐step PCR run and melt curve analysis. This assay should be a useful diagnostic alternative to aid clinical decisions about initiation and choice of antiviral therapy, especially in geographical regions with a high prevalence of HBV genotype D.

Impact of hepatitis B e antigen-suppressing mutations on the replication efficiency of entecavir-resistant hepatitis B virus strains.

Summary.  Hepatitis B e antigen (HBeAg)‐negative hepatitis B commonly requires long‐term treatment with nucleos(t)ide analogues aiming at persistently suppressing hepatitis B virus (HBV) replication to halt progression of liver disease and prevent complications. Entecavir (ETV) is widely used in HBeAg‐negative hepatitis B, but distinct HBV polymerase mutations can confer resistance against ETV, in conjunction with lamivudine resistance. Precore (PC) and basal core promoter (BCP) mutations that underlie HBeAg‐negativity enhance replication of lamivudine‐resistant mutants. To comprehensively analyse the impact of PC or BCP mutations on viral replication of ETV‐resistant HBV mutants, replication‐competent HBV constructs were generated harbouring lamivudine resistance (rtM204V/rtL180M, rtM204I) plus ETV resistance (rtS202G, rtS202I or rtT184G) on wild‐type (WT)‐, PC‐ and BCP‐backgrounds. Functional consequences on viral fitness and susceptibility to antivirals were assessed in vitro. The presence of any ETV resistance drastically reduced viral replication when compared to WT HBV. In rtS202G mutants (plus lamivudine resistance), addition of either PC or BCP mutations moderately enhanced the reduced replication, without reaching WT HBV levels. In rtS202I or rtT184G mutants, PC and BCP mutations did not significantly improve viral fitness. All ETV‐resistant constructs, independently of PC or BCP mutations, showed resistance towards ETV and lamivudine, but remained susceptible to tenofovir. Our data demonstrate that HBeAg‐suppressing PC or BCP mutations cannot restore the strongly reduced replicative capacity of ETV‐resistant HBV mutants to WT level, although they moderately increase replication of rtS202G combination mutants. ETV resistance thereby differs from lamivudine resistance alone, corroborating that ETV is in short term a safe option for HBeAg‐negative patients.

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

Summary.  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.

HBV genotype fluctuation (shifting and reversion) after liver transplantation

OBJECTIVES: Exclusive genome diversity of HBV has led to classification into more than 8 different genotypes and 40 subgenotypes. It has been demonstrated that the HBV genotype, subgenotype and subtype diversity is constant during viral transmission and chronic infection course. Unexpectedly, HBV genotype changes/shifts have rarely been observed in patients, in which the first detected genotype changed to another genotype during a longstanding course of disease. The mechanism behind genotype shifting remains unclear. However, initial co-infection with two different genotypes of HBV as well as hepatitis B e antigen (HBeAg)-seroconversion to hepatitis B e antibody (antiHBe) positivity might be prerequisites for HBV genotype switching. Also, it is noted that shifting can be triggered by antiviral or interferon therapy. In this longitudinal study, we report an unprecedented patient who experienced two sequential HBV genotype shifts. To the best of our knowledge, this is the first report of a host in which two sequential HBV genotype changes occurred, outside the context of co-infection or antiviral treatment. MATERIALS AND METHODS: A 68-years-old Caucasian man was referred to the University Hospital Leuven, in 2001. According to the clinical and Laboratory tests the patient was diagnosed as fulminant hepatitis B infection. Because of neurological deterioration, the patient received two times liver transplant from two HBsAg and anti-Hbc negative donors. After both procedures, the patient received 7 days of intravenous (IV) hepatitis B immune globulin and a standard immunosuppressives. Serum samples from 2001 to 2008 belonging to a HBV infected patient were sequentially investigated. HBV full-length genomes were isolated and sequenced and. Phylogenetically were analyzed. RESULTS: The four complete genome sequences belonged a sample at the first contact (2001), a sample 3 months after transplantation (2002) and two samples about three (2004) and five (2006) years post-transplantation were amplified and sequenced. HBV genotype from the first sample revealed subgenotype A2. Three months after transplantation, surprisingly the genotype had switched to genotype G. Of note, this strain encompassed 3 nucleotides insertion and also showed recombination with genotype A. Interestingly, HBV genotype A strains were isolated from next two samples (2004 and 2006) of this patient. [Figure 1] CONCLUSION: we described the first case of an HBV infected host showing genotype shifting and reversion. Possible underlying mechanisms include co-infection or superinfection and subsequent selection of a dominant genotype by immunosuppressive agents or HBIG administration.