Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

Heterologous replacement of the supposed host determining region of avihepadnaviruses: high in vivo infectivity despite low infectivity for hepatocytes.

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22-90 (Du-He4) or its subsegments 22-37 and 37-90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 10(8) viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants.

Evidence from nature: interspecies spread of heron hepatitis B viruses.

Heron hepatitis B viruses (HHBVs) in three subspecies of free-living great blue herons (Ardea herodias) from Florida, USA, were identified and characterized. Eight of 13 samples were positive in all assays used, whereas sera from egrets, which are also members of the family Ardeidae, were negative in the same assays. Comparative phylogenetic analysis of viral DNA sequences from the preS/S region of previously reported and novel HHBV strains isolated from captive grey herons (Germany) and free-ranging great blue herons (USA), respectively, revealed a strong conservation (95 % sequence similarity) with two separate clusters, implying a common ancestor of all strains. Our data demonstrate for the first time that different subspecies of herons are infected by HHBV and that these infections exist in non-captive birds. Phylogenetic analysis and the fact that the different heron species are geographically isolated populations suggest that lateral transmission, virus adaptation and environmental factors all play a role in HHBV spreading and evolution.

Inactivation of duck hepatitis B virus by a hydrogen peroxide gas plasma sterilization system: laboratory and 'in use' testing.

Human hepatitis B virus (HBV) is an important cause of nosocomial infections and can be transmitted by contaminated instruments. However, tests of the efficacy of sterilization of materials and equipment contaminated by HBV are difficult to perform because the virus cannot be cultured in the laboratory. In this study, we aimed to evaluate the capability of a low temperature, hydrogen peroxide gas plasma sterilizer (Sterrad, Advanced Sterilization Products, Irvine California,) to inactivate duck hepatitis B virus (DHBV). In laboratory efficacy studies using DHBV dried on to glass filter carriers and exposed to one-half of the hydrogen peroxide gas plasma sterilization process, there was a 10(7) or greater decrease in the viral titer, with no infectivity detected on the carriers after treatment. In-use studies were performed using a laparoscope that was experimentally contaminated with DHBV to mimic the possible transmission of infection between successive patients. Following exposure to the hydrogen peroxide gas plasma sterilization process no transmission of DHBV infection from the laparoscope occurred despite obvious visual soiling with blood (N = 8) while the transmission rate for the unprocessed laparoscope (positive control) was 100% (26/26), and that for instruments after a water wash was 63% (7/11). In conclusion the hydrogen gas plasma sterilization process completely inactivates DHBV a representative of the hepadna group of viruses.

Oral famciclovir against duck hepatitis B virus replication in hepatic and nonhepatic tissues of ducklings infected in ovo.

Detection of hepadnaviral DNA in extrahepatic tissues of human and animal models of hepatitis B virus (HBV) has raised the question of whether virus replication in organs other than the liver could be targeted for the treatment of chronic hepatitis B. Since duck hepatitis B virus (DHBV) replication is dynamic in the liver, kidney, pancreas, and spleen of newly hatched ducklings infected in ovo, we used the duck model and the new antiherpesvirus agent, famciclovir (FCV), to determine whether antiviral effect of nucleoside analogues on DHBV replication is pluripotential. Day-old ducklings hatched from eggs laid by a DHBV-carrier duck were bled and administered FCV (25 mg/kg/bd) orally for periods of 1, 2, 3, 6, 9, and 12 days. Seventeen (17) hours after the last dose of each regimen the duckling(s) was bled and postmortem samples of liver, kidney, pancreas, and spleen were snap-frozen and stored at -70 degrees C. Analysis of plasma samples of ducklings treated for 2 days and longer by dot-blot hybridisation showed that levels of DHBV DNA were reduced significantly compared to levels in samples collected before treatment begun. Southern blot hybridisation of tissue DNA corroborated these results and showed that DHBV DNA replicative intermediates in all the tissues examined were reduced to levels that reflected the amount of virus released into the blood of each treated duckling. It is concluded from these results that if antiviral agents could be transformed to active metabolites in any infected tissues including the liver, replication of hepadnaviruses would be inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical disinfection of duck hepatitis B virus: a model for inactivation of infectivity of hepatitis B virus.

The susceptibility of duck hepatitis B virus (DHBV) to the virucidal effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) was compared to hepatitis B virus (HBV) with the aim of using the duck as a model for studying HBV disinfection. Using viral DNA polymerase (DNAP) as a target, inhibition of DNAP activity by chlorine disinfectants was found to be concentration-dependent but independent of contact time. Two minute exposure of minimal effective concentrations of sodium hypochlorite (domestic bleach: 3600 ppm and industrial bleach: 3180 ppm) and sodium dichloroisocyanurate (3000 ppm available chlorine) to DHBV- and HBV-rich plasma totally inhibited DNA polymerase activity. DHBV particles in DHBV-carrier duck plasma (10(4.5) ID50/mL) were treated with these concentrations and inoculated intravenously into 18 one-day old ducklings (six animals/disinfectant). Analysis of plasma (0, 7 and 14 days post-infection) and post-mortem liver (14 days post-infection) by DNA hybridization techniques showed that DHBV DNA was undetectable in samples from all animals inoculated with disinfected virus particles. However, post-inoculation plasma and liver of 18 of 18 control ducklings inoculated with untreated virions were positive for DHBV DNA. These results show for the first time that total inhibition in vitro of hepadnavirus DNA polymerase activity by chemical disinfectants is predictive of inactivation of infectivity in vivo.