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.

Transgenic mice replicating hepatitis B virus but lacking expression of the major HBsAg.

Hepatitis B Virus (HBV) transgenic mice replicating the viral genome at high level but lacking expression of the small envelope protein (HBsAg) have been produced using a terminally redundant viral DNA construct (HBV 1.4). The generation of viable infectious progeny was dependent on sex and age of mice. Viral mRNA was abundant in liver and kidneys and at low levels in other organs of the mice. No viral particles or HBV envelope proteins could be detected in sera of mice. Despite expression of non-secreted LHBs and MHBs proteins in the liver, there was no accumulation of viral particles in the endoplasmic reticulum of hepatocytes and no necroinflammatory hepatitis was observed. Therefore, these mice represent an excellent model for studies of the role of HBsAg in viral assembly, antiviral immune responses, the further understanding of HBV immunopathogenesis, and the development of antiviral vaccines.

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

Characterization of duck leucocytes by monoclonal antibodies.

cDNAs encoding CD3epsilon, CD4 and the alpha-chain of CD8 of ducks were cloned. Monoclonal antibodies against Pekin duck CD4 and CD8alpha revealed that the antigens were expressed by the majority of thymocytes and by subpopulations of CD3+ cells in peripheral tissues. CD8alpha cell surface expression was also found on 90% of bursal cells. The B cell specificity of a newly developed mab to the immunoglobulin light-chain (L-chain) was confirmed by double labelling studies with the chicken B-cell cytokine BAFF. Using these tools and a mab reacting with the cytoplasmic domain of CD3epsilon, we demonstrated that the CD8alpha molecule is expressed to high levels in CD3-positive T cells and at lower levels in CD3-negative bursal and peripheral B cells. Mab 2-4, which recognizes the chicken CD28 molecule, was found to react with CD4-positive duck lymphocytes and with CD8-positive duck T cells but not with CD8-positive B cells. Mab K1, which recognizes a common antigen on chicken thrombocytes and monocytes/macrophages, was found to react with duck thrombocytes and macrophages. Thus, a range of mabs is now available which will allow to phenotype the major leucocyte populations in ducks, a surrogate infection model for hepatitis B virus.

Phenotyping hepatitis B virus variants: from transfection towards a small animal in vivo infection model.

The existence of vaccine escape and drug resistant hepatitis B virus (HBV) variants is now well established, and various of the underlying prototypic mutations have been defined. Genotypic detection of such variants allows to predict their clinical phenotype. However, the relevance of non-predefined mutations occurring during therapy can be assessed only by phenotypic assays. The fundamental properties of a functional virus are the ability to replicate the genome, to form infectious virions, and to cope with the host defense in order to establish and maintain infection; a virus meeting all these criteria is biologically fit. At present, HBV DNA transfection provides a reliable method to address replication-competence and physical formation of complete virus particles. The inherent inter-experiment variability of transient transfection can be overcome by stable cell lines expressing wild-type and prototypic variant HBVs. Such cell lines provide important tools for studying basic aspects of HBV replication as well as for drug discovery. Phenotypic assays measuring HBV infectivity are less advanced but several surrogate systems obviating the need for primary human hepatocyte cultures are being established. The ultimate, and most desirable, phenotypic assay system would be a small, immuno-competent experimental animal in which human HBV can establish chronic infection. Only this would allow to fully address the fitness of HBV variants, and thus to assess the risk of their spreading in the general population. Various ways towards this goal can be envisaged but recent model studies in the duck HBV system indicate that much more has to be learned on the molecular determinants of hepadnaviral host-range to rationally design such experiments.

The interferon system of non-mammalian vertebrates.

Genetic analysis in mice and other mammals showed that the interferon (IFN) system is important for resistance to viral and bacterial infections. IFN is thought to play a similar role in non-mammalian species, although knowledge is lagging behind. Molecular cloning revealed low but significant conservation of IFN genes in birds and fish. Whereas chickens and other birds have multiple genes for IFN-alpha and single genes for IFN-beta and IFN-gamma like most mammals, fish appear to have fewer IFN genes. Nonetheless, regulation of IFN-alpha and IFN-beta genes in response to viral and non-viral activators is well conserved in birds and fish, and similar sets of genes are induced in response to IFN in mammalian and non-mammalian vertebrates. IFN-like activities were also observed in reptiles and amphibians, but genes encoding these activities have not yet been cloned. Insights into the IFN system of non-mammalian species may lead to improved prophylactic and therapeutic approaches against virus infections in poultry and fish.