Sequence of events in natural infection of Pekin duck embryos with duck hepatitis B virus.

The major mode of natural infection of duck hepatitis B virus (DHBV) in Pekin ducks is vertical transmission, with 95 to 100% of the embryos from DHBV-infected dams eventually becoming infected. Maternally transmitted virus is present in large quantities in the yolk of unincubated eggs and is taken up by the embryo during early development. Synthesis of DHBV DNA in the embryo begins at about 6 days of incubation and coincides with the formation of the liver. DHBV DNA synthesis is incomplete, however, until 8 to 10 days of incubation, as shown by comparing the electrophoretic patterns of DHBV-specific nucleic acid species from embryonic livers at successive stages of development. From 8 days of incubation and continuing throughout embryonic development, subviral particles, which resemble viral replication intermediates isolated from infected livers of post-hatch ducklings, appear in the circulation. These particles contain a polymerase activity that utilizes an RNA template to synthesize viral DNA. Our results suggest that certain host functions, which appear during embryonic development, may be required for DHBV replication and assembly. It is possible that in mammals a similar developmental process occurs. The failure to find human hepatitis B virus in the circulation of most babies, born to hepatitis B virus carrier women, in the first few weeks after birth may reflect such a process.     

In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus.

Duck hepatitis B virus (DHBV) obtained from the serum of congenitally infected ducks was used to infect primary duck hepatocyte cultures 1 to 4 days after plating. Virus replication was demonstrated by the appearance, beginning at 2 days after infection, of intracellular covalently closed-circular and single-stranded DHBV DNA replicative intermediates which were not present in the inoculating virus preparation. With increasing time after infection there was further amplification of intracellular relaxed circular, covalently closed-circular, and single-stranded DHBV DNA. Cultures of primary duck hepatocytes are competent for infection with DHBV only during the first 4 days of culture. Synthesis of DHBV core antigen and DHBV surface antigen was detected by immunofluorescence in 10% of the hepatocytes in culture. De novo synthesis and release of infectious virus was also demonstrated. Therefore, all stages of viral replication were carried out by these experimentally infected primary hepatocyte cultures. This system makes it possible to study DHBV replication in vitro.     

Cellular receptor traffic is essential for productive duck hepatitis B virus infection

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.     

Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step

Elevated levels of prostaglandins (PGs), products of cyclooxygenases (COXs), are found in the plasma and stool of rotavirus-infected children. We sought to determine the role of COXs, PGs, and the signal transduction pathways involved in rotavirus infection to elucidate possible new targets for antiviral therapy. Human intestinal Caco-2 cells were infected with human rotavirus Wa or simian rotavirus SA-11. COX-2 mRNA expression and secreted PGE2 levels were determined at different time points postinfection, and the effect of COX inhibitors on rotavirus infection was studied by an immunofluorescence assay (IFA). To reveal the signal transduction pathways involved, the effect of MEK, protein kinase A (PKA), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inhibitors on rotavirus infection was analyzed. In infected Caco-2 cells, increased COX-2 mRNA expression and secreted PGE2 levels were detected. Indomethacin (inhibiting both COX-1 and COX-2) and specific COX-1 and COX-2 inhibitors reduced rotavirus infection by 85 and 50%, respectively, as measured by an IFA. Indomethacin reduced virus infection at a postbinding step early in the infection cycle, inhibiting virus protein synthesis. Indomethacin did not seem to affect viral RNA synthesis. Inhibitors of MEK, PKA, p38 MAPK, and NF-kappaB decreased rotavirus infection by at least 40%. PGE2 counteracted the effect of the COX and PKA inhibitors but not of the MEK, p38 MAPK, and NF-kappaB inhibitors. Conclusively, COXs and PGE2 are important mediators of rotavirus infection at a postbinding step. The ERK1/2 pathway mediated by PKA is involved in COX induction by rotavirus infection. MAPK and NF-kappaB pathways are involved in rotavirus infection but in a PGE2-independent manner. This report offers new perspectives in the search for therapeutic agents in treatment of severe rotavirus-mediated diarrhea in children.     

Monoclonal antibodies to a 55-kilodalton protein present in duck liver inhibit infection of primary duck hepatocytes with duck hepatitis B virus.

As an approach to identifying hepatocyte receptors for the avian hepadnavirus duck hepatitis B virus (DHBV), hybridomas were prepared from mice immunized with permissive duck hepatocytes. Monoclonal antibodies (MAbs) were screened for the ability to inhibit binding of DHBV particles to primary duck hepatocytes and to block infection. We identified two MAbs which partially blocked binding and caused marked inhibition of infection of primary duck hepatocytes with DHBV. Lack of cross-reactivity with DHBV envelope proteins suggested that inhibition of infection was due to specific interaction between the antibodies and a host cell surface molecule. Both MAbs immunoprecipitated a 55-kDa protein (p55) expressed in duck liver and several other duck tissues. p55 homologs were also identified in other birds and mammals. We predict from our data that only a small proportion of total cellular p55 molecules are expressed at the surfaces of hepatocytes and that p55 is involved in some early step in the infectious pathway.     

Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo.

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.     

Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBV-infected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.     

Identification and expression of glycine decarboxylase (p120) as a duck hepatitis B virus pre-S envelope-binding protein.

A 120-kilodalton protein (p120) was identified in the duck liver that binds to several truncated versions of duck hepatitis B virus (DHBV) pre-S envelope protein, suggesting p120 may serve as a DHBV co-receptor. The amino acid sequences of tryptic peptides from purified p120 were found to be the duck p protein of the glycine decarboxylase complex (DGD). DGD cDNA cloning revealed extensive protein conservation with the chicken homologue except for several insertions in the N-terminal leader sequence. The DGD cDNA contained no in-frame AUG codon at the predicted initiation site of the open reading frame, and site-directed mutagenesis experiments established an AUU codon as the translational initiator. The DGD protein expressed in rabbit reticulocyte lysates bound truncated DHBV pre-S protein identical to that of p120 derived from duck liver confirming DGD as p120. Moreover, transfection studies in liver- and kidney-derived cells revealed both cell surface and cytoplasmic expression of the protein. Cloning of the glycine decarboxylase cDNA will permit a direct test of whether it functions as a cell surface co-receptor or as a co-factor in the DHBV replication cycles.     

Conditional replication of duck hepatitis B virus in hepatoma cells

To facilitate investigations of replication and host cell interactions in the hepadnavirus system, we have developed cell lines permitting the conditional replication of duck hepatitis B virus (DHBV). With the help of this system, we devised conditions for core particle isolation that preserve replicase activity, which was not found in previous preparations. Investigations of the stability of viral DNA intermediates indicated that both encapsidated DNA and covalently closed circular DNA (cccDNA) were turned over independently of cell division. Moreover, we showed that alpha interferon reduced the accumulation of RNA-containing viral particles. The availability of a synchronized replication system will permit the biochemical analysis of individual steps of the viral replication cycle, including the mechanism and regulation of cccDNA formation.     

Immunology of hepatitis B virus and hepatitis C virus infection

More than 500 million people worldwide are persistently infected with the hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite many common features in the pathogenesis of HBV- and HCV-related liver disease, these viruses markedly differ in their virological properties and in their immune escape and survival strategies. This review assesses recent advances in our understanding of viral hepatitis, contrasts mechanisms of virus-host interaction in acute hepatitis B and hepatitis C, and outlines areas for future studies.     

Cloned duck hepatitis B virus DNA is infectious in Pekin ducks

Approximately 10% of German-bred Pekin ducks were found to be chronically infected with duck hepatitis B virus (DHBV). The genomes of three German DHBV isolates analyzed were closely related but showed substantial restriction site polymorphism compared with U.S. isolates. We tested the infectivity of three sequence variants of cloned DHBV DNA by injecting them into the liver of virus-free ducklings. Most of these animals injected with double-stranded closed-circular or plasmid-integrated dimer DHBV DNA developed viremia, demonstrating the infectivity of all three cloned DHBV DNA variants. The cloned viruses produced were indistinguishable from those from naturally infected animals, implying that our experimental approach can be used to perform a functional analysis of the DHBV genome.     

Immunopathogenesis of hepatitis B virus infection

Hepatitis B virus (HBV) infection is a non-cytopathic hepatotropic virus that can lead to severe liver disease including acute hepatitis, cirrhosis and hepatocellular carcinoma. Successful clearance of the virus as well as the establishment of liver disease is largely driven by a complex interaction between the virus and the host immune response. In this review, the immunological events, including both the innate and adaptive immune response are discussed in the setting of both acute and chronic HBV infection and liver disease.     

Dynamics of hepatitis B virus infection

Mathematical models of the dynamics of HIV and hepatitis C virus infection have proven to be of great utility in understanding pathogenesis and designing better treatments. Here, we review the state of the art in modeling and interpreting data obtained from hepatitis B virus infected patients treated with antiviral agents.