The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.evolution | zoonosis | virome | metagenomics | reverse genetics M ore than 40% of the human population has been infected with the hepatitis B virus (HBV), giving rise to 240 million chronic HBV carriers and ca. 620,000 HBV-associated deaths annually (1). A prophylactic vaccine containing the small HBV genotype A2 surface antigen (SHB) is part of the worldwide Expanded Program on Immunization. Because of the general success of SHBs-based vaccination, global eradication of HBV has been considered achievable (2, 3). Potential for the virus to be eradicated is supported by the fact that there are no known animal reservoirs. However, recent studies addressing the distribution of pathogens related to human viruses in wild animals, including mumps-and measles-related viruses in bats, have uncovered surprising putative novel reservoirs for human-pathogenic viruses (4). SignificanceHepatitis B virus (HBV) is the prototype hepadnavirus; 40% of humans have current or past infection. In a global investigation of viral diversity in bats, we discovered three unique hepadnavirus species. The relatedness of these viruses to HBV suggests that bats might constitute ancestral sources of primate hepadnaviruse...

Quantification of MHBs, particularly LHBs represents a novel tool for the identification of the IC stage.

Hepatitis B virus (HBV) is one of the smallest enveloped DNA viruses and the prototype member of the family of Hepadnaviridae that causes acute and chronic infections of mammals (including human) and birds. HBV has evolved an extreme adaptation and dependency to differentiated hepatocytes of its host. Despite its very limited coding capacity with only four open-reading frames, HBV is able to evade the immune system of the host and persist lifelong within infected hepatocytes. During active replication, HBV produces enormous viral loads in the blood and a massive surplus of subviral surface antigen particles in the serum of infected patients without killing their hepatocytes. Together with the use of a reverse transcriptase during replication, it provides an enormous genetic flexibility for selection of viral mutants upon selective pressure, for example, by the immune system or antiviral therapy. In addition, viral wild-type and mutated genomes are stably archived in the nucleus of the infected hepatocyte in an episomal DNA form that provides independence from cellular replication or integration within the host genome. We are just beginning to understand the delicate molecular and cellular interactions during the HBV replicative cycle within infected hepatocytes, so further studies are urgently needed to provide a better basis for further diagnostic and therapeutic options.

SummaryThe viral and cellular determinants leading to binding and entry of hepatitis B virus (HBV) are still not fully understood. We found that HBV infection of primary hepatocyte cultures is dependent on the presence of cholesterol in the viral envelope. Extraction of cholesterol from HBV purified from plasma of HBV-infected patients with methyl-betacyclodextrin (MbCD) leads to a strongly reduced level of infection. The cholesterol-depleted virions showed higher buoyant density (1.23 versus 1.17 g ml), a smaller diameter (39 versus 48 nm), but maintained particle integrity, antigenicity and ability to bind to hepatocytes. Although addition of exogenous cholesterol and cholesterol analogues restored the physical appearance of cholesteroldepleted virions, infectivity was only regained by cholesterol add-back. Infectivity of HBV produced from cell culture in the presence of inhibitors of cholesterol-synthesis is severely impaired. Interestingly, cholesterol extraction from cellular membranes, incubation with filipin and the protein tyrosine kinase inhibitor genistein showed no effect on HBV infection, excluding a role of lipid rafts for the infection process of HBV. In summary, presence of cholesterol within the viral envelope is not important for viral binding, but indispensable for the entry process of HBV and might be important for a later step in viral uptake, e.g. fusion in a yet unknown compartment.

The Taormina Consensus Conference defined ‘occult hepatitis B virus (HBV) infection’ (OBI) as the ‘presence of HBV DNA in the liver of individuals testing HBsAg-negative with currently available assays’. Most occult is the so-called ‘window period’ after exposure before HBV DNA appears in the blood. We identified two blood donors whose donations tested HBsAg- and HBV DNA-negative, but transmitted HBV. Both subsequently developed HBsAg and acute hepatitis. However, such cases are not considered as true OBI. A true transient OBI remains HBsAg-negative during the entire course. One case of acute OBI showed a peak viremia of 15,000 IU/ml HBV DNA and sub-borderline HBsAg, suggesting a ratio of virions to subviral particles of 1:10, whereas ‘normal’ cases show at peak viremia a ratio of 1:3,000. Blood donors with OBI may transmit HBV. We studied 5 blood donors with OBI and 55 of their recipients. In 22 recipients, transmission was probable, but they remained healthy. However, in 3 recipients, who were immunosuppressed at the time of transfusion, fatal fulminant hepatitis B developed. The majority of anti-HBc-positive healthy individuals have HBV DNA in the liver which may start replication under severe immunosuppression. Nine such cases are described here. OBI or reactivated HBV infections often lead to selection of HBsAg escape mutations as we could show in 11 of 14 cases. Infection of vaccinated individuals favors development of OBI as we observed in 6 blood donors. HB vaccination may solve the problem of overt HBV infection but may favor OBI.

The acute resolving occult HBV infection was caused by an HBeAg-negative variant, which otherwise was almost normal. The negative HBsAg result was probably due to an unusually low production of surplus HBsAg. The absence of the viral immunomodulator HBeAg and the early appearance of anti-HBs suggested a rapid noncytolytic HBsAg-specific T-cell response leading to low expression of HBsAg.

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We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common “a” determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.