Efficient Infection of Primary Tupaia Hepatocytes with Purified Human and Woolly Monkey Hepatitis B Virus

Abstract

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.With an estimated 350 million chronically infected people worldwide, hepatitis B virus (HBV) infection represents a major health care problem. Every year nearly 1 million individuals succumb to HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma (9, 18). Although an efficient and safe vaccine is available, HBV is spreading, especially in Asia and Africa (10). Treatment of chronic HBV infection is still unsatisfactory. At present, alpha interferon and lamivudine are the only therapeutic options available in clinical practice. However, alpha interferon yields a sustained suppression of viral replication in only about one-third of patients, and lamivudine resistance with mutations in the viral polymerase gene is a frequent event in lamivudine-treated patients (3, 7). Based on transfection studies of hepatoma cell lines, the mechanisms of HBV replication have been elucidated in great detail (11,13). By contrast, little is known about the early events of the viral life cycle. Unfortunately, permanent cell lines are not permissive to HBV infection, and primary human hepatocytes are not easily available for in vitro infection studies. Furthermore, the quality of liver tissue obtained at surgery for the preparation of primary human hepatocytes is highly variable (5). Therefore, alternative experimental systems for studying HBV infection are urgently needed.Two reports have described transient HBV infection of the Asian tree shrew, Tupaia belangeri, in vivo (17,20). Furthermore, successful infection of tupaias with human herpes simplex virus and hepatitis C virus has been described (2, 19). Tupaias are squirrel-like animals that are closely related to primates and are endemic to subtropical areas of southeast Asia (12). The animals are easily bred in captivity. In addition, cultures of primary tupaia hepatocytes (PTH) can be prepared using well-established liver perfusion protocols.We previously described HBV infection of PTH (17); infection efficiency was too low, h...

Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes†

“…[21][22][23][24] We and others have demonstrated that primary Tupaia hepatocytes (PTHs) represent a convenient and suitable in vitro model to study HBV infection and replication. [25][26][27] In this study, we demonstrate that HBV core promoter mutants associated with fulminant hepatitis exhibit a phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with mutations.…”

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

“…Further studies in the HBV and WHV system will have to establish whether mammalian core protein phosphorylation is essential for infectivity as well. The recently described infection of primary tupaia hepatocytes (34) might represent a suitable model to address this question. Interestingly, phosphorylation of capsid protein by capsid-associated kinase activities has also been suggested to play a key role during the early steps of HIV infection (35).…”

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

“…Currently, primary human hepatocytes (PHHs) (Lempp et al, 2017;Ni and Urban, 2017;Shimura et al, 2017), HepaRG cells (Gripon et al, 2002;Schulze et al, 2012), and primary tupia hepatocytes (PTHs) (Kock et al, 2001;Ren and Nassal, 2001) are the main cell models used to study the early steps of HBV infection. PHHs, the natural host of HBV, are regarded as the ideal cellular model for HBV infection.…”

“…However, the addition of DMSO and polyethylene glycol 8000 (PEG8000) during the infection of PTHs does not significantly promote the infectivity of HBV (Weizsäcker and Roggendorf, 2005). In addition, human serum competes with HBV particles for binding to the cell membrane (Kock et al, 2001), leading to a reduced experimental confidence level. Novel HBV infection models are thus urgently needed.…”