In Vitro transmission of duck hepatitis B virus to primary duck hepatocyte cultures

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato-and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (ϳ10 3 virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.The hepatitis B virus (HBV) is a 3.2-kb, noncytopathic DNA virus that induces chronic hepatitis in approximately 5% of the global population and is the most common infectious cause of hepatocellular carcinoma (15,26). Infection with the closely related woodchuck hepatitis virus (WHV) in the eastern American woodchuck (Marmota monax) has been validated as a valuable virologic and pathogenic model of HBV infection (17,37). Although liver inflammation is the paramount pathological consequence of HBV and WHV infections and hepatocytes are the site of the most active hepadnaviral replication, both viruses also invade the host lymphatic system. HBV DNA, including replicative intermediates and covalently closed circular DNA (cccDNA), as well as virus RNA transcripts and DNA sequences integrated with the cellular genome, have been detected in peripheral blood mononuclear cells (PBMC) from patients with active chronic hepatitis B (13,29,30,34).

Section: Discussionsupporting

confidence: 92%

In Vitro and In Vivo Infectivity and Pathogenicity of the Lymphoid Cell-Derived Woodchuck Hepatitis Virus

Lew¹,

Michalak

2001

“…DHBV-positive PDHs were obtained by infecting day-old Pekin-Aylesbury ducklings with DHBV (strain D16). Seven days later, duckling livers were collagenase perfused as previously described (31). PDHs were maintained in Williams E medium with 10 mM Tris, pH 7.6, 6 mM HEPES, 0.02% sodium bicarbonate, 1% penicillin, 1% streptomycin, 1% glutamine, 0.02% glucose, 10 M hydrocortisone 21-hemisuccinate, 0.01% insulin, and 1.5% dimethyl sulfoxide.…”

Section: Methodsmentioning

confidence: 99%

Hepatocytes Traffic and Export Hepatitis B Virus Basolaterally by Polarity-Dependent Mechanisms

Bhat

Snooks

Anderson

2011

Viruses commonly utilize the cellular trafficking machinery of polarized cells to effect viral export. Hepatocytes are polarized in vivo, but most in vitro hepatocyte models are either nonpolarized or have morphology unsuitable for the study of viral export. Here, we investigate the mechanisms of trafficking and export for the hepadnaviruses hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in polarized hepatocyte-derived cell lines and primary duck hepatocytes. DHBV export, but not replication, was dependent on the development of hepatocyte polarity, with export significantly abrogated over time as primary hepatocytes lost polarity. Using Transwell cultures of polarized N6 cells and adenovirus-based transduction, we observed that export of both HBV and DHBV was vectorially regulated and predominantly basolateral. Monitoring of polarized N6 cells and nonpolarized C11 cells during persistent, long-term DHBV infection demonstrated that newly synthesized sphingolipid and virus displayed significant colocalization and fluorescence resonance energy transfer, implying cotransportation from the Golgi complex to the plasma membrane. Notably, 15% of virus was released apically from polarized cells, corresponding to secretion into the bile duct in vivo, also in association with sphingolipids. We conclude that DHBV and, probably, HBV are reliant upon hepatocyte polarity to be efficiently exported and this export is in association with sphingolipid structures, possibly lipid rafts. This study provides novel insights regarding the mechanisms of hepadnavirus trafficking in hepatocytes, with potential relevance to pathogenesis and immune tolerance.

“…Primary duck hepatocytes (PDH) were used to quantify the infectious virus titer according to a modification of a previously described method (1). PDH were prepared by collagenase perfusion of 7-day-old PekinAylesbury ducklings, as described previously (52). DHBV-negative PDH were grown on glass coverslips for 48 h. Apical and basolateral supernatants from BeWo transcytosis experiments were clarified by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus Translocates across a Trophoblastic Barrier

Bhat

Anderson

2007

Mother-infant transmission of hepatitis B virus (HBV) accounts for up to 30% of worldwide chronic infections. The mechanism and high-risk period of HBV transmission from mother to infant are unknown. Although largely prevented by neonatal vaccination, significant transmission continues to occur in high-risk populations. It is unclear whether HBV can traverse an intact epithelial barrier to infect a new host. Transplacental transmission of a number of viruses relies on transcytotic pathways across placental cells. We wished to determine whether infectious HBV can traverse a polarized trophoblast monolayer. We used a human placenta-derived cell line, BeWo, cultured on membranes as polarized monolayers, to model the maternal-fetal barrier. We assessed the effects of placental maturity and maternal immunoglobulin on viral transport. Intracellular viral trafficking pathways were investigated by confocal microscopy. Free HBV (and infectious duck hepatitis B virus) transcytosed across trophoblastic cells at a rate of 5% in 30 min. Viral transport occurred in microtubule-dependent endosomal vesicles. Additionally, confocal microscopy showed that the internalized virus traverses a monensin-sensitive endosomal compartment. Differentiation of the cytotrophoblasts to syncytiotrophoblasts resulted in a 25% reduction in viral transcytosis, suggesting that placental maturity may protect the fetus. Virus translocation was also reduced in the presence of HBV immunoglobulin. We show for the first time that transcytosis of infectious hepadnavirus can occur across a trophoblastic barrier early in gestation, with the risk of transmission being reduced by placental maturity and specific maternal antibody. This study suggests a mechanism by which mother-infant transmission may occur.