Repeated Passage of Wild-Type Woodchuck Hepatitis Virus in Lymphoid Cells Does Not Generate Cell Type-Specific Variants or Alter Virus Infectivity

Abstract

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus, infects the liver but also invariably establishes persistent infection in the lymphatic system. Although the dose of invading virus appears to be the main factor in determining whether WHV infection is restricted to the lymphatic system or also engages the liver, the nature of WHV lymphotropism remains unclear and a role for a specific lymphotropic variant was not excluded. The availability of woodchuck lymphocyte and hepatocyte cultures susceptible to WHV infection allows investigation of this issue in vitro. We hypothesized that repeated passage of wild-type WHV in lymphoid cells should lead to enrichment of a lymphotropic virus variant, if in fact such a variant exists. For this purpose, wild-type WHV with a homogeneous sequence was used as the inoculum, while lymphoid cells from a single healthy woodchuck donor and a normal woodchuck WCM-260 hepatocyte line served as infection targets. The serial passage of the wild-type virus repeated up to 13 times for both cell types did not lead to the emergence of cell type-specific WHV variants, as revealed by sequence analysis of the virus envelope and the core and X gene sequences. Moreover, the virus passaged in both cell types remained infectious for naive woodchucks, produced infection profiles that depended upon virus dose but not on virus cellular origin, and retained its initial DNA sequence. These results imply that WHV lymphotropism is a natural propensity of the wild-type virus and is not a consequence of infection with a viral variant.Accumulated evidence indicates that hepadnaviruses are capable of replication not only in hepatocytes but also in cells of the immune system. In regard to symptomatic, chronic hepatitis B virus (HBV) infection, which is characterized by the continuous presence of HBV surface antigen (HBsAg) in serum and protracted liver necroinflammation, long-term persistence of virus has been shown in both the liver and the lymphatic system (6,21,26,35,42,43,45). However, liver and lymphoid cells have also been found to be the sites of HBV genome carriage in serum HBsAg-negative patients in whom acute hepatitis (AH) had apparently resolved completely when sensitive PCR-based assays were applied to detect the virus genome (3,4,30,37,40,46). This occult form of HBV infection might be a source of infectious virus available for transmission to healthy individuals through blood or organ donations (1,12,21,24,27), as well as a potential cause of diseases of seemingly unknown etiology engaging the liver and the lymphatic system. In this regard, retrospective studies of hepatocellular carcinoma (HCC) of previously undetermined etiology showed low levels of HBV genomes in 63.5% of liver tissue samples tested (38). In some of those cases, integration of viral DNA into the host's genome was detected. This implies that trace amounts of HBV, identifiable by molecular assays of a sensitivity greater than those currently used in clinical laboratories, may retain pathogeni...

Primary Occult Hepadnavirus Infection Induces Virus-Specific T-Cell and Aberrant Cytokine Responses in the Absence of Antiviral Antibody Reactivity in the Woodchuck Model of Hepatitis B Virus Infection

“…In summary, woodchucks infected, challenged, or rechallenged with 50 vge of otherwise pathogenic WHV acquired sustained, serologically, biochemically, and histologically silent but molecularly evident POI. The animals were not protected from infection with a high dose of the same virus and acquired acute hepatitis, as previously reported (10,50,56). POI and serologically apparent WHV infection are accompanied by WHV-specific T-cell proliferative responses that were comparable in magnitude.…”

“…In general, POI is characterized by the presence of WHV DNA in serum at levels compatible with those occurring in SOI (Յ200 vge/ml) and in peripheral blood mononuclear cells (PBMC) (Յ10 3 vge/g of total DNA), but usually not in the liver, the absence of serological (immunovirological) markers of infection, including WHV surface antigen (WHsAg), antibodies against WHsAg (anti-WHs) and WHV core antigen (anti-WHc), and by normal liver morphology (reviewed in references 49 and 54). The WHV nucleotide sequence in POI has been found to be the wild type (50,56). Further, the virus persisting in POI remains biologically competent, since when WHV derived from serum or lymphoid cells of animals with POI was administered to naive woodchucks, symptomatic AH was induced (10,50).…”

“…injected with 50 DNase digestion-protected vge of WHV/tm3 inoculum. It was previously established that WHV doses below 1,000 virions of WHV/tm3 inoculum, as well as other WHV inocula, consistently induce POI in WHV-naive, immunologically competent woodchucks (50,56). After 26 w.p.p.i., the animals were challenged with a high dose of WHV/tm3 (1.1 ϫ 10 10 vge; group LH [n ϭ 3]) or a low dose of WHV/tm3 (50 vge; group LL [n ϭ 3]) or injected with 0.5 ml of PBS (group LN; n ϭ 2) (Fig.…”

“…Moreover, the understanding of the natural course, virological properties, requirements of transmission, and potential pathological consequences of OBI is owed to a large degree to studies in the woodchuck model of HBV infection (reviewed in reference 49). Among others, it was established that replication of hepadnavirus in SOI progresses not only in the liver but also in the immune system (10,50,53,56; reviewed in reference 49). In woodchucks, this infection persists for life, and virus replicative intermediates, including WHV covalently closed circular DNA and mRNA, are detectable by highly sensitive assays employing PCR combined with identification of the resulting amplicons by nucleic acid hybridization (NAH), i.e., PCR/NAH (10,53).…”

Repeated Exposure to Trace Amounts of Woodchuck Hepadnavirus Induces Molecularly Evident Infection and Virus-Specific T Cell Response in the Absence of Serological Infection Markers and Hepatitis

“…injection of a single WHV dose containing less than 1,000 DNase digestion-protected virions invariably induce POI in WHVnaïve, healthy woodchucks (11,12,37,40). This was consistently observed after administration of low doses of different WHV inocula, i.e., WHV/ tm3 (GenBank accession number AY334075) (12,40), WHV/tm4 (GenBank accession number GU734791) (38), and WHV/tm5 (Mulrooney-Cousins and Michalak, unpublished). In the current study, four woodchucks (3 males and 1 female) were i.v.…”