scholarly journals Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

2001 ◽  
Vol 75 (8) ◽  
pp. 3811-3818 ◽  
Author(s):  
Mengji Lu ◽  
Gero Hilken ◽  
Dongliang Yang ◽  
Thekla Kemper ◽  
Michael Roggendorf
Keyword(s):  
Hepatitis Virus ◽  
Viral Population ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Lymphoproliferative Response ◽  
Hepatocyte Cultures ◽  
Liver Tissues

ABSTRACT Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

scholarly journals Coadministration of Gamma Interferon with DNA Vaccine Expressing Woodchuck Hepatitis Virus (WHV) Core Antigen Enhances the Specific Immune Response and Protects against WHV Infection

2001 ◽  
Vol 75 (11) ◽  
pp. 5036-5042 ◽  
Author(s):  
Felix Siegel ◽  
Mengji Lu ◽  
Michael Roggendorf
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Dna Vaccines ◽  
Hepatitis Virus ◽  
Dna Vaccination ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Lymphoproliferative Response ◽  
Virus Challenge ◽  
Cellular Immune

ABSTRACT DNA vaccinations are able to induce strong cellular immune responses in mice and confer protection against infectious agents. However, DNA vaccination of large animals appears to be less effective and requires repeated injections of large amounts of plasmid DNA. Enhancement of the efficiency of DNA vaccines may be achieved by coapplication of cytokine-expressing plasmids. Here we investigated, with woodchucks, whether coadministration of an expression plasmid for woodchuck gamma interferon (IFN-γ), pWIFN-γ, can improve DNA vaccination with woodchuck hepatitis virus core antigen (WHcAg). Animals were immunized with pWHcIm (a plasmid expressing WHcAg) alone or with a combination of pWHcIm and pWIFN-γ using a gene gun. Six weeks postimmunization, all animals were challenged with 105 genome equivalents of woodchuck hepatitis virus (WHV). The antibody and lymphoproliferative immune responses to WHV proteins were determined after immunization and after challenge. Vaccination with pWHcIm and pWIFN-γ led to a pronounced lymphoproliferative response to WHcAg and protected woodchucks against subsequent virus challenge. Two of three animals vaccinated with pWHcIm alone did not show a detectable lymphoproliferative response to WHcAg. A low-level WHV infection occurred in these woodchucks after challenge, as WHV DNA was detectable in the serum by PCR. None of the pWHcIm-vaccinated animals showed an anti-WHcAg antibody response after DNA vaccination or an anamnestic response after virus challenge. Our results indicate that coadministration of the WIFN-γ gene with pWHcIm enhanced the specific cellular immune response and improved the protective efficacy of WHV-specific DNA vaccines.

scholarly journals Infection Patterns Induced in Naive Adult Woodchucks by Virions of Woodchuck Hepatitis Virus Collected during either the Acute or Chronic Phase of Infection

2015 ◽  
Vol 89 (17) ◽  
pp. 8749-8763 ◽  
Author(s):  
Natalia Freitas ◽  
Tetyana Lukash ◽  
Louise Rodrigues ◽  
Sam Litwin ◽  
Bhaskar V. Kallakury ◽  
...  
Keyword(s):  
Chronic Infection ◽  
Hepatitis Virus ◽  
Acute Infection ◽  
Chronic Phase ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Circular Dna ◽  
The Individual ◽  
Cell Spread

ABSTRACTThe infectivity of hepadnavirus virions produced during either acute or chronic stages of infection was compared by testing the ability of the virions of woodchuck hepatitis virus (WHV) to induce productive acute infection in naive adult woodchucks. Serum WHV collected during acute infection was compared to virions harvested from WHV-infected woodchucks during either (i) early chronic infection, when WHV-induced hepatocellular carcinoma (HCC) was not yet developed, or (ii) late chronic infection, when established HCC was terminal. All tested types of WHV inoculum were related, because they were collected from woodchucks that originally were infected with standardized WHV7 inoculum. Despite the individual differences between animals, the kinetics of accumulation of serum relaxed circular DNA of WHV demonstrated that the virions produced during early or late chronic infection are fully capable of inducing productive acute infection with long-lasting high viremia. These findings were further supported by the analysis of such intrahepatic markers of WHV infection as replicative intermediate DNA, covalently closed circular DNA, pregenomic RNA, and the percentage of WHV core antigen-positive hepatocytes measured at several time points over the course of 17.5 weeks after the inoculation. In addition, the observed relationship between the production of antibodies against WHV surface antigens and parameters of WHV infection appears to be complex. Taken together, the generated data suggest thatin vivohepadnavirus virions produced during different phases of chronic infection did not demonstrate any considerable deficiencies in infectivity compared to that of virions generated during the acute phase of infection.IMPORTANCEThe generated data suggest that infectivity of virions produced during the early or late stages of chronic hepadnavirus infection is not compromised. Our novel results provided several lines of further evidence supporting the idea that during the state of chronic infectionin vivo, the limitations of hepadnavirus cell-to-cell spread/superinfection (observed recently in the woodchuck model) are not due to the diminished infectivity of the virions circulating in the blood and likely are (i) related to the properties of hepatocytes (i.e., their capacity to support hepadnavirus infection/replication) and (ii) influenced by the immune system. The obtained results further extend the understanding of the mechanisms regulating the persistence of hepadnavirus infection. Follow-up studies that will further investigate hepadnavirus cell-to-cell spread as a potential regulator of the chronic state of the infection are warranted.

NO IN VIVO EFFECT OF TRISODIUM PHOSPHONOFORMATE ON WOODCHUCK HEPATITIS VIRUS PRODUCTION

2009 ◽  
Vol 90B (1-6) ◽  
pp. 449-451
Author(s):  
Erik Nordenfelt ◽  
Anders Widell ◽  
Bengt GÖran Hansson ◽  
Bengt LÖfgren ◽  
Carsten MÖller-Nielsen ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Virus Production ◽  
Woodchuck Hepatitis Virus

scholarly journals Inhibition of Cellular Proteasome Activities Enhances Hepadnavirus Replication in an HBX-Dependent Manner

2004 ◽  
Vol 78 (9) ◽  
pp. 4566-4572 ◽  
Author(s):  
Zhensheng Zhang ◽  
Ulrike Protzer ◽  
Zongyi Hu ◽  
James Jacob ◽  
T. Jake Liang
Keyword(s):  
Recombinant Adenovirus ◽  
Hepatitis Virus ◽  
Proteasome Inhibitors ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type

ABSTRACT The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.

scholarly journals Antiviral Effect of Oral Administration of Tenofovir Disoproxil Fumarate in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection

2005 ◽  
Vol 49 (7) ◽  
pp. 2720-2728 ◽  
Author(s):  
Stephan Menne ◽  
Paul J. Cote ◽  
Brent E. Korba ◽  
Scott D. Butler ◽  
Andrea L. George ◽  
...  
Keyword(s):  
Oral Administration ◽  
Tenofovir Disoproxil Fumarate ◽  
Hepatitis Virus ◽  
Antiviral Effect ◽  
Wild Type Virus ◽  
Woodchuck Hepatitis Virus ◽  
Wild Type ◽  
Hepatic Expression ◽  
Chronic Hbv

ABSTRACT Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.

P.050 Appearance of core antigen negative hepatocytes in woodchucks chronically infected with woodchuck hepatitis virus

2006 ◽  
Vol 36 ◽  
pp. S76
Author(s):  
C. Xu ◽  
T. Yamamoto ◽  
T. Zhou ◽  
C. Aldrich ◽  
J.M. Cullen ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus

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

2001 ◽  
Vol 75 (4) ◽  
pp. 1770-1782 ◽  
Author(s):  
Yuan-Yee Lew ◽  
Tomasz I. Michalak
Keyword(s):  
Lymphoid Cell ◽  
Lymphatic System ◽  
Hepatitis Virus ◽  
Lymphoid Cells ◽  
Woodchuck Hepatitis Virus ◽  
Dna Viruses ◽  
B Virus ◽  
Total Pool

ABSTRACT 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 (∼103 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.

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