Use of the cross-reactivity with hepatitis B virus antigens and antibodies for the demonstration of a woodchuck hepatitis virus ‘e’ antigen-antibody system

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SUMMARYPrimary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virusspecific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3.3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Section: Methodsmentioning

confidence: 99%

Maintenance of Woodchuck Hepatitis Virus Activity in Woodchuck Hepatocyte Primary Culture

Thézé

Gripon

Fourel

et al. 1987

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“…So far, documented proof for the existence of a WHV e antigen has never been reported although the presence of an e antigen/anti-e antigen system in the blood of WHV-infected Eastern woodchucks was demonstrated by indirect immunological methods (Hantz et al, 1983). We have hence studied the expression and the processing of the product of the entire WHV C ORF.…”

mentioning

confidence: 99%

Characterization and biosynthesis of the woodchuck hepatitis virus e antigen

Carlier

Jean-Jean²,

Rossignol

1994

The biosynthesis of the secretory core gene product of the woodchuck hepatitis virus (WHV) was studied in human cells. We have shown that the WHV e antigen was a N-glycosylated (most likely a diglycosylated) protein, with an apparent M r of 24K. To demonstrate that the WHV precore protein was correctly processed in human cells, we engineered chimeric proteins in which signal peptides or arginine-rich domains of WHV and hepatitis B virus (HBV) precore proteins were exchanged. Our results showed that both the signal peptide and the arginine-rich region of WHV precore protein were cleaved off during the secretion pathway, as previously reported for precore protein of human HBV and duck HBV. These observations demonstrate that the maturation process of the e antigen is conserved in hepadnaviruses. In addition, on the basis of inhibition experiments, we suggest that the cleavage of the carboxy terminus of the WHV precore protein occurred in a post-endoplasmic reticulum compartment, most likely beyond the medial Golgi, and that this cleavage was catalysed by an aspartyl protease.

“…The cells were incubated for 30 min at 4 °C with the appropriate quantity of antibody (at saturating concentration), then washed three times in cold PBS and brought to a concentration of 106 cells/ml in a 1 ~ formaldehyde saline solution. Anti-WHs was obtained from a woodchuck which had recovered from WHV infection and was subsequently boosted with purified WHsAg (Hantz et al, 1983). This serum showed anti-WHs reactivity even at dilutions beyond the anti-WHc endpoint as demonstrated by immunofluorescence analysis of WHV-infected liver (Frommel et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

“…Serum was obtained from the remaining 4 ml of blood and stored at -20 °C. Sequential samples were tested in parallel in DNA or RNA by PCR as described below, and for WHsAg by radioimmunoassay (RIA) using HBsAg commercial kits, available from Abbott Laboratories, taking advantage of the cross-reactivity between WHsAgs and HBsAgs (Hantz et al, 1983;Frommel et al, 1984).…”

Section: Methodsmentioning

confidence: 99%

“…For neutralization experiments, serum-purified WHsAg (Hantz et al, 1983) and purified recombinant HBcAg (Biogen) were used. Undiluted conjugated antibodies (1 mg/ml) were incubated for 30 min at 4 °C with different concentrations (10, 1 and 0.1 ~tg/ml) of their corresponding antigens or with the same volume of PBS and centrifuged for 10 min at 14000 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

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Demonstration of woodchuck hepatitis virus infection of peripheral blood mononuclear cells by flow cytometry and polymerase chain reaction

Chemin

Baginski

Vermot-Desroches

et al. 1992

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Peripheral blood mononuclear cells (PBMCs) from 10 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of WHV surface (WHs) and core (WHc) antigens (WHsAg and WHcAg) by cytofluorometry using fluorescein isothiocyanate-conjugated anti-WHs and anti-HBc-purified immunoglobulins from woodchuck and human sera. The presence of viral DNA and RNA was detected in the serum and PBMCs from the same blood samples by polymerase chain reaction (PCR) with two primer sets located in the S and C genes of the WHV genome. Seven animals were found positive for both WHsAg and WHcAg on the surface of PBMCs: four WHVchronic carriers, two WHsAg-positive animals with acute WHV infection, and one woodchuck which was bled during the incubation phase of WHV infection and which became WHsAg-positive only 1 month later. Sixteen to 71% of the studied leukocyte population expressed WHsAg with a low density of expression whereas 7 to 72% expressed WHcAg with a high density of expression. Only two cases were positive for WHsAg without WHcAg on PBMCs, one WHV chronic carrier and one anti-WHs-positive animal. All woodchucks positive for WHcAg and/or WHsAg by cytofluorometry were positive also for WHV DNA and RNA in PBMCs by PCR. The tenth animal was found negative for both viral antigens as well as for WHV DNA and RNA in PBMCs despite the presence of persistent viral DNA in the serum as detected by PCR. Five healthy woodchucks devoid of WHV serological markers served as negative controls. These results obtained with a novel approach further confirm, in the woodchuck model, that a significant proportion of PBMCs are probably permissive for WHV replication. The possible immunopathogenic implications of the phenomenon are discussed.