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

Abstract

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.

Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

“…WHV DNA and RNA have been identified in PBMCs and lymphatic organs of woodchucks with WHsAg-reactive chronic hepatitis (29,30) and in convalescent animals shortly after seroconversion to anti-WHs (31). In our laboratory, pathogenic virus has been documented in lymphoid cells throughout the life-span of woodchucks after apparent complete recovery from AH (19).…”

Applications of Flow Cytometry to Clinical Microbiology

“…In another work, Chemin et al demonstrated the selective detection of HBsAg and HBcAg on B lymphocytes and natural killer cells from chronically HBV-infected patients (52). Hepatitis C virus (HCV), woodchuck hepatitis virus, and varicella-zoster virus were also detected by FCM in PBMCs using monoclonal antibodies to HCV core antigen (34), polyclonal antibodies to woodchuck hepatitis virus (51), and antibodies to the gpI glycoprotein from varicella-zoster virus (296), respectively. Thus, FCM detection of viral antigens offers a potentially useful automated assay for the clinical diagnosis of multiple blood-borne viruses.…”

Monitoring of early events of experimental woodchuck hepatitis infection: Studies of peripheral blood mononuclear cells by cytofluorometry and PCR

“…The aim of the present study was to investigate prospectively the early events in PBMc following experimental infection of woodchucks. Since WHV and HBV infections of PBMC are characterized by low levels of viral DNA/RNA and antigens [5], two highly sensitive methods were used: flow cytometry [6,7] to detect WHV antigens on lymphocytes surface and PCR [8,9] to detect WHV DNA and RNA in both serum and PBMC.…”