Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis

Abstract

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8-and 7.5-kilobase species, while 2.3-and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.

Distribution of hepatitis B virus DNA sequences in different peripheral blood mononuclear cell subsets in HBs antigen‐positive and negative patients

“…In six out of 15 cases, HBV-DNA was present in at least two different cell types. HBV infection of T and B lymphocytes, and among T lymphocytes, of both T4 and T8 subsets [9], has been previously reported, but infection of monocytes has not been detected despite the use of an equally sensitive technique [9,10]. Our data suggest that HBV-DNA may also be present in monocytes.…”

Treatment of chronic woodchuck hepatitis virus infection in the eastern woodchuck ( marmota monax ) with nucleoside analogues is predictive of therapy for chronic hepatitis B virus infection in humans

“…Values above 1 ϫ 10 7 WHV genome equivalents per milliliter of serum (WHVge/mL) were analyzed by dot-blot hybridization (four 10-µL replicates per sample) as previously described. 5,22 Samples containing WHV DNA below this sensitivity cutoff were analyzed using a quantitative polymerase chain reaction (PCR)based method. 23 Serum samples were analyzed in duplicate.…”

“…The contents were transferred to 1.1-mL plastic tubes, and 300 µL of 1:1 37% formaldehyde/20ϫ SSC was added. 22 Following 45 minutes of incubation at 60°C, the samples were applied to nitrocellulose membranes and rinsed with 20ϫ SSC using a 96-well dot-blot manifold (GIBCO-BRL, Gaithersburg, MD), and hybridized to a 32 P-labeled, 3.2-kb cloned WHV-DNA fragment as previously described. 5,22 Assessments of the level of WHV genome equivalents for the PCR analyses were determined by direct comparisons to parallel PCR amplification of a dilution series (1,000,000 to 1,000 WHVge/mL) of the standardized serum pool (WHV7p1) used to infect these animals that contains a known WHV genome content.…”

“…22 Following 45 minutes of incubation at 60°C, the samples were applied to nitrocellulose membranes and rinsed with 20ϫ SSC using a 96-well dot-blot manifold (GIBCO-BRL, Gaithersburg, MD), and hybridized to a 32 P-labeled, 3.2-kb cloned WHV-DNA fragment as previously described. 5,22 Assessments of the level of WHV genome equivalents for the PCR analyses were determined by direct comparisons to parallel PCR amplification of a dilution series (1,000,000 to 1,000 WHVge/mL) of the standardized serum pool (WHV7p1) used to infect these animals that contains a known WHV genome content. 5,22 The dilution standards were included, in duplicate, with each run of the thermocycler and were blotted, in duplicate, on each hybridization membrane containing the test samples amplified in the same thermocycler run.…”

“…5,22 Assessments of the level of WHV genome equivalents for the PCR analyses were determined by direct comparisons to parallel PCR amplification of a dilution series (1,000,000 to 1,000 WHVge/mL) of the standardized serum pool (WHV7p1) used to infect these animals that contains a known WHV genome content. 5,22 The dilution standards were included, in duplicate, with each run of the thermocycler and were blotted, in duplicate, on each hybridization membrane containing the test samples amplified in the same thermocycler run. Two negative controls were also included, in duplicate, in the standards: uninfected woodchuck serum used for the dilutions and the water/buffers used for the PCR components.…”