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Woodchucks (Marmota monax) infected with woodchuck hepatitis virus (WHV) represent a highly valuable laboratory model of hepatitis B virus (HBV) infection, in which molecular, immunological and pathological events occurring in infected humans are adequately reflected. To advance studies on T cell immune responses and propagation of hepadnavirus in T lymphocytes in this animal model, we determined the complete sequence of woodchuck interleukin-2 (wIL-2) cDNA by utilizing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) reaction. The wIL-2 sequence revealed a single open reading frame encoding for the predicted precursor protein comprised of a signal peptide and a 134 amino acid-long mature protein. The mature wIL-2 protein produced in the Escherichia coli expression system, designated as ec-rwIL-2, was found to be immunogenic but not biologically active. In contrast, precursor wIL-2 protein cloned into baculovirus transfer vector and expressed in Sf9 cells, designated as bac-rwIL-2, demonstrated functional competence. Further, bac-rwIL-2 was able to stimulate proliferation and to induce multiple daughter cell generations in woodchuck T cells, as well as facilitated the survival of standard IL-2-dependent mouse CTLL-2 cells in culture. Western blot analysis of bac-rwIL-2 using antibodies generated against ec-rwIL-2 revealed a single protein band of 15.5kDa. The availability of biologically active recombinant wIL-2 should facilitate ex vivo studies on functional competence of woodchuck T lymphocytes derived from different stages of hepadnaviral hepatitis and assist in recognizing their contribution to the pathogenesis of liver injury in the woodchuck model of hepatitis B.

Real-time polymerase chain reaction (PCR) assays were developed for woodchuck leukocyte cluster of differentiation (CD) and cytokine mRNA expression. Plasmid DNA standards of each marker (CD3, CD4, CD8, IL-2, IFN-gamma, TNF-alpha, IL-4, IL-10), and RNA standards from mitogen-stimulated woodchuck peripheral blood mononuclear cells (PBMCs) were used to validate and optimize the assays for TaqMan 7700 and iCycler PCR instruments. The complementary DNAs (cDNAs) produced by reverse transcription (RT) of RNA were quantified by real-time PCR against the plasmid DNA standards (6-8 log range) with detection of as few as 10-50 copies of amplicon cDNA per reaction. Analysis of unstimulated and concanavalin A-stimulated woodchuck PBMC demonstrated increased CD and cytokine mRNA expression following mitogenic activation. A liver sample from a woodchuck hepatitis virus (WHV) infected woodchuck with histologically confirmed acute hepatitis had increased intrahepatic CD and cytokine mRNAs compared to liver from an uninfected control woodchuck. The real-time PCR assays were highly specific for the woodchuck markers in PBMC and liver samples and were equally applicable for use in alternate real-time PCR instrumentation. These assays will enable the high-throughput analyses of mRNA markers during WHV infection, and thereby facilitate continued modelling of the immunopathogenesis and immunotherapy of human hepatitis B virus (HBV) infection.