Further Characterization of Marek's Disease Virus-infected Lymphocytes. I. In Vivo Infection

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.