Detection of equine arteritis virus (EAV)-specific cytotoxic CD8(+) T lymphocyte precursors from EAV-infected ponies

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a Cr-51 release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain Cr-51 efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection