Analysis of ovine lentivirus infectivity and replication by using a focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay

Abstract

A focal immunoassay and an antigen-capture enzyme-linked immunosorbent assay (antigen-capture ELISA) were developed to quantify infectious ovine lentivirus (OvLV) and OvLV capsid protein (CA) (p27), respectively. The in vitro kinetics of replication and cytopathogenicity of distinct biological clones of OvLV (rapid/high and slow/low phenotypic variants) were assessed. Both viruses were detected by focal immunoassay within 48 h postinfection, 2 days before syncytia were observed in goat synovial membrane cells infected with rapid/high OvLV and 4 days before they appeared in cultures infected with slow/low OvLV. CA was first detected by antigen-capture ELISA in supernatants of cells infected with rapid/high OvLV 4 days postinfection, and it reached a plateau by 10 days, 4 days after peak syncytium formation. In contrast, in cultures infected at the same multiplicity of infection with slow/low OvLV, CA was detected 8 days postinfection, and the titer gradually increased over the following 12 days while the number of syncytia gradually decreased. Peripheral blood mononuclear cells (PBMC) from seropositive sheep treated with phorbol 12-myristate 13-acetate (PMA) generally expressed CA earlier and at higher levels than PBMC treated with either phytohemagglutinin or concanavalin A. Serum CA levels above 3 ng/ml were found in 58% (18 of 31) of seropositive sheep. However, there was no correlation between PMA-induced CA expression and levels of antigenemia. Viral heterogeneity may account for variations both in CA expression in cultures of PBMC and in antigenemia, humoral immune response, and viral pathogenicity in infected animals.