Detection of Proviral DNA of Bovine Immunodeficiency Virus in Bovine Tissues by Polymerase Chain Reaction (PCR) and PCRin SituHybridization

AbstractIn this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCRin situhybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conservedpolgene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIVpolgene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed byin situamplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected toin situhybridization with an internal biotinylated probe and detected with streptavidin–gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissuesin vivoand has a broad cell tropism