Detection of latent pseudorabies virus by in situ hybridization

The presence of pseudorabies virus (PRV) nucleic acid in brain tissue sections of acutely and latently infected swine was detected by an in situ hybridization procedure using 35S-nick translated PRV DNA probes. PRV nucleic acid was detected in three of four naturally infected pigs. Tissues found to be infected with PRV were the pons/medulla region, frontal cerebral cortex, and cerebellum. In a related study, an attempt was made to increase the sensitivity and specificity of a dot blot hybridization assay with the use of synthetic oligonucleotides as probes for the detection of PRV DNA. Three 25-base-long oligonucleotides, complementary to three DNA sequences within the Sph 1 subfragment p7 of BAM H1 fragment 2, were end-labeled with gamma 32P-ATP using T4 polynucleotide kinase. The oligonucleotide probes were hybridized to swine cellular DNA and purified PRV-DNA that had been bound to a nitrocellulose paper. Hybridizations were performed at varying temperatures and at various salt concentrations. The three oligonucleotide probes, which were rich in guanine and cytosine, hybridized to swine cellular DNA at all temperatures under varying conditions of stringency