Detection of latent pseudorabies virus in porcine tissue, using a DNA hybridization dot-blot assay

A DNA-hybridization dot-blot technique was developed to detect the presence of pseudorabies virus (PRV) DNA in porcine tissue. Seven 32P-nick translated probes of high specific activity were prepared from transformed Escherichia coli plasmids into which Bacillus amyloliquefaciens H (Bam HI) restriction fragments of PRV-DNA had been inserted. Samples of DNA that had been extracted from porcine tissue or from PRV grown in tissue culture were transferred to nitrocellulose paper, using a microsample filtration manifold and were hybridized to the probes under high-stringency conditions. Under optimal hybridization conditions, the minimum detection amount of PRV-DNA was 10(-11) g, which is equivalent to 1 copy of the PRV genome/80 host cells. Four probes did not show cross hybridization with DNA extracted from tissues of known PRV-negative swine, and these were subsequently used to detect PRV-DNA in infected porcine tissues. Generally, correlation between virus isolation and hybridization data was good for tissues from swine that had died of acute PRV infection. Furthermore, PRV-DNA was present in specific tissues of all 4 seropositive swine that had recovered from pseudorabies and in which no infective virus or viral products were detected at necropsy. Pseudorabies virus DNA was present in the rostralis cerebral cortex (n = 2) or in the medulla oblongata (n = 1) and trigeminal ganglion (n = 1). This probably indicated the portal of entry of the virus into the CNS. In another seropositive pig, there was evidence of a productive infection in the tonsils, although virus was not isolated in a tissue culture system.(ABSTRACT TRUNCATED AT 250 WORDS)