Enzymatic amplification of latent pseudorabies virus nucleic acid sequences

Abstract

To investigate various aspects of the latency of pseudorabies virus in swine (PRV, suid herpesvirus 1) we developed in vitro nucleic acid amplification methods based upon the polymerase chain reaction. Primers flanking a 156-bp region of the pseudorabies virus gp II gene were annealed to purified PRV DNA as well as DNA isolated from the trigeminal ganglia of swine latently infected with PRV and subjected to PCR amplification. Following amplification, 100 fg of PRV DNA was visualizable on stained gels and 1 fg (equivalent to 6 viral genome copies) was detectable when amplification was combined with blot hybridization. PRV-specific DNA sequences which remained undetectable by direct blot hybridization assays were amplified to levels visualizable on ethidium-bromide-stained gels in 5 of 5 experimental latently infected animals. In addition, oligonucleotide primers specific for a 223-bp region of the PRV immediate-early gene (IE 180) were capable of amplifying overlapping latency associated transcripts (LATs), via a cDNA intermediate, in 6 of 6 latently infected swine. These nucleic acid amplification methods should be applicable to the investigation of PRV latency, and gene expression during latency and reactivation, in which few cells harbor latent virus.