Genomic detection methods for detection of pseudorabies virus infection in Indian pigs

Abstract

Pseudorabies or Aujeszky’s disease is an alpha-herpes virus infection that causes significant economic losses due to piglet mortality, respiratory disease in feeder pigs and reproductive losses in breeding age swine. Latency is an integral part of the ecology of herpes viruses and reactivation of latent pseudorabies virus (PRV) genome results in renewed replication and potential transmission to susceptible pigs. In India, the status of the disease is not clear except for 2 isolated reports in early 70’s. Peripheral blood lymphocytes (PBLs), tonsil and brain tissues collected at slaughter and nasal swabs from Indian native pigs with respiratory illness were processed for PRV genomic detection by PCR using different sets of specific primers. All the samples were negative for the presence of PRV genomic sequences. Total DNA samples extracted from these samples were also confirmed to be negative by Southern hybridization using a P32–dATP labeled PRV gII region specific probes. Attempts to isolate the virus in PK–15 and IBRS–2 cell-lines were unsuccessful. SYBR green real-time PCR was developed using a PRV reference clone. The conserved region, glycoprotein gII (gB), of PRV of the clone was targeted with specific primers to amplify 318 bp product. The minimum detection limit estimated using varying dilutions of standard reference clone (10–1 to10–15) was 1.4 to 14 copies per reaction. In the present study, no pseudorabies virus genome could be detected in the tissue/nasal swab samples screened from Indian native pigs even by real-time PCR assay, that can detect as few as 14 copies of PRV genomic sequences.