Molecular double-check strategy for the identification and characterization of Suid herpesvirus 1

Large scale vaccination with glycoprotein E (gE)-deleted marker vaccines and the rapid and reliable differentiation of wild-type and marker vaccine strains are important aspects in eradication programs for Suid herpesvirus 1 [SuHV-1, syn. Aujeszky's disease virus (ADV) or pseudorabies virus (PrV)]. Therefore, two multiplex real-time PCR (qPCR) assays for the genetic differentiation of wild-type and gE-deleted vaccine SuHV-1 strains have been developed. In the first multiplex qPCR SuHV-1 gB-gene specific detection was combined with a gE-gene specific assay and an internal control based on heterologous DNA. In the second system, a SuHV-1 UL19 (major capsid protein gene) assay, a different gE-gene specific assay and an internal control based on the beta-actin gene were combined. The gB-gene, UL19 as well as both gE-gene specific assays had an analytical sensitivity of less than 10 genome copies per reaction in the respective multiplex approaches. A series of reference strains including field isolates obtained from domestic and wild animals, and gE-deleted SuHV-1 were reliably detected, while genetically related non-SuHV-1 herpesviruses tested negative. Both newly developed triplex SuHV-1-specific qPCR assays are specific and sensitive methods for the rapid genetic differentiation of wild-type viruses and gE-deleted vaccine strains in a single reaction.