Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV)

A double in situ hybridization method for the simultaneous detection of por cine reproductive and respiratory syndrome virus (PRRSV) and porcine circov irus (PCV) genomes in the same tissue section was applied to lung tissues f rom 9 pigs in which PRRSV and PCV coinfection had been previously demonstra ted. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-label ed antisense RNA probe for PCV, and hybridization was detected with anti-di goxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/dia minobenzidine, respectively. PRRSV and PCV genomes were identified in the s ame pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.