A new method to identify cell types that support porcine circovirus type 2 replication in formalin-fixed, paraffin-embedded swine tissues

Abstract

Porcine circovirus type 2 (PCV2) is detected in high amounts within the characteristic microscopic lesions of postweaning multisystemic wasting syndrome (PMWS) affected pigs. In spite of recent advances on disease pathogenesis, the precise cell types that support viral replication are still a major issue of scientific discussion. In this study, a new methodology to detect cell types that support PCV2 replication was designed. For this purpose, two in situ hybridisation (ISH) methods were developed and applied on tissues of PMWS naturally affected pigs using two probes designed from the ORF1 sequence of the virus. While the complementary probe (CP) detected ssDNA, ORF1 mRNA and replicative form (RF) of PCV2, the RF probe (RFP) exclusively hybridised with the RF of the virus, thus, only labelling cells where PCV2 replication is taking place. Both probes demonstrated to be specific and equally sensitive by an in vitro Southern blot hybridisation assay. ISH labelling with the CP was extensive in lymphoid tissues and of variable amount in other non-lymphoid tissues. With this probe, mainly macrophage-like cells were labelled but also other cell types such as hepatocytes and other epithelial cells. Tissues in which RFP labelling was found more frequently were lung, inguinal and mesenteric lymph nodes, tonsil and liver. Labelling with the RFP was always nuclear, and found in the same cell types as with the CP, although in a relatively low proportion of them; labelling of macrophage-like cells was infrequent. Therefore, the results indicate that at least a certain proportion of macrophages may support PCV2 replication, but main cells where PCV2 replicates are of epithelial/endothelial origin. In summary, the present study permitted the study of cell types that support PCV2 replication by the use of ISH on formalin-fixed, paraffin-embedded tissues of PMWS affected pigs.