Detection of African swine fever virus in infected pig tissues by immunocytochemistry and in situ hybridisation

The techniques for determining cellular sites of establishment and persistence of African swine fever virus (ASFV) were established in susceptible domestic pigs and the resistant African reservoir hosts, the warthog and bushpig. Detection, both in vitro and in vivo, was achieved by in situ hybridisation and immunocytochemistry, focusing principally on specific probes for vp73, a major capsid protein. Hybridisation of radio-labelled probes for DNA and RNA was relatively insensitive and time consuming whereas hybridisation of non-radioactive DNA probes was quicker and more sensitive. Detection of vp73 protein by immunocytochemistry was as sensitive as non-radioactive DNA hybridisation, offering in addition improved speed, ease and morphology. Both non-radioactive DNA hybridisation and immunocytochemistry were therefore used to detect ASFV DNA and protein in a range of porcine cells infected in vitro with different ASFV isolates. Malta and Malawi isolates infected both alveolar and bone marrow macrophages, but infected negligible numbers of endothelial (<1%) and kidney cells (IBRS2 cells). Attenuated Uganda isolate, however, infected a high percentage of endothelial cells and IBRS2 cells as well as alveolar and bone marrow macrophages. When used to investigate the cell tropism of ASFV in tissues from pigs infected with the highly virulent Malawi isolate of ASFV, both techniques identified virus principally in cells of the mononuclear phagocytic system. In the lung, double staining revealed that pulmonary intravascular macrophages, but not alveolar macrophages, were infected.