Assembly of African Swine fever virus: quantitative ultrastructural analysis in vitro and in vivo.

African swine fever virus is an icosahedral double-standed DNA virus which replicates in the cytoplasm of porcine monocytic cells. Progeny virus particles, like poxviruses and iridoviruses, are assembled in cytoplasmic inclusions, termed virus factories. The formation of these structures in susceptible cells infected in vitro with pathogenic and attenuated ASFV isolates has been studied by semiquantitative and qualitative electron microscopy. Recognizable virus factory elements were detected by B hr postinfection (hpi) and increased significantly in profile area between 12 and 18 hpi. The number of virus particles associated with the virus factories also increased significantly between 12 and 24 hpi. These included particles with ("full") and without ("empty") a nucleo-protein core. Similar results were obtained for both pathogenic (Malawi) and attenuated (Uganda) virus isolates, but the replication of pathogenic virus in the macrophage was more efficient than that of the attenuated virus in a porcine kidney cell line, where a relatively higher percentage of defective "empty" particles were observed. Analysis of virus factory formation was also done directly on lung and liver tissue from a pig infected with the highly pathogenic Malawi virus isolate. The in vivo data for virus factory area and particle number in both pulmonary intravascular macrophages and liver Kupffer cells at Day 4 postinfection were similar to those observed in vitro. As expected, using postembedding immunoelectron microscopy, DNA (both cellular and viral) was detected in the cell nucleus, cytoplasmic virus, and mature extracellular virus. More interestingly, DNA was absent in the cytoplasm, but readily observed in virus factories. This DNA, which we presume to be viral, was found in close association with membrane-like material and "empty" particles, suggesting that the virus DNA may enter a partially formed capsid which is then sealed in order to develope into a fully assembled particle. According to this hypothesis, ASFV morphogenesis involves the initial formation of "empty" particles within the virus factory. The adjacently formed nucleo-protein material is then inserted into the "empty" particles, as has been described for poxviruses. These particles then mature in to the "full" particles and leave the virus factory as a prelude to release from the cell by budding.