INTRACELLULAR VIRUS-DNA DISTRIBUTION AND THE ACQUISITION OF THE NUCLEOPROTEIN CORE DURING AFRICAN SWINE FEVER VIRUS PARTICLE ASSEMBLY - ULTRASTRUCTURAL IN-SITU HYBRIDIZATION AND DNASE-GOLD LABELING

African swine fever virus (ASFV) is a large complex icosahedral double -stranded DNA virus that replicates in the cytoplasm of susceptible ce lls. Assembly of new virus particles occurs within the perinuclear vir oplasm bodies known as virus factories. Two types of virus particle ar e routinely observed: ''fulls,'' which are particles with an electron- dense DNA-containing nucleoid, and ''empties,'' which consist of the v irus protein and membrane icosahedral shell but are without the incorp oration of the virus genome. The objective of this study was to unders tand ASFV morphogenesis by determining the distribution of intracellul ar viral DNA in the virus factory and during virus particle assembly. The ultrastructural localisation of DNA within ASN-infected cells was achieved using two complementary methods: with an ASFV-specific DNA pr obe to the major capsid protein (p73) gene (B646L) hybridised in situ or through detection of all forms of DNA (viral and cellular) with gol d-labelled DNase. Conditions for in situ hybridisation at the electron microscopic level were optimised for infected cells in two Lowicryl r esins (K4M and HM20) and using two nonradioactive probe labels (digoxy genin and biotin). The morphological data indicate that the viral DNA, perhaps from specialised storage sites within the factory, begins to condense into a pronucleoid and is then inserted, at a single vertex, into an ''empty'' particle. Further maturation of the viral particle, including closure of the narrow opening in the icosahedron, gives rise to ''intermediate'' particles, where the nucleoprotein core undergoes additional consolidation to produce the characteristic mature or ''fu ll'' virions. The site of particle closure may represent a ''weak poin t'' at one vertex, but the mechanisms and structures involved in the p ackaging and release of the virus genome via such a port are yet to be determined. (C) 1998 Academic Press.