Intracellular Virus DNA Distribution and the Acquisition of the Nucleoprotein Core during African Swine Fever Virus Particle Assembly: Ultrastructuralin SituHybridisation and DNase-Gold Labelling

Abstract

African swine fever virus (ASFV) is a large complex icosahedral double-stranded DNA virus that replicates in the cytoplasm of susceptible cells. Assembly of new virus particles occurs within the perinuclear viroplasm bodies known as virus factories. Two types of virus particle are routinely observed: “fulls,” which are particles with an electron-dense DNA-containing nucleoid, and “empties,” which consist of the virus protein and membrane icosahedral shell but are without the incorporation of the virus genome. The objective of this study was to understand ASFV morphogenesis by determining the distribution of intracellular viral DNA in the virus factory and during virus particle assembly. The ultrastructural localisation of DNA within ASFV-infected cells was achieved using two complementary methods: with an ASFV-specific DNA probe to the major capsid protein (p73) gene (B646L) hybridisedin situor through detection of all forms of DNA (viral and cellular) with gold-labelled DNase. Conditions forin situhybridisation at the electron microscopic level were optimised for infected cells in two Lowicryl resins (K4M and HM20) and using two nonradioactive probe labels (digoxygenin 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 “full” virions. The site of particle closure may represent a “weak point” at one vertex, but the mechanisms and structures involved in the packaging and release of the virus genome via such a port are yet to be determined.