Figure 1 Electron micrograph of a cryosection of ASFV-infected Vero cells. The electron micrograph shows an intracellular virus particle (IV) and three extracellular virus particles (EV). pm, plasma membrane. Bar = 200 nm. (Courtesy of G. Andres.)

has been classified for many years as an Iridovirus. However, several properties of ASFV are similar to those of poxvirus. The genome of ASFV resembles that of poxviruses in the presence of covalently closed ends and terminal inverted repetitions (see later). In contrast, the genomes of frog virus 3 and two other iridoviruses are both circularly permuted and terminally redundant. In addition, ASFV particles contain, like those of poxvirus, all the enzymatic machinery necessary for the synthesis and modification of early mRNA. Frog virus 3 particles do not contain RNA polymerase, and RNA synthesis in cells infected with frog virus 3 is sensitive to the host RNA polymerase II inhibitor a-amanitin, whereas transcription of the ASFV genome is resistant to this inhibitor.

ASFV has recently been assigned to a new family, Asfarviridae, as a member in the genus Asfivirus.

Virion Structure and Proteins

The ASFV particles have an icosahedral morphology and consist of a DNA-containing nucleoid of about 80 nm, a thick protein layer of about 30 nm, designated the core shell, a lipid envelope surrounding the core shell, the capsid, and, in the case of the extracellular virus, an outer membrane derived by budding through the plasma membrane (Fig. 1). The extracellular virions have a diameter of 200 nm. The capsid consists of a hexagonal arrangement of capsomers that appear as 13 nm long hexagonal prisms, each with a central hole. The intercapsomer distance is about 8 nm and the triangulation number is estimated to be between 189 and 217, corresponding to a number of capsomers between 1892 and 2172.

Two-dimensional gel electrophoresis analysis of purified virions has resolved 54 structural proteins with molecular weights ranging from 10000 to 150000. Some of these proteins have been localized in the virion by immunoelectron microscopy. The virus attachment protein pl2 and protein p24 are present in the external region of extracellular virions, while proteins pl50, p37, p34 and pl4, products of polyprotein pp220, are found in the core shell. Protein p72, located in the surface of unenveloped intracellular virions, is the capsid protein.

ASFV particles contain a DNA-dependent RNA polymerase and other activities involved in the poly-adenylation, methylation and capping of RNA. Other enzymes present in the virions are a protein kinase, two nucleoside triphosphate phosphohydrolases, an acid phosphatase, and a deoxyribonuclease active on single-stranded DNA.

The genes coding for 15 virion proteins have been identified and sequenced (Fig. 3). Two of these genes code for polyprotein precursors. Thus, gene CP2475L encodes a polyprotein of 220 kDa, which, after proteolytic cleavage, gives rise to four major structural proteins: pl50, p37, p34 and pl4; gene CP530R encodes polyprotein pp62, the precursor of proteins p35 and pl5.

Properties of Genome

The genome of ASFV is a linear double-stranded DNA molecule with a size that oscillates between 170 and 190 kbp, depending on the virus isolate. The two DNA strands are covalently closed, at both ends, by a 37 nucleotide-long hairpin loop, composed, almost entirely, of incompletely paired A and T residues. The loops at each DNA end are present in two equimolar forms (fast and slow components) that, when compared in opposite polarities, are inverted and complementary (flip-flop), as in the case of poxvirus DNA (Fig. 2A, B). There is no homology between the hairpin loops of ASFV and vaccinia virus but, close to the hairpin loops, both viruses have a sequence 16 nucleotides long with a homology of about 80%. Following the hairpin loops there is a perfect terminal inverted repeat (TIR) with a length of 2134 nucleotides, which consists of unique sequences interspersed with five different sets of repeated sequences (Fig. 2C). The central region of the TIR contains 33 direct repeats in tandem of 34 nucleotides.

In addition to the TIRs, two types of internal repetitions have been described: short (10-50 bp) direct repeats in tandem at both intergenic and intragenic positions, and long (200 bp) tandemly repeated units found at the left end of the genome. The latter repeated sequences share similarities with eucaryotic scaffold-associated region (SAR)-like sequences.

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