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The entry of African swine fever virus into Vero cells has been investigated by both biochemical and morphological techniques. A quantitative electron microscopy analysis of the early steps of the infection has shown that African swine fever virus enters Vero cells by a receptor-mediated endocytosis mechanism. The internalization of virus particles is a temperature- and energy-dependent process, since it did not take place at 4 degrees or in the presence of NaF and 2,4-dinitrophenol. To determine the involvement of acidic intracellular vacuoles in the virus entry pathway we have tested the effect of lysosomotropic agents in the infection. Chloroquine, dansylcadaverine, amantadine, methylamine, and ammonium chloride inhibited African swine fever virus production in Vero cells. Dansylcadaverine and chloroquine did not inhibit virus adsorption and internalization; however, in the presence of these drugs, virus particles were retained in cytoplasmic vacuoles and early viral RNA and protein synthesis were not detected, indicating that these compounds inhibit an early step in the infectious cycle, probably the uncoating of the virus particle.

Purified preparation of porcine lymphocytes were infected with three isolates of virulent African swine fever virus (ASFV). Electron microscopy showed the presence of small numbers of mature virus particles in degenerating cells. The titres of infective virus released were low and reached a maximum by 24 h after infection.

African swine fever virus emerges from infected Vero cells either from areas of smooth cell surface or from microvilli. The two patterns may occur at different sites on the same cell and are unique for this virus. The scanning electron micrographs supplement regular thin section views.

Extracellular and intracellular African swine fever virus (ASFV) was purified using a two-phase aqueous polymer system. Both the structure of the virus and the polypeptides present during the purification procedure were studied. After PEG/dextran phase separation and centrifugation through 20% (w/v) Ficoll, 79% of input infectivity was recovered as semi-purified virus. The density of the virus after equilibrium centrifugation in sucrose was 1.19 g/ml. The envelope of the virion consisting of a unit membrane was removed from the virion after centrifugation in sucrose. Removal of envelope was associated with the loss of a 230 kilodalton (kd) glycoprotein from the virion. Disruption of the viral surface structure resulted in a loss of infectivity. Eighteen of the most prominent of the 33 polypeptides of extracellular or cell free (CF) virus were those with molecular weights of 230, 195, 165, 155, 150, 125, 116, 97, 92, 73, 62, 58, 50, 45, 35, 33, 25 and 11 kd, while the fourteen most prominent polypeptides in intracellular or cell associated (CA) virus were 103, 97, 92, 84, 73, 62, 58, 54, 47, 45, 35, 33, 25 and 17 kd. The 45 kd polypeptide may be actin which copurifies with the virus. No major differences were found in the number or size of proteins among three isolates of ASFV. Electron micrographs of thin sections of ASFV show the capsid to consist of a distinct double layer of closely packed capsomeres enclosed on both sides with a semi-transparent layer. Cell associated virus measured from side-to-side 188 nm and vertex-to-vertex 212 nm. The capsid encloses an inner core composed of a dense nucleoid surrounded by a 40-48 nm layer of core protein.

The association of African swine fever virus (ASFV) with swine erythrocytes in vivo, in high titers, was verified by inoculating 30 pigs with 17 ASFV isolates and assaying their plasma and washed erythrocyte fractions for residual virus. Viral antigens were specifically localized on the surface of in vitro and in vivo swine erythrocytes, using the fluorescent antibody technique and 3 monoclonal antibodies specific for ASFV. The same monoclonal antibodies immunoprecipitated virus-specific polypeptides of molecular weights 13 kd and 73 kd from ASFV-infected Vero cells. Erythrocytes from viremic swine infected with Lisbon-60, Dominican Republic, Badajoz-M98, or Cameroon isolates of ASFV were studied by transmission electron microscopy. Virus was found in membrane depressions at the surface of erythrocytes. These surface depressions resembled stages of smooth surfaced pits. Erythrocytes from viremic pigs were fragile osmotically.

The association of African swine fever virus (ASFV) with the cytoskeleton was investigated. Immunofluorescent studies of ASFV infected cells with anti-ASFV serum showed a temporal and spatial development of viral inclusions which moved from a peripheral to a perinuclear location and fused to give a single large perinuclear factory. The migration and fusion of viral inclusions was inhibited by colchicine suggesting a function for microtubules in assembly site organization not previously described. Accumulation of virions outside the inclusions and inhibition of viral release was also observed in colchicine treated cells. Viral antigens and structural elements were retained on the cytoskeleton fraction of Triton X-100 extracted cells. Reorganization of cytoskeletal elements around the assembly sites was demonstrated by transmission electronmicroscopy and by immunofluorescent studies using monoclonal antibodies against actin, tubulin and vimentin. Intermediate filaments accumulated around the viral factories, microtubules were greatly decreased in number and microfilaments were reorganized in association with the plasma membrane. Bundles of 15 nm tubules of unknown origin were also observed around the assembly sites. The distribution of viral proteins in soluble, cytoskeleton and detergent insoluble nuclear fractions was studied by pulse-chase experiments with [35S]methionine. SDS-PAGE analysis showed the presence in the cytoskeletal and nuclear fractions of 150, 72, 38, 28, 19 and 15 kDa virus structural proteins which increased after a 5 h chase. Our results indicate a close association of ASFV replication with the cytoskeleton similar to events described during FV3 replication but which differ from those occurring in poxvirus-infected cells.

The role of the host cell RNA polymerase II in African swine fever (ASF) virus growth has been examined using inhibitors of this enzyme. The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of mRNA precursor synthesis in mammalian cells, strongly inhibits the production of infectious progeny virus in Vero cells, but does not significantly affect the synthesis of virus-specific macromolecules. On the other hand, virion assembly seems to proceed normally in the presence of DRB, as virus particles can be seen in electron micrographs with a morphology indistinguishable from that observed in the absence of the inhibitor. However, taking into account the inhibition of the infectivity caused by the drug, most of these particles must be defective. In contrast with this effect of DRB on ASF virus replication, the toxin alpha-amanitin does not inhibit the production of infectious ASF virus in Vero cells or porcine alveolar macrophages. This result indicates that the host RNA polymerase II does not transcribe viral genes and that active transcription of the cell genome is not needed for ASF virus replication.

African swine fever virus infected most, if not all, of the macrophages (monocytes) and ca. 4% of the polymorphonuclear leukocytes from porcine peripheral blood. B and T lymphocytes, either resting or stimulated with phytohemagglutinin, lipopolysaccharide, or pokeweed mitogen, were not susceptible to the virus. All of the mitogens used inhibited African swine fever multiplication in susceptible cells. The number of virus passages in vitro and the virulence degree of the virus did not affect the susceptibility of porcine B or T lymphocytes to African swine fever virus.

Replicating and mature viral particles were detected with the transmission electron microscope in blood platelets of pigs infected with virulent haemadsorbing and non-haemadsorbing African swine fever virus isolates. Although platelet numbers decreased terminally in infected pigs, the most noticeable morphological damage to these cells apparent in the last 2 days of the disease included cytoplasmic swelling, vacuolation, fragmentation and loss of dense granules.

A Vero cell line persistently infected with African swine fever virus was established by infecting the cells in the presence of 10 mM NH4Cl (Vero-P cell line). The virus derived from the Vero-P cultures infected Vero cells, and virus titers were comparable to those obtained in Vero cells acutely infected with African swine fever virus. The structural proteins of the virus from Vero-P cells were similar to those of the virus produced in lytic infections. Virus production was low when the Vero-P cells were growing logarithmically and increased considerably in confluent cultures when lysis appeared in a fraction of the cell population.

A simple method of obtaining analogue images from traced models of biological specimens is presented. It consists of the photographic defocusing of traced models and it is illustrated with negatively stained cylindrical forms of the ASFV; the black lines of the trace in the model correspond to the negative stain surrounding the viral morphological subunits as seen in the electron micrograph. The photographic defocusing is the means by which the traced model is filtered and is used to introduce grey levels on an otherwise black and white image. The right amount of defocusing is attained when the width of the trace of the model equals the width of the rim of the negative stain appearing between the morphological subunits in the electron micrograph.

The structure of African swine fever virus particles has been examined by electron microscopy. The analysis of virions prepared by negative staining, thin sectioning, and freeze-drying and shadowing showed that the virus particle was composed of several concentric structures with an overall icosahedral shape. The inner region of the virus particles was a nucleoid that was surrounded by a membrane covered by the capsid. The capsid had side-to-side dimensions of 172 to 191 nm and was built up by capsomers arranged in an hexagonal lattice. Computer-filtered electron micrographs of either negatively stained or freeze-dried and shadowed capsids revealed capsomers with a hexagonal outline and a hole in the center. The intercapsomer distance ranged from 7.4 to 8.1 nm. The triangulation number of the capsid was estimated to be 189 to 217, indicative of 1892 to 2172 capsomers. Extracellular African swine fever virus particles had an external membrane that resembled the cytoplasmic unit membrane.

Monoclonal antibodies specific for African swine fever (ASF) viral proteins of 14, 32, 73, 174, and 240 kDa were produced and characterized. Immunoelectron microscopy detected the 73 kDa but not the 14-, 32-, or 240-kDa proteins at the surface of the virion. The 32-kDa protein was detected by radioimmunoassay 2 hr after infection of porcine monocytes and Vero cells, was detected in the seven widely divergent ASFV isolates tested, and stained brilliantly virus-infected cells in indirect immunofluorescence suggesting that monoclonal antibodies directed against this protein may be useful in ASFV diagnosis. Two monoclonal antibodies detected heterogeneity between ASF viruses.