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Virus subpopulations with variable virulence, immunogenicity, and infectivity to pigs were readily generated by passaging Tengani isolate of African swine fever virus, either biologically cloned or uncloned, in Vero cell cultures. Avirulent virus populations which account for more than 99% of virus in an uncloned preparation of the 27th passage are laboratory artefacts, perhaps do not exist in nature. Furthermore, attenuation of virulence did not occur uniformly in all subpopulations newly generated, and a continuous modulation of virus populations differing in immunogenicity and virulence took place in the same individuals inoculated with the 27th passage virus. The same virus preparation, appearing to be slightly virulent in pigs, contained at least a virulent subpopulation that was manifested only by further inoculating susceptible pigs with viremic blood collected at various times during the clinical course. A cloned virus after 23 passages in cell cultures generated a subpopulation (99.9%) which induced subclinical infection in pigs; however, the infection did not confer a solid immunity to homologous challenge with Tengani isolate in these pigs. The Tengani isolate contained subpopulations of virus with immunogenicities shared by the Lisbon '60 isolate and also contained at least one subpopulation specific for the Tengani only.

The virus capsomeres of the outer and inner layers of capsids were effectively released simultaneously from purified virions by lipase digestion and were purified by a linear gradient ultracentrifugation. The capsid consisted of an array of double layers of uniformly arranged individual capsomeres where a lipid(s) served as a matrix in between the capsomeres.

The sedimentation coefficient of the infective unit of African swine fever in tissue culture harvest fluids was measured in a preparative ultracentrifuge. The boundary locator method used also permitted making an estimate of heterogeneity. The sedimentation coefficient ranged from 3,000 to 8,000 Svedberg units, representing many classes of infective particles. Electron microscopy on culture fluids from infected cells showed many kinds of virus-containing units. Sucrose-CsCl gradient centrifugation was used to concentrate and to purify (partly) African swine fever virus for analytical ultracentrifugation. The optical patterns of the physical particles revealed a range of coefficients from 1,800 to 3,200 Svedberg units in tris-buffered saline solution at 20 C and buoyant densities from 1.19 to 1.24 g/ml in CsCl. The disparity of these values from those obtained by preparative ultracentrifugation indicates a change in the virus structure or a selection of viral populations on purification (or both).

A microplaque assay for Vero cell-adapted Lisborn '60 strain of African swine fever virus (L'60-uncloned) and a large plaque-forming strain cloned from the L'60-uncloned strain was developed by an immunoperoxidase method. The immunoperoxidase method can be used to stain microplaques of 3 days after inoculation, whereas the conventional plaque assay requires 5 to 7 days to develop visible plaques. A linear relationship between viral concentration in the inoculum and plaque numbers was observed. Viral titers obtained by both microplaque assay and conventional plaque assay were comparable, and both methods were reproducible and reliable. The viral titer obtained by either one of the plaque assay methods was approximately 0.9 log10 lower than that obtained by the hemadsorption test.

An African swine fever virus is an heterogeneous population, consisting of clones having different biological characteristics in respect to hemadsorption, virulence, infectivity, plaque size, and antigenic determinants. The following observations were made: Nonhemadsorbing virus (NHV) have been segregated from field isolates from Haiti (HT-1) and a bone marrow- and buffy coat-passaged Portuguese isolate (L'60BM89BC1) and appear as a major, minor, or equal mixture with hemadsorbing viruses in the virus population. Biological characteristics of the virus inoculated into pigs often differed from viruses isolated later from the same pigs. Virulence and nonhemadsorbing characteristics of isolated clones were genetically stable. The lethal effect of 2 NHV clones of L'60BM89BC1 virus was dose-dependent; small doses of virus induced immunologic deaths or recoveries from the clinical disease in pigs, and large doses induced acute deaths. The NHV of Lisbon isolate of 1960 (L'60) and HT-1 isolate share the same antigenic determinants for inducing protection. Tengani isolate contained clones of distinctly different antigenic determinants, not shared by L'60 or HT-1 isolate that enabled it to overcome the protection induced by the other clones. Passaging of an African swine fever virus isolate in pigs or cell cultures may readily alter the proportions of the different clones in the population and thereby change its overall characteristics. A new virus population with atypical hemadsorption was found in HT-1 field isolate and L'60BM89BC1 virus.

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.

African swine fever (ASF) is caused by an icosahedral cytoplasmic, double stranded DNA virus. In the acute form of the disease, pigs die from disseminated intravascular coagulation (DIC) with extensive damage of the free and fixed macrophage systems and the reticular epithelial cells of the thymus; mortality is virtually 100%. In recent years, subacute and chronic forms of ASF have become more prevalent in the field, especially in outbreaks occurring outside the continent of Africa, and virus isolated from these outbreaks have often been of lesser virulence. In pigs experimentally infected with such isolates, a number of immunopathological manifestations have been encountered, e.g. hypergammaglobulinemia associated with necrotizing pneumonia, persistent infection in the presence of ASF-specific antibodies, and lack of demonstrable virus neutralizing antibodies. Nevertheless, the immune systems of pigs that have clinically recovered have not been impaired by the infection. We suggest that the heterogeneous composition of the virus population in a given isolate may be one of the causes of the anomalous immune responses. When a number of biological markers, i.e., hemadsorption characteristics, plaque size, infectivity, virulence, antigenic determinants, and genomic structure, were used to characterize the virus clones derived from various ASF virus (ASFV) isolates, considerable heterogeneity was apparent. In the present investigation, 20 monoclonal antibodies (MAb), which specifically identified the 14 kDa viral protein within the cytoplasmic membrane of the infected cells, were used to determine epitopic differences among a number of virus clones derived from various isolates. All of the non-African isolates examined contained two epitopically different groups of virus clones, and the reaction profiles obtained were distinctly different from those obtained with the clones of an African isolate (Tengani). It was concluded that an ASFV isolate is composed of a biologically diverse virus population with distinctly different members which are only identified after cloning.

DNA from African swine fever (ASF) virus was isolated and was characterized by two restriction enzymes, SmaI and EcoRI. Although both enzymes can distinguish Vero cell-adapted ASF isolates by characteristic restriction endonuclease cleavage patterns, all ASF isolates examined exhibited a high degree of similarity, as measured by co-migration of most of the DNA fragments. The molecular weight of ASF DNA, based on size estimates of DNA fragments from cleavage patterns, ranged from 93 x 10(6) to 100 x 10(6). Virus genome heterogeneity was observed in uncloned, cell culture-adapted ASF isolates as well as in a plaque-purified virus after serial passage in Vero cells. In contrast to the rather minor differences in restriction pattern among the Vero cell-adapted isolates, a major alteration in restriction endonuclease cleavage sites was observed during adaptation of the wild-type virus to cell culture.

Infection-specific, nonstructural, African swine fever virus antigens, as visualized by the immunofluorescence technique, appeared as fine stipplings, evenly distributed in the cytoplasm and nucleus of Vero cells and in monocytes by postinoculation hour (PIH) 3. Cytoplasmic inclusion bodies (IB) appeared at PIH 4 and continued to increase in size up to PIH 8. The viral DNA was solely synthesized within the IB of infected monocytes, as evidenced by an autoradiographic chase experiment; maximum synthesis occurred at PIH 5. The 2,300-S and 2,500-S (sedimentation coefficient) structural viral antigens were visualized in the IB at PIH 5 and 6, respectively. The infective new progeny of African swine fever virus were formed between PIH 7 and 8, and the cell surface viral antigens were produced between PIH 8 and 9. Free virus was released between PIH 10 and 11. Infected cells detached from the glass surface from PIH 13. The 5-iodo-2'-deoxyuridine interfered with the formation of infective virus, but not interfere with the production of viral antigens.

A method of measuring and expressing the virulence of African swine fever virus in numerical terms was developed. Seventeen viruses (13 hemadsorbing and 4 nonhemadsorbing) were tested and classified into 3 groups: highly infectious and highly virulent, highly infectious and moderately virulent, and slightly infectious and slightly virulent. This classification was based on the number of 50% hemadsorption unit (HA50) or TCID50 required to produce 1 LD50 for swine, the number of HA50 or TCID50 required to produce one 50% pig infectious dose (PID50), and the number of PID50 required for each LD50. The virulent virus (group 1) required less than or equal to 10 virus units (HA50 or TCID50) for 1 PID50 and LD50 (highly infectious and highly lethal), respectively, and had a ratio of 1.0 for PID50/LD50, ie, all infected pigs died from acute African swine fever. Tengani, L'60, and DR-I isolants and nonhemadsorbing viruses of Haiti-1 isolant belong to this group. The moderately virulent virus (group 2) required less than 10 virus units for 1 PID50 and 20 to 562 virus units for 1 LD50 (highly infectious and moderately lethal), respectively, and recoveries from the clinical disease and immunologic deaths were frequent. Madrid '75, Haiti-1, DR-II, and BR-I isolants belong to this group. The slightly virulent virus (group 3) required 56 to 10,000 virus units for 1 PID50 and 56,200 to 3,120,000 virus units for 1 LD50 (slightly infectious and less lethal).(ABSTRACT TRUNCATED AT 250 WORDS)