[Polypeptides of African swine fever virus]. / Polipeptidy virusa afrikanskoi chumy svinei.

The published data on the characteristics and properties of structural and nonstructural polypeptides of the African porcine virus are reviewed. Localization of the viral proteins in virions and infected cells, kinetics of biosynthesis, glycosylation, phosphorylation and the antigenicity of the proteins are discussed.

African swine fever virus: purification of capsomeres released by lipase.

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.

Phosphorylation of African swine fever virus proteins in vitro and in vivo.

Highly purified African swine fever virus contains a cyclic AMP-independent protein kinase which phosphorylates endogenous virus proteins with a specific activity of about 0.45 pmol/microgram of virus protein. The major substrates for the virion protein kinase in vitro were the structural proteins p10 and p9. Both proteins were phosphorylated preferentially at serine residues. A possible relationship between protein p10 phosphorylation and RNA synthesis in vitro by the virion-associated RNA polymerase is suggested by the finding that N-alpha-tosyl-L-lysyl-chloromethyl ketone inhibited both phosphorylation of p10 and transcription. Two phosphoproteins, with molecular masses of 35 and 17 kDa, were found in African swine fever virus purified from infected Vero cells labeled with [32P]phosphate. A phosphopolypeptide with a molecular mass of about 35 kDa was found in the cytoplasm of infected Vero cells.

Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32.

Localization of structural proteins in African swine fever virus particles by immunoelectron microscopy.

Seven African swine fever virus structural proteins were localized in the virion by immunoelectron microscopy. African swine fever virus-infected cells were incubated, before or after embedding and thin sectioning, with monoclonal antibodies specific for different structural proteins, and after labeling with protein A-gold complexes, the samples were examined in the electron microscope. Proteins p14 and p24 were found in the external region of the virion, proteins p12, p72, p17, and p37 were found in the intermediate layers, and protein p150 was found in the nucleoid and in one vertex. A monoclonal antibody that recognized protein p150 as well as p220, a virus-induced, nonstructural protein, could also bind to a component present in the nucleus of both uninfected and virus-infected cells.

Mechanism of thrombocytopenia in African swine fever.

Pigs were inoculated with an African swine fever (ASF) isolate of moderate virulence, and the changes in the number of circulating blood platelets during infection were correlated with the appearance of antiviral antibody and fluctuations in total plasma hemolytic complement concentrations. Thrombocytopenia was detected by postinoculation days (PID) 7 and 8, and antiviral antibody was detected by PID 7, using an indirect immunofluorescence technique. The total hemolytic complement concentration was moderately and transiently decreased from PID 5 to 9, but was consistently low from PID 18 to 26. Pigs inoculated with an ASF virus isolate of greater virulence had a decrease in platelet counts on PID 6 and 7, and the total plasma hemolytic complement levels decreased in all pigs by PID 6 to 7. Antibody to ASF virus was not detected in pigs inoculated with the more virulent isolate. Pigs sensitized to ASF viral antigen with an inactivated-virus vaccine or by previous infection with ASF were challenge exposed. Sensitized pigs became clinically ill and thrombocytopenic by 24 to 72 hours earlier than did inoculated, nonsensitized pigs. Vaccinated pigs inoculated with homologous virus had lower blood virus concentrations than did nonvaccinated pigs. African swine fever virus-sensitized pigs inoculated with heterologous virus had a higher fatality rate than did nonsensitized pigs, and the pigs died peracutely, with only a few gross lesions in evidence. In vitro experiments demonstrated that ASF virus antigen induced platelet aggregation in platelet-rich plasma from recovered, nonviremic pigs. Viral antigen, antibody, or complement was not demonstrable on the surface of platelets from pigs inoculated with ASF virus isolate, by direct immunofluorescence testing.

Purification and properties of African swine fever virus.

We describe a method for African swine fever (ASF) virus purification based on equilibrium centrifugation in Percoll density gradients of extracellular virions produced in infected VERO cells that yielded about 15 +/- 9% recovery of the starting infectious virus particles. The purified virus preparations were essentially free of a host membrane fraction (vesicles) that could not be separated from the virus by previously described purification methods. The purified virus sedimented as a single component in sucrose velocity gradients with a sedimentation coefficient of 3,500 +/- 300S, showed a DNA-protein ratio of 0.18 +/- 0.02 and a specific infectivity of 2.7 X 10(7) PFU/micrograms of protein, and remained fully infectious after storage at -70 degrees C for at least 7 months. The relative molecular weights of the 34 polypeptides detected in purified virus particles ranged from 10,000 to 150,000. Some of these proteins were probably cellular components that might account for the reactivity of purified virus with antiserum against VERO cells.

Proteins specified by African Swine Fever virus. IV. Glycoproteins and phosphoproteins.

African Swine Fever virus infected MS cells labeled with radioactive 14C-amino acids, 32Pi or [3H]-glucosamine were examined by high resolution sodium dodecylsulfate polyacrylamide gel electrophoresis and showed 43 infected cell polypeptides. Twenty-one of these proteins were present in the nuclear fraction of infected cells. At least 22 of the infected cell polypeptides induced antibodies during natural infections in swine. The pattern of infected cell polypeptides modified by incorporation of showed prosthetic groups that at least 8 polypeptides were phosphorylated and at least three specific viral glycoproteins (A, B and C) were detected by immunoprecipitation. The most highly glycosylated polypeptide corresponds to the structural viral protein VP51.

Sedimentation coefficient of African swine fever virus.

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).

Proteins specified by African swine fever virus. II. Analysis of proteins in infected cells and antigenic properties.

Infection of MS cells with African swine fever virus (ASFV) produces inhibition of protein synthesis which is detectable from 4.5 hours after infection. At least 34 viral polypeptides have been indentified with molecular weights ranging between 9500 and 243,000 daltons. Three of these proteins show affinity for the cell nucleus and nine are in both the nuclear and cytoplasmic fractions. Ten early proteins were found, and most of the structural proteins were late proteins. Most of the proteins are synthesized within the first 8 hours after infection. At least nine proteins induced antibodies in the natural infection. Six of these proteins are structural proteins. The antigenic determinants of VP172, VP162, VP146, VP73, VP34, and IP23.5 are in the primary structure of the proteins.

Proteins specified by African swine fever virus. I. Analysis of viral structural proteins and antigenic properties.

At least 28 polypeptides have been identified in intracellular virus, with molecular weights ranging from 11,500 to 243,000 daltons. By treatment with Nonidet P-40 and 2-mercaptoethanol it is possible to obtain subviral particles that have lost some proteins and have a density in CsCl of 1.31 g/cm3 which is higher than that of the complete virus (1.23 g/cm3). After addition of NaCL the virus loses its major protein VP73 which indicates that it is localized in the viral envelope. Cores obtained after this treatment are made up of at least 14 proteins. Incorporation of 3H-fucose and 3H-glucosamine in intracellular virus occurs in three minor components. The protein VP42 is possibly the cell actin and appears to be strongly associated with the virus. It is not possible to eliminate it under conditions where the viral envelopes disappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.

Purification and physicochemical characteristics of African swine fever virus.

Methods for the purification of African swine fever virus (ASFV) and its dissection into two fractions are described. The difficulties which have been encountered previously in attempts to purify the virus, namely contamination with large amounts of cellular constituents and aggregation of the virus particles, have been overcome by treatment with Tween 80 and by the use of 1 M-NaCl in the sucrose gradients. Five major polypeptides, mol. wt. 10(3) X 125 (VP1), 76 (VP2), 50 (VP3), 44 (VP4) and 39 (VP5) were found in the purified particles. The virus was dissected by treatment with Nonidet NP 40 into (a) a fraction which had the appearance of an empty capsid shell and capsid shell and contained the polypeptides VP2 and VP3 and (b) a structure containing VP1 and VP4. The location of VP5 was not ascertained.

Isolation and properties of the DNA of African swine fever (ASF) virus.

African swine fever (ASF) virus was grown either in swine macrophages or in VERO cells and purified free of cell DNA. Virus DNA was isolated from virions as a molecule with a sedimentation coefficient of 60S and a contour of 58 +/- 3 mum. .these two values give a mol. wt. of 102 +/- 5 X 10(6) and 107 +/- 5 X 10(6), respectively, for the genome of ASF virus. Denatured DNA fragments from ASF virus reassociate with a C0t1/2 value of 0-60 +/- 0-05 MS, which compared with the corresponding value for T4 DNA gives for the molecular mass of ASF virus DNA a value of 102 +/- 8 X 10(6) daltons. Only virus DNA is synthesized ASF virus-infected swine macrophages.