Mapping and sequence of the gene coding for protein p72, the major capsid protein of African swine fever virus

The gene encoding protein p72, the major structural protein of African swine fever virus and one of the most immunogenic proteins in natural infection has been mapped and sequenced. The gene was mapped by using oligonucleotide probes deduced from amino acid sequences of tryptic peptides obtained from purified protein p72. This allowed the location of the gene in fragment EcoRI B of African swine fever virus DNA. The nucleotide sequence obtained from this region revealed an open reading frame encoding 646 amino acids corresponding to a protein with a calculated molecular weight of 73,096 Da. This open reading frame contains the coding information for all the sequenced tryptic peptides from protein p72. A search at the National Biomedical Research Foundation Data Bank did not reveal any significant homology with other described proteins.     

Proteins specified by African swine fever virus

Summary 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 of³H-fucose and³H-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 desappear morphologically. At least the proteins VP172, VP162, VP146 and VP73 act as antigens in the natural infection.With 5 Figures     

Proteins specified by African swine fever virus

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.     

DNA-binding proteins specified by African swine fever virus

[35S]Methionine-labeled proteins from total or cytoplasmic extracts of Vero cells infected with African swine fever virus were chromatographed on native and denatured DNA-cellulose and DNA-binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), by DNA binding to Western blots, or by two-dimensional electrophoresis. Thirteen virus-specific DNA-binding proteins were detected by one-dimensional analysis. Major species have molecular mass 44,000 (44K), 38K, 20K, 18K, 14K, 13K, and 12K. The remaining DNA-binding proteins are proteins with molecular mass 130K, 110K, 35K, 33K, 17K, and 14.5K. When viral DNA used in the binding assay the results were very similar but the 13K protein did not bind viral DNA. Seven other minor virus-specific DNA-binding proteins could be detected by two-dimensional analysis. This technique also enabled the assignment of virus-specific proteins. Seven of the virus-specific DNA-binding proteins are structural proteins. Twelve are late proteins, the remaining being early proteins synthesized before viral DNA replication. Most of the virus-specific DNA-binding proteins bind both to double-stranded and to single-stranded DNA. The 110K, 29K, and 18K DNA-binding proteins bind only to single-stranded DNA. Two virus-specific enzymatic activities, DNA polymerase and RNA polymerase, were present in the fractions separated by DNA-cellulose chromatography. The virus-specific single-stranded DNA nuclease did not bind to DNA.     

Strong sequence conservation of African swine fever virus p72 protein provides the molecular basis for its antigenic stability

Summary The major capsid protein p72 of African swine fever virus (ASFV) has long been considered an important immunodominant antigen for serologic diagnosis. Here we describe the cloning and sequence analysis of two p72-coding genes from ASFV strains Uganda (UGA) and Dominican Republic-2 (DR2). Sequence comparison of these genes, together with those from two other ASFV strains (BA71V and E70), demonstrated that the p72 proteins are highly conserved (97.8% to 100% amino acid sequence identity) in strains isolated from different parts of the world. These results support previous observations indicating that p72 is antigenically stable, and provide a useful molecular basis for further development of ASFV serologic tests using this important antigenic molecule.     

Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection

Although antibody-mediated immune mechanisms have been shown to be important in immunity to ASF, it remains unclear what role virus neutralizing antibodies play in the protective response. Virus neutralizing epitopes have been identified on three viral proteins, p30, p54, and p72. To evaluate the role(s) of these proteins in protective immunity, pigs were immunized with baculovirus-expressed p30, p54, p72, and p22 from the pathogenic African swine fever virus (ASFV) isolate Pr4. ASFV specific neutralizing antibodies were detected in test group animals. Following immunization, animals were challenged with 10(4) TCID(50) of Pr4 virus. In comparison to the control group, test group animals exhibited a 2-day delay to onset of clinical disease and reduced viremia levels at 2 days postinfection (DPI); however, by 4 DPI, there was no significant difference between the two groups and all animals in both groups died between 7 and 10 DPI. These results indicate that neutralizing antibodies to these ASFV proteins are not sufficient for antibody-mediated protection.     

Epitopic diversity of African swine fever virus

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.     

Phylogenetic analysis of swine fever virus strains

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.     

Characterization of African swine fever virus antigenic proteins by immunoprecipitation

Summary African swine fever virus is a large, complex virion in which numerous proteins have been identified by biochemical techniques. Few of these proteins have been shown to react with antibodies from recovered swine, leading to speculation that the immunological unreactivity of some viral proteins might explain the inability of immune sera from surviving animals to neutralize the virus. We used immunoprecipitation of radiolabeled viral proteins to examine these sera in more detail. Gradient sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis of these immunoprecipitates revealed that at least 37 viral proteins participated in antigen-antibody reactions in this system. Differences in the molecular weights of some immunoprecipitable proteins were noted between different isolates of virus, between the same isolate grown in different cells, and between an isolate adapted to Vero cells and one not adapted to these cells.With 3 Figures     

A gene homologous to topoisomerase II in African swine fever virus.

A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases. The gene is located within EcoRI fragments P and H of the African swine fever virus genome. Sequencing of this region has revealed a long open reading frame, designated P1192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543. Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA. The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29%. The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established. The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged.     

Plaque assay for African swine fever virus on swine macrophages

Summary.  A plaque assay developed to detect the infection of African Swine Fever Virus on swine macrophages is described. Plaques were generated by all of the virus isolates tested. The method is suitable not only for virus titration but also for the selection of clones in protocols for isolation/purification of recombinant viruses. Received December 28, 2001; accepted February 20, 2002 Published online April 26, 2002     

Molecular cloning of African swine fever virus DNA

African swine fever virus DNA (about 170 kbp) was cleaved with the restriction endonuclease EcoRI and most of the resulting 31 fragments were cloned in either the phage vector λWES.λB or the plasmid pBR325. Three fragments were not cloned in those vectors, the largest fragment EcoRI-A (21.2 kbp) and the two crosslinked terminal fragments, EcoRI-K′ and D′. Endonuclease SalI cut fragment EcoRI-A into three pieces which were cloned in plasmid pBR322. The two terminal EcoRI fragments were cloned after removal of the crosslinks with nuclease S1 and addition of EcoRI linkers to the fragment ends. The complete library of the cloned fragments accounted for about 98% of ASF virus genome, the missing sequences being those removed by the nuclease S1 in the process of cloning the terminal fragments.     

Glycosylated components of African swine fever virus particles

Extracellular African swine fever (ASF) virus particles were specifically agglutinated by several lectins, suggesting the presence of surface glycosylated component(s) containing at least glucose, mannose, or both; galactose, N-acetylgalactosamine, or both; N-acetylneuraminic acid and N-acetylglucosamine, but not fucose. When virions were purified from infected Vero cells labeled with [14C]glucosamine, [14C]galactose and analyzed by polyacrylamide gel electrophoresis, no major structural glycoproteins were detected. However, several species of glycolipids were found when virions were extracted with organic solvents and analyzed by thin layer chromatography. These, plus two minor glycosylated structural components, of apparent mol wt 230K and 95K, could account for the agglutination of ASF virions with concanavalin A.     

Polypeptides and structure of African swine fever virus

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.     

Interaction of African swine fever virus with macrophages

Morphological data obtained by electron microscopy have shown that African swine fever virus adapted to VERO cells enters swine macrophages, its natural host cell, by a mechanism of receptor-mediated endocytosis. Binding studies with 3H-labeled virus and competition experiments with UV-inactivated virus have shown that the virus entry that leads to a productive infection in swine macrophages is mediated by saturable binding sites on the plasma membrane. The virus also penetrated into rabbit macrophages that do not produce infectious virus and initiated the synthesis of some early viral proteins; however, the viral replication cycle was aborted since viral DNA synthesis did not occur. The interaction of ASF virus particles with rabbit macrophages was mediated by nonsaturable binding sites, suggesting that the lack of specific receptors in these cells may be related to the absence of a productive infection. A similar abortive infection was detected in macrophages from other virus-resistant animal species.     

The structural protein p54 is essential for African swine fever virus viability

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of β-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and β-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and β-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.     

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.