Modulation of immune cell populations and activation markers in the pathogenesis of African swine fever virus infection.

African swine fever (ASF) virus induces immune cell alterations that may be detected by changes in peripheral blood cells phenotypic antigens and activation markers which were examined by flow cytometry, analyzing both cell proportion and/or expression intensity of superficial antigens. These studies were conducted in pigs with experimental acute of chronic ASF infection to determine whether changes among important surface activation markers and phenotypic antigens, and their correlative lymph node status, reflected similar or disparate aspects of immune pathology. In acute infection produced by virulent viruses, macrophage and B lymphocyte populations decreased in peripheral blood after a short activation period at the beginning of the infection. A significative decrease of interleukin 2 receptor (IL 2R) expression was also observed in those pigs. These variations correlated with lymph node cell depletion due to an intense lymphoid cell death by apoptosis, affecting mainly the B lymphocyte subpopulation as determined by immunohistochemistry. Nevertheless, pigs infected with an attenuated isolate undergoing chronic persistent infection, presented a distinct pattern of modification, according with a different clinicopathological evolution. Changes consisted in systemic immune activation coincident with the highest viremia titer, with an augmentation in CD8+ T lymphocyte, macrophage, and B cell populations, and MHC (major histocompatibility complex) antigens. Percentage elevation of circulating immune subpopulations was accompanied by cell accumulation with lymphoid hyperplasia but a conserved distribution of B lymphocytes in lymphoid organs of chronically infected pigs.

Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.

Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus.

A recombinant baculovirus harboring the hemagglutinin (HA) gene of African swine fever virus, with homology to the T-lymphocyte surface antigen CD2, was constructed. The efficient expression of the HA gene was determined by immunofluorescence and Western blot studies on insect cells infected with the recombinant baculovirus. The baculovirus-expressed HA showed hemadsorption and erythrocyte-agglutinating activities characteristic of the CD2 homolog protein induced by the virus in infected macrophages. Pigs immunized with the recombinant HA developed hemagglutination-inhibition and temporary infection-inhibition antibodies that recognize a 75-kDa structural protein and were protected against lethal infection.

Characterization of a soluble hemagglutinin induced in African swine fever virus-infected cells.

Hemadsorption (Had) of erythrocytes to the surface of African swine fever virus (ASFV)-infected cells is a well-known phenomenon but hemagglutination of pig erythrocytes in the supernatant of ASFV-infected cells has not been reported before. We report here the discovery of a pig erythrocyte-agglutinating activity released to the in vitro cell culture medium by cells infected with some isolates of ASFV. This finding allowed the identification and characterization of a soluble hemagglutinin (HA) molecule that could be separated from the ASFV particles either by ultracentrifugation or by gel-permeation chromatography. The HA was inactivated by agents known to affect protein conformation such as heat, beta-mercaptoethanol, urea, and guanidine isothiocyanate. Glycosylation seemed to be of importance since treatment of HA with glycosidase F inhibited the hemagglutinating activity and HA could be partially purified by affinity chromatography on immobilized concanavalin A. When native it had an estimated molecular weight of 300 kDa by gel-permeation chromatography yielding 51-kDa protein monomers under denaturing conditions as identified by immunoblotting. Preliminary attempts to correlate the induced anti-HA serum antibodies with viremia or infection-inhibition serum antibodies after infection of pigs with attenuated ASFV or immunization with purified HA are also reported.

Cell culture propagation modifies the African swine fever virus replication phenotype in macrophages and generates viral subpopulations differing in protein p54.

We have detected 86 African swine fever (ASF) virus-induced proteins in infected pig macrophages by two-dimensional electrophoresis. No differences among protein patterns of wild-type viruses could be observed by this methodology. However, during cell culture adaptation and propagation we have characterized changes in the molecular weight of the ASF virus specified protein p54, which show direct correlation with both size and number of viral subpopulation variants generated during cell culture propagation. Passages in culture appear to select for viral subpopulations that specify p54 proteins with higher molecular weights than the wild-type virus. The virus propagation in cell culture also affected its replication phenotype in pig macrophages decreasing the viral titers in these cells between passage 44 and 81. Nevertheless, the changes observed in p54 did not imply differences in biological properties, such as infectivity, virulence or host cell range among viral clones isolated, each one specifying for only one p54 form with different molecular weight. This protein becomes then a valuable quantification marker to follow evolution and generation of ASF virus diversity in vitro.

African swine fever virus-induced proteins on the plasma membranes of infected cells.

The African swine fever virus-induced proteins on plasma membranes of infected cells have been studied by two different procedures, iodination and incubation of infected cells labeled with [35S]methionine with a specific antiserum, obtained from pigs immunized with a monkey stable cell-adapted African swine fever virus. The combined use of both procedures identified proteins IP56, IP51, IP35, IP34, IP31, IP30, IP25.5, IP23.5, IP16, IP15, IP14, and IP12 as viral antigens exposed on the surface of infected cells. Proteins IP16, IP15, and IP14 were recognized by the immune serum from survivor pigs, obtained after challenge with homologous virulent virus, but not by the immune serum from the same pigs immunized only with the cell-adapted virus.

Genetic stability of African swine fever virus grown in monkey kidney cells. Brief report.

Viral DNA subpopulations were produced when the ASFV was grown in monkey kidney MS cells. They were detected after 44 passages but not during the first 14 passages or in the unadapted ASFV E 70 strain grown in pig leukocytes. Different viral variants were isolated and their genomes were characterized. Restriction enzyme site variations were detected in both terminal fragments, Cla I-M and Sal I-F, and in the internal fragments Clal-O and Sma I-H. These variations result in changes in the size of the viral genome which ranges from 156 Kbp to 170 Kbp.

Neutralization of African swine fever virus by sera from African swine fever-resistant pigs.

Sera from African swine fever-resistant pigs with infection-inhibitory activity decreased virus replication in infected porcine buffy coat cultures. This same effect was observed even after virus was adsorbed. The infection-inhibition was not reversed by removing the immune serum from the assay cultures. Reduction of African swine fever virus replication by immune sera was demonstrated by fluorescent focus assay on MS cell line cultures. Virus-neutralization tests showed a persistent fraction of non-neutralized virus, which was not demonstrable by infection-inhibition tests. One hypothesis for explaining this difference is proposed.

Inhibition of African swine fever infection in the presence of immune sera in vivo and in vitro.

In assay cultures, sera from African swine fever convalescent pigs inhibited infection by homologous African swine fever virus. The infection-inhibition capacity did not correspond with the virus-neutralizing capacity. The serum did not prevent infection by heterologous virulent viruses. Sera from pigs challenge inoculated with the homologous virulent virus and later with a heterologous virulent virus inhibited the infection by different heterologous virulent viruses. These sera did not interfere with the infection by pseudorabies virus. The specificity of the reaction indicated that the infection inhibition was caused by antibody.

Lymphocyte function and cell-mediated immunity in pigs with experimentally induced African swine fever.

Twenty-five pigs inoculated with African Swine fever virus were studied for the development of changes in lymphocyte numbers and function. The results indicated that lymphopenia and decreased percentage of circulating T-lymphocytes occurred as early as 7 days after the pigs were inoculated. These were accompanied by depressed lymphocyte function, as measured by mitogen- or antigen-induced blastogenesis. There were proportionately greater decreases in T-lymphocyte numbers and function than were found for B-lymphocytes. Studies of phagocytic cell function revealed decreased phagocytic function for peripheral blood monocytes and unchanged (normal) neutrophil function. Depressed T-lymphocyte function occurred in pigs with acute and subacute infections, indicating depression of cellular immune function.

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.