African swine fever is a significant disease of domestic swine, with mortality rates approaching 100%. No vaccine is currently available, making quarantine and slaughter the only effective control strategy (44).African swine fever virus (ASFV), the causative agent of African swine fever, is a unique and complex DNA virus that infects cells of the mononuclear-phagocytic system, including fixed-tissue macrophages and specific lineages of reticular cells. Affected tissues show extensive necrosis following infection with highly virulent viral strains (13,36,38). Moderately virulent ASFV strains also appear to infect these cell types, but the degree of tissue involvement and the resulting tissue damage are much less severe (13,36,38). The abilities of ASFV to replicate and efficiently induce marked cytopathology in monocytes-macrophages in vivo appear to be critical factors for ASFV virulence.ASFV is the sole member of the family Asfarviridae and the only known DNA arbovirus (14,38). ASFV is a large, icosahedral virus that contains a linear double-stranded DNA genome (170 to 190 kbp) encoding approximately 165 genes (50; C. A. Balinsky et al., unpublished data). The availability of complete ASFV genome sequences has revealed that, similar to poxviruses, ASFVs encode proteins with functions essential for viral replication, including those involving structure and assembly of the virion and those responsible for biogenesis of mRNA and DNA. A large number of ASFV genes are of unknown function and may be involved in aspects of viral virulence and host range (46,50; Balinsky et al., unpublished).Pathogenic ASFV genomes contain 11 to 15 multigene family 360 (MGF360) genes and either 9 or 10 multigene family 530 (MGF530) genes (Balinsky et al., unpublished). Recently, we have identified MGF360 and MGF530 genes as novel macrophage host range determinants necessary for efficient growth in macrophages (54). Infection of macrophage cell cultures with MGF360-MGF530 (MGF360/530) gene deletion mutant Pr4⌬35 (six MGF360 and two MGF530 genes deleted) resulted in a 2-to 3-log reduction in virus titers and early cell death, suggesting a direct or indirect role for these genes in some aspect of infected-cell survival (54) (L. Zsak, unpublished data). In addition, a swine virulence determinant (VAD) containing MGF360/530 genes was mapped by using in vivo marker rescue to the left variable region of the ASFV genome (37).The mode of action of the ASFV MGF360/530 genes is unknown. Homology searches reveal no homology to other known genes. MGF360/530 genes have a conserved motif of 100 amino acids (28% amino acid identity) at the amino ter-

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.

An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo,8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

A fluorogenic probe hydrolysis (TaqMan) PCR assay for African swine fever virus (ASFV) was developed and evaluated in experimentally infected swine. This sensitive and specific one-step single-tube assay, which can be performed in 2 h or less, detected viral DNA in tonsil scraping samples 2 to 4 days prior to onset of clinical disease. Thus, the assay would have application for preclinical diagnosis of African swine fever and surveillance and/or emergency management of a disease outbreak