Deletion of a CD2-like gene, 8-DR, from African swine fever virus affects viral infection in domestic swine

Abstract

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 (delta8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, delta8-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 delta8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with delta8-DR. A delay in spread to and/or replication of delta8-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 delta8-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 delta8-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.

FIG. 1

Characterization of Δ8-DR and 8-DR.R. (A) Diagram of the 8-DR gene regions in the parental Malawi Lil-20/1 isolate, the deletion mutant virus Δ8-DR, and its revertant 8-DR.R. (B) Southern blot analysis of Malawi Lil-20/1 (lane 1), Δ8DR (lane 2), and 8-DR.R (lane 3). Purified viral DNAs were digested with EcoRI, electrophoresed, blotted, and hybridized with a 3.0-kbp probe including 8-DR gene sequences and flanking regions on either side (3 kb), an 8-DR gene probe (8-DR), and a GUS gene probe (Bgus). (C) PCR analysis of 8-DR.R (lane 1) and Δ8-DR (lane 2) viral DNAs for p72, 8-DR, and GUS sequences.

FIG. 2

Growth characteristics of ASFV Malawi Lil-20/1, Δ8-DR, and 8-DR.R viruses in primary swine macrophages infected with each virus at an MOI of 0.01. At indicated times, duplicate samples were collected and titrated for intracellular (A) and extracellular (B) virus yield. Data represent means and standard errors of two independent experiments.

FIG. 3

8-DR is necessary for erythrocyte-associated viremia. Shown are titers (mean values from five animals) of Δ8-DR and 8-DR.R viruses in whole blood, plasma, PBMC, and erythrocytes (RBC) from pigs at various times.

FIG. 4

Detection of Δ8-DR and 8-DR.R viruses in the draining internal iliac lymph node at 2 dpi by in situ hybridization. Magnification, ×450.

FIG. 5

Mitogen-induced proliferation in PBMC cultures infected with Δ8-DR and 8-DR.R. PBMC (10⁵/ml) from 4 to 10 naive swine were either infected (MOI = 10) with Δ8-DR or 8-DR.R or mock infected in the presence of the mitogen PHA, ConA, PWM, or TPA for 3 days and then pulsed with 1 μCi of [³H]thymidine per ml for 16 h. Bars indicate mean values of five replicates.

FIG. 6

Electron micrographs of PHA-treated PBMC cultures infected with 8-DR.R (A) and Δ8-DR (B) at 72 h p.i. Note the extensive macrophage cytopathology (arrows) and the normal appearance of lymphocytes in cultures infected with both viruses. The bar represents 5 μm.