The African swine fever virus thymidine kinase gene is required for efficient replication in swine macrophages and for virulence in swine

Abstract

African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter-beta-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK- ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK+ revertant virus. Swine surviving TK- ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.

FIG. 1

Diagram of the TK region of the Malawi LiL-20/1 genome, showing the placement and orientation of the TK gene and adjacent genes p10 (DNA binding protein 5-AR) (33), K205R, K145R, and K421R (Ba71V open reading frames) (46); structure of the TK gene deletion transfer vector, p5.3, showing insertion sites into the pBluescript multiple cloning site; and structure of plasmid pLR-TK, containing a 2,164-bp PCR fragment of the intact TK gene and flanking ASFV DNA.

FIG. 2

Southern blot analysis of parental, recombinant, and revertant ASFV DNA digested with BglII. ASFV Malawi (A and B) and ASFV Haiti (C) were probed with a TK probe (HindIII fragment of pLR-TK) (A and C) or a GUS probe (SmaI/SacI fragment of the p72GUS cassette) (34) (B). Lanes 1, swine spleen isolate; lanes 2, Vero-adapted isolates; (lanes 3, TK deletion mutant viruses; lanes 4, revertant virus.

FIG. 3

Growth characteristics of ASFV Malawi and Haiti parental (closed circles) and recombinant (open circles) viruses on primary swine macrophages. Cells were infected at MOIs of 0.01 (A and B) and 10 to 20 (C and D) with the appropriate viruses (which were absorbed for 2 h at 37°C), rinsed twice with growth medium, and incubated. At the indicated times, cultures were harvested and lysates were titrated for total virus yield on Vero cells by the immunoperoxidase method (49). (A and C) Growth of ASFV Malawi (parent) and TK deletion mutant v5.3; (B and D) growth of ASFV Haiti H811 (parent) and TK deletion mutant vH53. Data represent the TCID₅₀ titers ± standard errors of the means (14) assayed for two or three independent experiments.

FIG. 4

Virus titers obtained on successive passages of parental (closed circles) and TK deletion mutant (open circles) viruses on primary swine macrophages. (A) ASFV Malawi viruses; (B) ASFV Haiti H811 viruses. Cells in T-25 flasks were inoculated with 1 ml (passage [pass] 1) or 2 ml (passages 2, 3, and 4) of virus (which was absorbed for 2 h), rinsed twice with growth medium, and incubated for 2 to 4 days. Cultures were observed for CPE, and cells and medium were harvested at 2 days (passage 1) or 4 days (passages 2, 3, and 4); then culture lysates were examined for GUS expression, and TCID₅₀ titers were determined by the immunoperoxidase method. Titers ± standard errors of the means are the results of three independent experiments.

FIG. 5

Plaques formed on primary swine macrophages by parental Malawi (A) and Haiti (B) ASFV and by TK deletion mutants Malawi v5.3 (C) and Haiti vH53 (D).