Novel swine virulence determinant in the left variable region of the African swine fever virus genome

Abstract

Previously we have shown that the African swine fever virus (ASFV) NL gene deletion mutant E70DeltaNL is attenuated in pigs. Our recent observations that NL gene deletion mutants of two additional pathogenic ASFV isolates, Malawi Lil-20/1 and Pr4, remained highly virulent in swine (100% mortality) suggested that these isolates encoded an additional virulence determinant(s) that was absent from E70. To map this putative virulence determinant, in vivo marker rescue experiments were performed by inoculating swine with infection-transfection lysates containing E70 NL deletion mutant virus (E70DeltaNL) and cosmid DNA clones from the Malawi NL gene deletion mutant (MalDeltaNL). A cosmid clone representing the left-hand 38-kb region (map units 0.05 to 0.26) of the MalDeltaNL genome was capable of restoring full virulence to E70DeltaNL. Southern blot analysis of recovered virulent viruses confirmed that they were recombinant E70DeltaNL genomes containing a 23- to 28-kb DNA fragment of the Malawi genome. These recombinants exhibited an unaltered MalDeltaNL disease and virulence phenotype when inoculated into swine. Additional in vivo marker rescue experiments identified a 20-kb fragment, encoding members of multigene families (MGF) 360 and 530, as being capable of fully restoring virulence to E70DeltaNL. Comparative nucleotide sequence analysis of the left variable region of the E70DeltaNL and Malawi Lil-20/1 genomes identified an 8-kb deletion in the E70DeltaNL isolate which resulted in the deletion and/or truncation of three MGF 360 genes and four MGF 530 genes. A recombinant MalDeltaNL deletion mutant lacking three members of each MGF gene family was constructed and evaluated for virulence in swine. The mutant virus replicated normally in macrophage cell culture but was avirulent in swine. Together, these results indicate that a region within the left variable region of the ASFV genome containing the MGF 360 and 530 genes represents a previously unrecognized virulence determinant for domestic swine.

FIG. 1.

(A) Diagram showing the locations of the eight MalΔNL cosmid clones used for in vivo marker experiments. (B) Locations of plasmid subclones of cosmid C4 (pS1, pP1, pE1, pM1, and pN1) are indicated. Solid boxes denote clones capable of restoring virulence to E70ΔNL.

FIG. 2.

(A) (l) Diagram showing the genomic location of cosmid C4 on the MalΔNL genome. (II and III) EcoRI restriction maps of cosmid C4 (II) and the corresponding region on virulence-rescued E70ΔNL (E70C4) following in vivo marker rescue with cosmid C4 (III). (IV) MalΔNL DNA sequence which is present in the rescued virulent virus is indicated (dashed lines). (B) Schematic representation of the MalΔNL fragment and MGF ORFs (MGF 360 genes open boxes and MGF 530 genes are hatched boxes) present in E70 M1 rescued virus. (C) Purified DNAs from the parental virus, E70ΔNL (lane 1); rescued viruses (E70C4 in panels l and lland lanes 2 and 3; E70Ml in panel lll and lanes 2 and 3); and MalΔNL (lane 4) were digested with EcoRI (panels I and II) or BglII (panel III), electrophoresed, blotted and hybridized with ³²P-labeled C4 DNA probe (panels Il and IIl). DNA fragments derived from MalΔNL are indicated.

FIG. 3.

Comparison of the left variable regions of the MalΔNL and E70ΔNL genomes. (A) ORFs corresponding to MGF 360 family members (hatched boxes) and MGF 530 genes (hatched boxes). The relative position and extent of the left variable region deletion of E70ΔNL is shown. (B) Comparative analysis of ORFs encoded in the left variable region of MalΔNL and E70ΔNL. Amino acid (AA) lengths and amino acid identities (ID) are indicated.

FIG. 4.

Characterization of MalΔSVD recombinants. (A) Diagram of the swine virulence determinant gene region in the parental MalΔNL isolate and the swine virulence determinant deletion mutant virus MalΔSVD. E denotes EcoRI restriction endonuclease site. (B) Southern blot analysis of parental virus (lane 1) and four independently isolated MalΔSVD viruses (lanes 2, 3, 4, and 5). Purified viral DNA was digested with EcoRI, electrophoresed, blotted, and hybridized with ³²P-labeled L3IL gene probe (panel I) or ³²P-labeled probe generated from the recombination transfer vector (panel II). Molecular size markers are in kilobase pairs at the left of panel ll. Panel III, PCR analysis of MalΔSVD (lanes 2 and 4) and parental MalΔNL (lanes 3 and 5) for L3IL gene sequences (lanes 4 and 5). Positive-control PCR for the MalΔSVD viral DNAs using primers for a region of the genome immediately flanking the swine virulence determinant region are included (lanes 2 and 3). Molecular size markers are shown in lane 1.

FIG. 5.

Growth characteristics of MalΔNL and MalΔSVD in swine macrophage cell cultures. Primary swine macrophages were infected with MalΔNL and MalΔSVD (MOI = 0.1). At indicated times, duplicate samples were collected and titrated for total virus yield. Titers are expressed as log₁₀ TCID₅₀. Data represent means and standard errors of two independent experiments.