An African swine fever virus ERV1-ALR homologue, 9GL, affects virion maturation and viral growth in macrophages and viral virulence in swine

Abstract

The African swine fever virus (ASFV) genome contains a gene, 9GL, with similarity to yeast ERV1 and ALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase-9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A 9GL gene deletion mutant of MAL (Delta9GL), exhibited a growth defect in macrophages of approximately 2 log(10) units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Delta9GL-infected macrophages. The Delta9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Delta9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Delta9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Delta9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV 9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.

FIG. 1

Alignment of the predicted amino acid sequence of the ASFV Malawi Lil-20/1 ORF 9GL product (ASF) with the yeast ERV1 and rat ALR motif-containing proteins. Homologues shown are as follows (with the GenBank accession number of the nucleotide sequence given in parentheses following each): ENTOMOPOX, M. sanguinipes entomopox virus ORF MSV093 product (AF063866); VACCINIA, vaccinia virus ORF E10R product (M35027); WORM, C. elegans gene product F56C11.3 (AF043697); DROSOPHILA, D. melanogaster homologue (AA696037); RAT, Rattus norvegicus ALR (D30735); YEAST, S. cerevisiae ERV1 (X60722); MALARIA, P. falciparum homologue (AL031745); CHLORELLA, chlorella virus ORF A465R product (U42580); IRIDOVIRUS, fish lymphocystis disease virus homologue (L63545); ARABIDOPSIS, Arabidopsis thaliana (AC003058). The consensus sequence was constructed with the Clustal W computer program (58) by using the Dayhoff PAM 250 symbol comparison table with a 0.5 cutoff value. Invariant amino acids in all proteins are shown on a solid background, while conservative substitutions are shaded. Periods in the sequence denote gaps introduced by the alignment program.

FIG. 2

Alignment of the predicted amino acid sequences encoded by 9GL homologues in ASFV isolates. MAL, Malawi Lil-20/1; KIL, Killean III; KIM, Kimakia; VI, Victoria Falls; TE, Tengani; K1, Fairfield/96/1. Isolates with identical amino acid sequences are represented as E70X (E70, Cameroon, E75, Haiti 811, La Granja, Lee, Lisbon 60, Uganda 61, Spencer, Zaire, and BA71V [GenBank accession no. U18466]) and PR4X (Pretoriuskop/96/4, Pretoriuskop/96/5, Wildebeeslaagte/96/1, Crocodile/96/1, Crocodile/96/3, and Chiredzi/83/1). The consensus sequence was constructed with the Genetics Computer Group (GCG; Madison, Wis.) program Pileup (12) by using the Dayhoff PAM 250 symbol comparison table with a 0.4 cutoff value. Residue differences are shown above the consensus sequence. Asterisks denote amino acid residues conserved in the ERV1-ALR family of proteins.

FIG. 3

Expression of p14 in ASFV-infected cell cultures. (A) Cell extracts of mock-infected (lane 1) and Malawi Lil-20/1-infected (lanes 2 to 7) swine macrophage cell cultures were pulse-labeled at 1 to 3 (lane 3), 3 to 5 (lane 4), 5 to 7 (lane 5), 7 to 9 (lane 6), and 9 to 18 (lanes 2 and 7) HPI and immunoprecipitated with anti-9GL monospecific serum (lanes 1 and 3 to 7) or a preimmune serum (lane 2). (B) Extracts of infected cells at 16 HPI maintained in the presence (lane 1) or absence (lane 2) of ara-C were immunoprecipitated with anti-9GL serum. Positions of the molecular size markers in kilodaltons are given on the left.

FIG. 4

Characterization of an ASFV 9GL gene deletion mutant, Δ9GL, and its revertant, 9GL-R. (A) Diagram of Malawi Lil-20/1 and recombinant viruses Δ9GL and 9GL-R. (B) (I and II) Southern blot analysis of DraI-digested parental and Δ9GL viral DNAs (lanes 1 and 2, respectively) probed with deleted 9GL sequences (panel I) or genomic regions flanking the deletion (panel II). (III) Southern blot analysis of SphI/SalI-digested DNAs from Δ9GL (lanes 1 and 3) and its revertant, 9GL-R (lanes 2 and 4), probed with the righthand terminal region (lanes 1 and 2) and a 9GL gene probe (lanes 3 and 4). (IV) Immunoprecipitation analysis of lysates from macrophage cell cultures infected with the parental, revertant, and mutant viruses (lanes 1, 2, and 3, respectively) by using monospecific anti-9GL serum.

FIG. 5

Growth characteristics of ASFV Malawi Δ9GL and the revertant, 9GL-R, in swine macrophage cell cultures. Porcine macrophage cell cultures were infected at an MOI of 0.1 (A) or 20 (B) with Δ9GL or 9GL-R. At indicated times postinfection, duplicate samples were collected and titrated for extracellular (EC) and intracellular (IC) virus yields. Data are means and standard errors of results from two independent experiments.

FIG. 6

Plaque formation of Malawi Lil-20/1 and Δ9GL on primary porcine macrophage cell cultures. Porcine macrophage cell cultures were infected with 100 PFU of Δ9GL (A) or Malawi Lil-20/1 (B), overlaid with 0.5% agarose, and incubated at 37°C for 5 days; then they were fixed with a 4% phosphate-buffered saline-paraformaldehyde solution prior to staining with 0.1% crystal violet.

FIG. 7

Budding virions and virus factories from Malawi Lil-20/1- and Δ9GL-infected macrophages at 16 HPI. (A and C) Malawi Lil-20/1-infected macrophages; (B and D) Δ9GL-infected macrophages. EP, empty particle. Magnification, ×42,536. Bar, 0.5 μm.