Sequential deletion of genes from the African swine fever virus genome using the cre/loxP recombination system

Abstract

A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus VΔMGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus VΔMGFΔGUS. The EP402R and EP153R genes were subsequently deleted from the genome of VΔMGFΔGUS, using the same GUS marker gene, to construct virus VΔMGFΔCD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.

Fig. 1

Schematic diagram showing generation of recombinant ASF viruses expressing GUS reporter gene and excision of the GUS gene by the bacteriophage Cre recombinase. First generation recombinant virus VΔMGF-GUS was created by homologous recombination between left and right flanking regions (FLANK L and FLANK R) from genes MGF360 8L and MGF 505 4R on the wild type BA71V genome and transfer vector plasmid pΔMGFloxPGUS resulting in the deletion of the truncated MGF360 9L and MGF505 genes 2R and 3R and the insertion of vp72GUS gene flanked by loxP sequences. A plasmid expressing the bacteriophage Cre recombinase protein under control of the ASFV vp72 gene promoter was transfected into cells infected with VΔMGF-GUS and second generation virus recombinant VΔMGFΔGUS was generated by site-specific recombination between the two loxP sites resulting in deletion of the vp72GUS marker gene and single loxP site and the retention of a single loxP site. Non viral sequences A and B were included to facilitate the construction of plasmid pΔMGFloxPGUS by overlapping PCR.

Fig. 2

Generation of successive recombinant ASFV viruses Panel A. Analysis of genomic viral DNA gene deletions and insertions by PCR. Viral DNA was extracted from wild type BA71V virus and the recombinant viruses VΔMGF-GUS, VΔMGFΔGUS, VΔMGFΔCD2vLectin-GUS, VΔMGFΔCD2vLectinΔGUS. Specific fragments were amplified by PCR and the products were analysed by electrophoresis on 1% agarose gels. The following primer sets were used in the lanes 1–3 (8LSEQ+4RSEQ, lanes 4–6 (8LSEQ+RGUS), lanes 7–9 (EXT8L+RGUS), lanes 10–12 (152RSEQ+364RSEQ), lanes 13–15 (152RSEQ+RGUS), lanes 16–18 (EXT152R+RGUS). The following viral genomic DNAs were used as templates in the lanes 1, 4, 7, 10, 13 and 16 (BA71V), lanes 2, 5 and 8 (VΔMGF-GUS), lanes 3, 6 and 9 (VΔMGFΔGUS), lanes 11, 14 and 17 (VΔMGFΔCD2vLectin-GUS), lanes 12, 15 and 18 (VΔMGFΔCD2vLectinΔGUS). Lane M1 contains a 1 kb DNA ladder and lane M2 a 100 bp DNA ladder. Panel B. Genomic maps of BA71V (170101 bp) and the four recombinant viruses showing the positions of deletions of the truncated MGF360 9GL, MGF505 2R and 3R genes located between positions 17725 and 20236. The two recombinant viruses VΔMGFΔCD2vLectin-GUS and VΔMGFΔCD2vLectinΔGUS show the additional deletion of the EP153R and EP402R genes located between positions 55,705 and 57,447. Recombinant viruses VΔMGF-GUS and VΔMGFΔCD2vLectin-GUS show insertion of the GUS gene flanked by two loxP (P) sequences and viruses VΔMGFΔGUS and VΔMGFΔCD2vLectinΔGUS show deletion of the GUS gene retaining one copy of the loxP (P) sequence.

Fig. 3

DNA sequence analysis of viruses BA71V, VΔMGF-GUS and VΔMGFΔGUS at insertion site. The primers 8LSEQ and 4RSEQ were used to amplify by PCR DNA fragments from genomic DNAs spanning the insertion and deletion junctions between the MGF360 8L and 9L and MGF 505 3R and 4R genes were cloned into the HindIII and XhoI sites of pCDNA3. The fragments were sequenced with the 8LSEQ primer. Non viral sequences A and B were derived from the transfer vector pΔMGFloxPGUS. The sequences underlined indicate the MGF360 8L sequence, loxP sequence and non-viral sequences A and B. Sequences of the flanking genes to the left and right (FLANK L and FLANK R) are also indicated.

Fig. 4

Replication kinetics of wild type BA71V and recombinant viruses. Vero cells were infected at a multiplicity of infection of 10 with parental BA71V strain or recombinant viruses VΔMGF-GUS, VΔMGFΔGUS, VΔMGFΔCD2vLectin-GUS, VΔMGFΔCD2vLectinΔGUS. At various hours post-infection, as indicated on the x axis, total virus was harvested and infectious virus titrated by plaque formation on Vero cells. The virus titre shown is the mean of triplicate samples. The titre obtained is indicated in plaque forming units per ml (pfu/ml) on the y axis. Titres obtained following infection with viruses BA71V □, VΔMGF-GUS ○, VΔMGFΔGUS +, VΔMGFΔCD2vLectin-GUS Δ and VΔMGFΔCD2vLectinΔGUS ⋆ are indicated.