Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus

Abstract

African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

Fig. 1

Schematic diagram showing generation of recombinant ASF virus OUR T88/3ΔDP2 with the deletion of the DP71L and DP96R genes. Recombinant virus OUR T88/3ΔDP2 was created by homologous recombination between the MGF360 18R and MGF360 19R genes on the wild type OUR T88/3 genome and transfer vector plasmid pΔDP2loxPGUS resulting in the deletion of the DP71L and DP96R genes and the insertion of vp72GUS gene.

Fig. 2

Analysis of genomic viral DNA gene deletions and insertions by PCR. Viral DNA was extracted from wild type OUR T88/3 virus and the recombinant virus OUR T88/3∆DP2. 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+2 (18RSEQ and 19RSEQ), lanes 3+4 (18RSEQ and RGUS), lanes 5+6 (EXT18R and RGUS). The following viral genomic DNAs were used as templates in the lanes 1, 3 and 5 (OUR T88/3) and lanes 2, 4 and 6 (OUR T88/3ΔDP2).

Fig. 3

Replication kinetics of OUR T88/3 and recombinant OUR T88/3ΔDP2 viruses. Pig bone marrow macrophages were infected at a high multiplicity of infection (m.o.i.) of 10 or low m.o.i. of 0.1 with parental OUR T88/3 strain or recombinant virus OUR T88/3ΔDP2. At various hours post-infection, as indicated on the x axis, total virus was harvested and infectious virus titrated on 96 well plates by analysis of infection on cultures of pig bone marrow macrophages. The virus titre (TCID₅₀/ml) is the mean of three individual observations. Titres obtained following infection with viruses OUR T88/3 high m.o.i. and low m.o.i. ; OUR T88/3ΔDP2 high m.o.i. and low m.o.i. are indicated.

Fig. 4

Clinical scores post-challenge with OUR T88/1. Clinical scores (y-axis) of individual pigs from the three separate groups at different days post-challenge (x-axis). Pigs were challenged at day 0 (C-0am). Clinical scoring system as designed by King et al. (2011).

Fig. 5

Viraemia estimated by qPCR for individual pigs post-inoculation and post-challenge. Viraemia estimated by qPCR and expressed as ASFV genome copy per ml of blood (y-axis) for individual pigs at different days post-inoculation and post-challenge (x-axis). Group 1 OUR T88/3 , Group 2 OUR T88/3ΔDP2 and Group 3 unvaccinated . Five pigs had no detectable ASFV DNA in blood post-immunisation (pig 1—Group 1, pigs 14, 15, 17 and 18—Group 2). Four pigs had no detectable levels of ASFV DNA in blood post-challenge (pigs 1 and 5—Group1, pigs 16 and 17—Group 2).