Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Abstract

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Lumpy Skin Disease Virus with Four Knocked Out Genes Was Attenuated In Vivo and Protects Cattle from Infection

“…After removal of selective pressure and intramolecular recombination of the viral genome, two types of viruses are formed: the original and recombinant with the deleted target gene. Intramolecular recombination also led to the removal of the gpt gene from the viral genome [ 20 , 22 ].…”

“…The LSDV066 gene was knocked out as a result of a translation frame failure due to the insertion of a construct for the expression of foreign genes under the control of a synthetic vaccinia virus promoter [ 20 ]. The pIN-LSDV066-IL18 integration plasmid used in these studies provided for the insertion into the viral genome of a sequence encoding bovine interleukin-18 (IL18) mRNA.…”

“…Recombinant viruses were generated by homologous recombination under temporary dominant selection conditions as previously described [ 20 ]. Briefly, a monolayer of LT cells was infected with LSDV Atyrau/KZ or its derivatives at a dose of 0.1 PFU/cell for 2 h, and then transfected with one of the integration plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.…”