Study On The Biological Characteristics Of A Recombinant Goat Pox Virus Expressing FMDV Vp1 Gene

Goat pox, on the A list infectious diseases of the World Organisation for Animal Health (OIE), is a highly contagious viral disease of goats, characterized by pyrexia, generalized skin, internal pox lesions and lymphadenopathy. The disease is caused mainly by goat pox virus (GPV), classified in the genus Capripoxvirus of the family Poxviridae. The disease inflicts substantial losses in terms of reduced productivity and lower quality of wool and leather. It poses a major obstacle in the intensive rearing of goats and also greatly hampers international trade. Goat pox is the most important of all pox diseases of domestic animals causing high mortality in kids and significant economic losses. In enzootic areas, both live attenuated and inactivated vaccines are useful in the prevention and control of goat pox, but inactivated vaccines give only short-term immunity. Live attenuated vaccines are highly immunogenic and cause long term of immunoprotection, but nowadays diagnostic method could not distinguished vaccinated animals by traditional activated vaccine with naturally infected animals.Foot-and-mouth disease (FMD), also on the A list infectious diseases of OIE, is a severe, highly communicable viral disease of cattle and swine caused by Foot-and-mouth disease virus (FMDV). It also infects sheep, goats, deer, and other cloven-hooved ruminants, results in huge economic loss. Although Goat and sheep are not as sensitive as pig and cattle, they play an important role during prevalent and spreading process of FMDV as carriers. Many studies show that the structural VP1 carries critical epitopes responsible for the induction of neutralizing antibodies. So VP1 played a key role in immune action and become firstly target antigen to develop genetic engineering vaccine.In this paper, we chose GPV vaccine AV41 strain widely used in China as parental virus, thymidine kinase gene as inserted site for foreign gene, and constructed recombinant GPV expressing FMDV VP1 gene without report gene by the way of reversal plaque selection. Above all, we cloned ORF64-ORF67 gene fragment from GPV AV41 genome as flanking sequences for homologous recombinant into GPV genome, which contain thymidine kinase (TK) gene. Then the gene fragment was subcloned into pMD 18-T simple vector, resultant plasmid was named pTK. The gene fragment was sequenced and analysis. ORF64 was composed of 396 base pairs encoding membrane protein. ORF65 was composed of 444 base pairs encoding membrane protein.ORF66 was composed of 534 base pairs encoding TK protein, which contained conserved ATP-binding motif (P-loop) and thymidine kinase cellular-type signature. ORF67 was composed of 594 base pairs encoding host range protein. Comparing with GPV reference strains, ORF64-ORF67 had some mutant, and homology of nucleotides and amino acids sequences was between 94.4% and 100%. Among others, the homology of nucleotides and amino acids sequences of TK was between 96.8% and 100%, and 100% with GPV isolates in China. The analysis result showed that GPV AV41 had high homology with isolates, so it could serve as parental virus to construct recombinant GPV. The LacZ reporter gene of E.coli under the control of vaccinia virus late promoter PH was released by enzymatic digestion and inserted into the plasmid pTK at Kpn I site of thymidine kinase gene, giving rise to a new GPV transfer vector, named as pTK-LacZ. Using Lipofection Reagent, the plasmid pTK-LacZ was transfected into bovine testes (BT) cells infected with GPV AV41. A recombinant GPV harboring LacZ reporter gene, designated as rAV41-LacZ, was obtained after five rounds of blue plaque purification and PCR identification. Furthermore, the products of PCR were sequenced. Then rAV41-LacZ was detected by PCR and TCID₅₀ during passaging to 25th in BT and lamb testes (LT) cells. The result showed that rAV41-LacZ was stable and LacZ gene also remain stabile, TCID₅₀ is similar between rAV41-LacZ and GPV AV41 and the absent TK gene had no obvious effect on multiplication of virus.After rAV41-LacZ was constructed successfully, FMDV type O vp1 gene was chose as target antigen. The vp\ gene under the control of vaccinia virus early/late promoter P7.5 was released by enzymatic digestion and inserted into the plasmid pTK at Kpn I site of thymidine kinase gene, giving rise to a new GPV transfer vector, named as pTK-VP1. Using Lipofection Reagent, the plasmid pTK-VP1 was transfected into bovine testes (BT) cells infected with rAV41-LacZ. A recombinant GPV harboring VP1 gene without LacZ reporter gene, designated as rAV41-VPl, was obtained after six rounds of reverse LacZ selection and PCR identification. vp\ gene amplified by PCR was sequenced for identification. The expression and immunogenicity of the VP1 were analyzed by growth kinetics analysis in BT and LT cells, Western blot, and indirect immunofluorescence test. The results showed that FMDV VP1 could be expressed authentically and efficiently, VP1 was expressed in intracellular. Compared with rAV41-LacZ, rAV41-VPl showed no obvious difference with respect to virus replication and size and shape of virus plaque, but there was little difference with cytopathogenic effects in BT cell between rAV41-VPl and rAV41-LacZ. Then the rAV41-VPl was detected by PCR, TCID₅₀ and Western blot during passaging in BT and LT cells. The result showed that the rAV41-VPl had stable genetic character and VP1 gene remain stabile, TCID₅₀ is similar between rAV41-VPl and rAV41-LacZ and The rAV41-VPl remained the character of parental vaccine strain.To sum up, rAV41-LacZ and rAV41-VPl will be a valuable tool with which to develop potential of recombinant GPV to deliver vaccine antigens to goat and sheep, this study provided a technical platform and basis for development recombinant vaccine of expressing foreign gene without reporter gene in GPV. In summary, the rAV41-LacZ is is a good candidate recombinant virus vaccine to control goat pox and FMD.