Construction And Identification Of The Recombination Pseudorabies Virus Expressing GP5 Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus (HuN4)

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae, is widespread among the swine population, causing reproductive failure in sows and respiratory disorders in pigs of all ages, and has an economic impact on the pig industry worldwide. An emerged highly pathogenic porcine reproductive and respiratory syndrome virus (HPPRRSV), occurred in the pigs in main swine industry provinces of mainland China in 2006, which made a big loss in the raise of pigs. As we koown, the vaccines against classic PRRSV can provide partly protection to pigs challenged with HPPRRSV. Therefore, it is urgent to develop an effective vaccine against HPPRRS. In our laboratory, attenuated vaccines against HPPRRS have been developing. This study attempted to develop a live vectored vaccine against HPPRRS.GP5 protein is the main envelope glycoprotein of PRRSV. It was reported that, GP5 protein of PRRSV by means of DNA immunization develop specific neutralizing and protecting antibodies. GP5, has the highest genetic diversity among isolates. We analyzed and compared the GP5 sequences of HPPRRSVs. As a result, all the Chinese isolates examined belong to the NA-type. The HPPRRSV isolates from China have highly homology, but has some key mutations with the classic PRRSVs for vaccine. Using the PRV vector to express the GP5 protein of CH-1a, the rPRV-GP5 can protect the pig counteract the attack of the highly pathogenic CH-1a. Because the amino acids homology of GP5 between CH-la and the HPPRRSV is only 93%, the rPRV-GP5 could not provide complete protection. Therefore, in this study, we aimed to construct a new rPRV, expressing the GP5 of HPPRRSV, and investigate the use in prevention and cure of the HPPRRS.In this study, we used the PRV Bartha-K61 strain as a vector, the ORF5 gene of HPPRRSV and EGFP gene were instrted to the PRV gG promoter downstream, then the expression vector pgG-HPGP5-EGFP was constructed. The result of PCR and restriction enzymes digestion suggested that the construction of pgG-HPGP5-EGFP was successful. Through fluorescent microscope detecting, we obtained the result that the best efficiency expression of the EGFP gene was 48 hours post-transfection. PRV Bartha-K61 DNA and the plasmid pgG-HPGP5-EGFP were co-transfected into Vero cells with calcium phosphate, 60-72 hours post-transfection, we screened out the rPRV with fluorescence microscopy, After 6 times plaque purification, we got a strain rPRV lasting express EGFP. The rPRV was analyzed by PCR, Western blot and IFA, the result confirmed that HPPRRSV GP5 protein had been expressed well in recombinant virus, and was named rPRV-HPGP5.