| Porcine reproductive and respiratory syndrome is an infectious disease caused by Porcine reproductive and respiratory syndrome virus,which is characterized by reproductive dysfunction in sows and respiratory diseases in pigs of all ages.The new PRRSV variant HP-PRRSV spread rapidly in China in 2006,causing huge economic losses to the domestic pig breeding industry.A new variant of PRRSV,NADC30-Like-PRRSV,appeared again in China in 2013.Although the pathogenicity of NADC30-Like-PRRSV was lower than that of HP-PRRSV,NADC30-Like-PRRSV was easily recombined with other types of strains.In recent years,NADC30-LikePRRSV has been prevalent in many large-scale pig farms in China and has become a new dominant strain.Porcine pseudorabies virus can infect pigs of all ages and cause reproductive disorders and diseases of nervous system and respiratory system of piglets.In 2011,a more virulent variant of PRV appeared in China,and new PR outbreaks also broke out in pig farms inoculated with Bartha-K61 vaccine,which seriously threatened the healthy development of the pig breeding industry in China.At present,the prevention and control of PRRS and PR mainly depends on vaccine immunization.However,the classical vaccines of PRRS and PR cannot provide complete protection to pigs,so there is an urgent need for safe and effective new vaccines to control and purify the two viruses.In order to prepare a safe and effective new vaccine that can simultaneously prevent and control PRRS and PR,this study intends to use the modified attenuated PRV strain as the carrier and the main antigen gene ORF5 of PRRSV as the exogenous gene to construct the recombinant virus strain.And,it is expected that the recombinant virus can protect swine against PRRSV and PRV,and become a candidate vaccine strain that can simultaneously control PRRS and PR.In order to investigate the current epidemic situation of PRRSV in Henan Province,A total of 110 serum and lung samples of suspected PRRSV infected pigs from several pig farms in Henan Province were collected forPRRSV detection and amplification of ORF5 gene to analyze their genetic and variability from 2019 to 2022.The results showed that the total positive rate of PRRSV was 29.09%,which showed an increasing trend from 2019 to 2022.Sixteen PRRSV ORF5 gene sequences amplified from positive samples showed 82.3%-99.3% nucleotide and 54.3%-99% amino acid homology with reference sequences,respectively.The results of genetic evolution analysis showed that the 15 PRRSV GP5 sequences obtained in this study were most closely related to the reference sequence Henan-XINX in Lineage1 group.One sequence belonged to Lineage 8.7 and was most closely related to HUN4.It is suggested that the main epidemic strains in Henan are HP-PRRSV and NADC30-Like PRRSV,and the prevention and control of dominant PRRSV strains should be strengthened.In addition,it is urgent to develop a novel and efficient PRRSV vaccine.In this study,the ORF5 gene of HP-PRRSV was amplified,and the transfer vector p G-GP5 /HP-EGFP was successfully constructed by connecting with the PRV transfer vector p G stored in the laboratory through Bam H I locus.The ORF5 gene was recombined into rPRV-g E-/g I-/TK-deletion strains by homologous recombination technique.10 rounds of plaque purification were performed to obtain the recombinant virus rPRV-GP5/HP-EGFP successfully.The recombinant virus rPRV-GP5/HP was obtained by using CRISPR/Cas9-EGFP knockout plasmid to cut part of EGFP gene.RT-PCR,indirect immunofluorescence and Western blot verified that ORF5 gene was successfully transcribed and expressed.In addition,in order to construct recombinant porcine pseudorabies virus expressing both HP-PRRSV and NADC30-Like-PRRSV ORF5 genes.The NADC30-Like-PRRSV ORF5 gene was amplified and the eukaryotic expression vector p BApo-EF1α_Pur_DNA was inserted into the Bam H I and Hind III sites to construct p BA-GP5/NA.p G-GP5/HP-EGFP was digested by Bst Z17 I,and the p BA-GP5/NA vector expression box was amplified.The transfer vector p G-GP5/HPGP5/NA-EGFP was successfully constructed by seamless cloning technology.Then,with the help of CRISPR/Cas9-g G vector,two ORF5 genes were recombined into rPRV-g E-/g I-/TK-three gene deletion strains by homologous recombination technology.Then obtain recombinant virus rPRV-GP5/HP.Through RT-PCR,indirect immunofluorescence and Western blot test,it was preliminarily determined that the ORF5 gene of the two loci was simultaneously transcribed and expressed.The physicochemical characteristics,growth characteristics and genetic stability of the two recombinant viruses were analyzed in this study.The results showed that the culture characteristics of the recombinant virus were similar to that of the parent strain,indicating that the insertion of exogenous genes and the complex recombination process had no significant effect on the virus itself,and the two recombinant viruses had good genetic stability.In this study,three-week-old piglets with double negative PRV and PRRSV antigen and antibody were used as animal models to immunize piglets with recombinant viruses rPRV-GP5/HP and rPRV-GP5/HP-GP5/NA,and PRRSV vaccine control group and DMEM blank group were set up.The two recombinant viruses can stimulate piglets to produce high levels of PRV ELISA antibody and neutralizing antibody,and the immune effect is the same as commercial vaccine.However,only certain ELISA antibodies could be detected after immunizing piglets with the two recombinant viruses,and the antibody levels were slightly lower than those of commercial vaccines.No neutralizing antibody was detected.The reason for this is unclear,which may be related to the protein expression level or insufficient leakage of GP5 protein epitopes.Further research is needed to modify GP5 to stimulate the production of high levels of neutralizing antibodies in piglets.The results of challenge protection test showed that the two recombinant viruses could provide some protection against PRV and PRRSV virulent attack,and could effectively inhibit the proliferation of viruses in each organ.In summary,two recombinant viruses rPRV-GP5/HP and rPRV-GP5/HPGP5/NA were successfully obtained in this study,and their biological characteristics and immunogenicity were explored,providing a powerful tool for the prevention and control of PRRS and PR,and provide theoretical support for the research and development of new vaccines. |
