The Preparation And Immune Protective Efficacy Of Novel Bivalent Vaccines Of Porcine Reproductive And Respiratory Syndrome Virus And Porcine Circovirus Type2

Porcine reproductive and respiratory syndrome virus(PRRSV)was an important pathogen that caused huge economic losses to the world pig industry.Typical clinical symptoms of PRRS are severe respiratory disease in infected newborn and growing pigs and reproductive failure in pregnant sows.Porcine circovirus type 2(PCV2)was considered to be the main pathogen resulting in post weaning multisystem wasting syndrome(PWMS).It's common that co-infection of PCV2 and PRRSV exists in the field.In this study,we successfully established co-infection model of PCV2 and PRRSV in piglets,then we prepared novel PCV2-PRRSV bivalent vaccines and analyzed their immune protective efficacy in pigs based on this model.The contents of this paper are including:1.Establishment of co-infection model of PRRSV and PCV2 in conventional pigsIn this study,nineteen conventional 5-week-old pigs free of PRRSV and PCV2 were randomly divided into four groups:pigs co-infected with PRRSV and PCV2(n=5);pigs infected with PRRSV alone;pigs infected with PCV2(n=5)alone;negative control(n=4),and observed for 21 days.The results showed that pigs co-infected with PRRSV and PCV2 showed obvious clinical symptoms,including growth retardation,severe respiratory distress,and two of the lesions included severe proliferative interstitial pneumonia in lungs and lymphoid depletion,necrotizing hepatitis,mild necrotizing bronchiolitis died at 19-20 dpi.The main microscopic lesions included severe proliferative interstitial pneumonia in lungs and lymphoid depletion,necrotizing hepatitis,mild necrotizing bronchiolitis and infiltrates of macrophages in lymphoid.In contrast,the pigs infected with PRRSV or PCV2 alone showed moderate gross lesions and histopathological changes of lungs and lymph nodes.Meanwhile,the loads of PRRSV and PCV2 in the pigs doubly inoculated were higher than those inoculated with PRRSV or PCV2 alone.This co-infection model should be very usefully to study on the pathogenesis of PRRSV and PCV2 and development of new vaccines against these diseases in the future.2.Construction and identification of recombinant baculovirus expressing GP5 and M proteins of PRRSVThe ORF6 gene and ORF5 gene(wild type or modified)of PRRSV were obtained and separately cloned into pFastBac donor plasmid or cloned into different promoters of the same donor plasmid.After identification by PCR,double enzymes digestion and sequencing analysis,the recombinant donor plasmids were all transformed into DH10Bac cells,and then the recombinant bacmids were screened by blue-white plaque assay twice.The correct recombinant bacmids were prepared and transfected into Sf9 insect cells,and then recombinant baculoviruses were obtained and confirmed by Western blot and indirect immunofluorescene assay(IFA).The results showed that PRRSV GP5 and M proteins were successfully expressed separately or in the fused way(GP5-M)by using the baculovirus expression vector system(BEVS)platform.This study laid the foundation for the development of PRRSV gene engineering subunit vaccines.3.Immune protective efficacy of PRRSV and PCV2 bivalent vaccines in pigletsIn this study,twenty 3-week-old pigs free of PRRSV and PCV2 were randomly divided into four groups:pigs concurrently vaccinated with a PRRSV recombinant GP5+M vaccine and a PCV2 recombinant Cap vaccine(group GP5+M+Cap);pigs concurrently vaccinated with a killed PRRSV vaccine and the recombinant PCV2 Cap vaccine as group GP5+M+Cap(group PRRSV+Cap);challenge control and negative control.After immunization twice,pigs were co-infected with PCV2 and PRRSV,except the negative control.The results showed that the group GP5+M+Cap developed higher specific anti-GP5 ELISA antibody and neutral antibody than group PRRSV+Cap,while they both developed similar titers of specific anti-Cap ELISA antibody and neutral antibody.After challenge,the vaccinations reduced mortality and improved RDWG compared with challenge control.Pigs in GP5+M+Cap group showed lower PCV2 and PRRSV viral load and viraemia than other groups,as well as reduced gross lesions and hisopathological changes of lung and lymph node.Therefore,it was indicted that the concurrently use of the PRRSV subunit vaccine and the PCV2 subunit vaccine may develop a potential bivalent vaccine against both PCV2 and PRRSV.