Establishment Of PCR Assay For Discrimination Variant Strain Of Pseudorabies Virus And Protective Efficacy Of Inactivated Pseudorabies Virus Deleted GE/gI Genes Vaccine

Pseudorabies,caused by Pseudorabies Virus(PRV),is an important infectious disease in swine.PRV infection is characterized by sows reproductive disorder and newborn piglets nervous system disorders and death.According to experience of eradicating PRV in the United States and Europe,Pseudorabies is well controlled by application of gE gene deleted vaccines in past 30years in China.However,the outbreak of Pseudorabies has caused devastating disease and economic losses in Chinese swine agriculture since the end of 2011.Several reports have proved that the virulence of PRV has increase greatly.In this study,after the serological epidemiological investigation,it was confirmed that Pseudorabies prevalent in East China.And a PCR assay for differentiation variant strains from classic strains of PRV was developed.And animal experiment results showed that new inactived variant PRV vaccine could provide protective efficacy against variant PRV challenge.It should be useful for the prevention and control of the disease.The contents of this study contain three parts as following:1.Epidemiological investigation and isolation of PRV in Mid-eastern ChinaIn order to understand the prevalence and infection of swine pseudorabies in Mid-eastern China,this study was carried out to the serological and pathogenic epidemiology investigation and the isolation of PRV epidemic strains.The results show that the gB antibody in serum maintained a high level in 2012-2015.The average positive rate reached 92.3%(7119/7716).Meanwhile,the positive rate of gE antibody increased from 0%in 2012 to 34.9%in 2014.And then it decreased to 31.6%in 2015.The average gE antibody positive rate was 30%.Among the four provinces of Jiangsu,Shandong,Zhejiang,Anhui,Zhejiang Province had the highest positive rate of 42.1%,and Anhui had the lowest of 24%.From 2012 to 2015,the rates of gE positive pig farms were 0%,40.8%,55.0%and 65.0%.And the average positive rate was 52.6%(162/308).And the rates of gE positive pig farms in 2014 in the 4 province were all 100%.The PRV PCR results showed that PRV infection rates were 14.1%,24.9%,28.9%in 2013,2014 and 2015.The average infection rate was 25.1%(121/481).Meanwhile,the positive rates of pig farms were 22.7%,36.5%and 40.5%in 2013,2014 and 2015.The average positive rate was 37.3%.Finally,4 PRV were isolated from PRV positive samples,and they are proved to be field strains.The data indicated that PRV widely spread in the region.And it provides a theoretical basis for the disease prevention and control.2.Establishment of PCR assay for differentiation variant strains from classic strains of porcine pseudorabies virusTo distinguish variant PRV strains from the classic,we developed 2-step PCR by designing 2 pairs of primes based on genome sequences alignment results of PRV variant and classic isolates.Variant PRV strains could be distinguished from classic strains except HB98 vaccine strain by using the primers based on UL44 gene sequences(1st PCR).PRV variant strain and HB98 could be differentiated further by using primers based on UL36.The detection sensitivity of 2-step PCR is 2-20 TCID50 and 50TCID50 PRV load.Meanwhile,no band is amplified by this method from the classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine circovirus type 2,Japanese encephalitis virus,encephalomyocarditis virus and porcine epidemic diarrhea virus cultural samples.The coincidence rate between this method and national standard PCR is 91%.This method can be used for rapid diagnosis of PRV in laboratory.3.Protective efficacy of inactivated PRV deleted gE/gI Genes vaccineTo evaluate the immune effects of inactivated PRV ZJ01 gE/gl deleted strain vaccine,rZJO1△gE/gl was inactivated by formalin and 3 inactivated vaccines were prepared by homogenized with adjuvants.Mice experiment results showed that rZJ01/F/15A vaccine could provide high immune responses.At three weeks after second vaccination,the mice in rZJ01/F/15A group had high levels of anti-gB antibody,and the neutralizing antibody titer was up to 1:158.Piglets experiment results showed that the anti-gB and neutralizing antibodies were highest in rZJ01/F/206 group at 3rd week post primary immunity.At 2nd week post secondary immunity,the neutralizing antibody titer was up to 1:256.After challenge with ZJ01 strain,the pigs in un-vaccinated group exhibited significantly clinical signs.But the pigs in rZJ01/F/206 group only had a transient rise in rectal temperature.And the virus loads in lung,liver and brain in in rZJ01/F/206 group were the lowest,compared with those in other groups.And the gross and histological lesions in rZJ01/F/206 group were also slight.It indicated that rZJ01/F/206 could provide protective effect against PRV ZJ01 challenge.It should be an ideal vaccine candidate for preventing this reemerged disease.