Molecular Epidemiological Investigation And The Immunogenicity Of Recombinant Adenovirus Vaccines Of PRRSV And PCV2

Porcine reproductive and respiratory syndrome virus(PRRSV)has become one of the most prevalent swine diseases in China,particularly after the unparalleled outbreaks of HP-PRRSV since 2006.While NA-type PRRSV strains have previously been detected in China,the EU-type PRRSV has also been several reported since 2011.PCV2 is a small,circular,non-enveloped DNA virus.PCV2 can be further divided into four subtypes PCV2a,PCV2b,PCV2c,PCV2d.PCV2 is the main pathogenic factor cause of porcine circovirus-related disease(PCVAD).PRRSV and PCV2 are the most common co-infection,suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality.Coinfection with PRRSV and PCV2 have resulted in up-regulation of PCV2 replication and increased severity of PRRSV-induced lesions in lung tissues.To investigate the epidemiological and genetic variability of PRRSV and PCV2 in Guangxi from 2015 to 2016.The recombinant adenovirus vaccines against PRRSV and PCV2 were constructed,which laid the foundation for the prevention and control of these diseases.The main results were as follows:(1)The molecular epidemiological investigation of PRRSV and PCV2 in Guangxi in 2015-2016,The positive rates of PRRSV and PCV2 were 63%-90%and 85%-93%,respectively.The positive rate of PRRSV antibody continued to decline from 2014 to 2016 in Yulin City.Herd immunity unevenness are more likely to cause the new virus mutation,causing a huge obstacle to prevent and eliminate of these diseases.Seven PRRSV strains belonged to two different subgroups highly pathogenic PRRSV strains.The GXB11-2015 isolate lacked 150 bases with 2 discontinuous missing bases,resulting the amino acid deletion type 1+20+29.In this study,27 PCV2 strains were sequenced in Guangxi province.The results showed that PCV2d could be the main genotype.According to the recombinant analysis,three gene recombination phenomenawere found.Screening stress analysis showed that ORF2 was more susceptible to positive screening.(2)In this study,the ORF3,ORF5 gene of highly pathogenic PRRSV GD strain,European type LV strain,classic PRRSV and PCV2 ORF2 gene were cloned into the RAPAd(?)universal adenovirus expression vector system.The recombinant plasmid and shuttle plasmid pacAd5 9.2-100 were co-transfected into HEK293 cells,and the recombinant adenovirus rAd-NAORF3-ORF5-IL18,European PRRSV and American PRRSV the recombinant adenovirus rAd-EUORF3-ORF5-NAORF3-ORF5,American type PRRSV and PCV2 recombinant adenovirus rAd-ORF2-NAORF3-ORF5,European type PRRSV and PCV2 recombinant adenovirus rAd-ORF2-EUORF3-ORF5 and rAd-NAORF3-ORF5-IL18 and rAd-EUORF3-ORF5-IL18 containing cytokines18.The recombinant adenovirus were identified by western-botting and IFA.(3)Mice immunized with the recombinant adenovirus vaccines were showed that ORF5 and ORF3 had immunological synergistic effect,which could promote the cellular immunity and humoral immunity.IL18 as a cytokine adjuvant can promote the immune effect of rAd-NA-ORF3-ORF5-IL18,rAd-EU-ORF3-ORF5-IL18,rAd-ORF2-IL18.The rAd-ORF2-NA-ORF3-ORF5,rAd-ORF2-EU-ORF3-ORF5 can enhance the immune response of Thl and Th2 cells.(4)Pigs immunization results indicate that the recombinant adenovirus induced humoral immunity and cellular immune response.The level of rAd-EU-ORF3-ORF 5-NA-ORF3-ORF 5 in CD4+T lymphocytes was significantly higher than the control group1.46 times(P<0.05).The viral load in the blood of the wild immunized group was 3.5 times higher than that of rAd-NA-ORF3-ORF5-EU-ORF3-ORF5.The recombinant adenovirus vaccines provided effective immunoprotection after challenge.In summary,the PRRSV and PCV2 strains in China continue to undergo new changes.The invasion of PRRSV exotic strains and PCV2 genotype drift have posesd a huge challenge to the prevention and control.This study enriched the current epidemiological knowledge of PRRSV and PCV2.The recombinant adenovirus against EU-type/NA-type PRRSV and PCV2 induced specific humoral and cellular immunity in mice and pigs.The challenge of PRRSV and PCV2 protection improved the piglets protection,these results of recombinant adenovirus of PRRSV and PCV2 provided the foundation for genetic engineer vaccines.