Isolation And Identification Of Porcine Circovirus Type 2 And Evaluation Of Immune Efficacy Of Six PCV2 Commercial Vaccines

Porcine circovirus disease（PCVD）is a general term for a variety of diseases in pigs caused by Porcine circovirus type 2（PCV2）,which can lead to immunosuppression in pigs and can cause severe developmental delays in pigs,which causes huge economic losses to the pig industry.Pigs are the main host of porcine circovirus type 2.It has also been shown that PCV2 can replicate and spread among mice,presenting an invisible infection.Vaccination is currently the main measure to prevent and control porcine circovirus disease.The types of PCV2 commercial vaccines include whole virus inactivated vaccines,subunit vaccines,chimeric vaccines,etc.In this study,the immunization effect of different types of PCV2 vaccines was evaluated by mouse animal models.1.Isolation and identification of Porcine circovirus type 2In this study,we used porcine kidney cells（PK15 cells）to isolate the virus from the kidney tissue of pigs suspected of PMWS in a pig farm in Jiangxi,and identified the isolate as porcine circovirus type 2 by PCR and whole-genome sequencing,named PCV2-ZQP strain.The whole genome sequencing results showed that the whole genome of this strain was 1768 bp in length,ORF1 gene was 945 bp in length and ORF2 gene was 702 bp in length.The homology and genetic evolution analysis with different genotypes of PCV2 strains included in Gen Bank showed that the isolate belonged to PCV2d type,and the homology with the whole genome nucleotide sequence of PCV2 reference strain ranged from 92.8%to 99.9%;the homology with the nucleotide sequence of ORF1 gene of PCV2 reference strain ranged from 97.1%to100%;the homology with the nucleotide sequence of ORF2 gene of PCV2 reference strain ranged from 84.3%to 99.6%.2.Establishment of a real-time fluorescent quantitative PCR assay for Porcine circovirus type 2 TaqMan probesIn this study,we designed and synthesized specific primers and probes based on the ORF1 gene sequence of porcine circovirus type 2（accession number:HM038017.1）in Gen Bank,screened the primers by SYBR Green I dye method and TaqMan probe method,and optimized the reaction conditions for fluorescence quantitative PCR.A real-time quantitative PCR detection method for PCV2 TaqMan probe was successfully established.The results showed that the linear relationship between the template concentration and the threshold cycle number（Ct value）was good when the template concentration was 7×10²～7×10⁷ copies/μL;the minimum detection amount of the method was 7×10⁰ copies/μL;the method had no specific amplification with PRV,Po RV,PRRSV and PEDV;the intra-group coefficient of variation and inter-group coefficient of variation of the method were less than 2%,and the reproducibility was good.The fluorescence quantitative PCR method established in this study can be used for the quantitative detection of PCV2 nucleic acid content.3.Evaluation of immune efficacy of six PCV2 commercial vaccinesBased on the fact that PCV2 is recessively infected in mice,this study was conducted to evaluate the immunization effect of six PCV2 commercial vaccines（named Vaccine A～Vaccine F）using Kunming mice as an animal model for PCV2infection.Female Kunming mice aged 6-8 weeks were immunized with different types of PCV2 vaccine,and each mouse was injected with 0.03 head parts（15μL～60μL）by intramuscular injection into the leg.Blood was collected weekly after immunization and serum levels of PCV2-specific antibodies were measured.The results showed that specific antibodies could be detected 2 weeks after vaccine B immunization,which could not be detected by other groups of vaccines,reached the peak of immunization at week 4,and maintained high antibody levels until the end of the experiment,which was significantly better than other groups of vaccines.The antibody levels after immunization of mice with six different PCV2 commercial vaccines were ranked as Vaccine B>Vaccine C>Vaccine D>Vaccine E>Vaccine F>Vaccine A.Four weeks after immunization of mice with PCV2 commercial vaccine,the PCV2-ZQP strain was used as the virulent strain for immune attack test.The viral solution was injected intraperitoneally at an attack dose of 1×10¹¹ copies/each,and the mice were slaughtered 15 days after infection,and the PCV2 viral load in the lungs of mice was determined by the PCV2 fluorescence quantitative PCR method established in this study to compare the immune protective effects of different vaccines.The results showed that the PCV2 viral load was 1.02×10⁷ copies/0.1g of lung after unimmunized attack mice.The PCV2 viral load after vaccine A to vaccine F immunization of attacked mice was 2.88×10⁶ copies/0.1g lung,2.72×10⁶ copies/0.1g lung,3.5×10⁶ copies/0.1g lung,7.89×10⁶ copies/0.1g lung,4.05×10⁶ copies/0.1g lung,4.31×10⁶ copies/0.1g lung,with vaccine B providing the best immune protection.The immunoprotective effects of the six different PCV2 commercial vaccines were ranked as Vaccine B>Vaccine A>Vaccine C>Vaccine E>Vaccine F>Vaccine D.