The Study On Complete Genomic Sequence And Real-time Quantitative PCR Detection Of Porcine Circovirus 3 And Prokaryotic Expression Of Cap Protein

Porcine circovirus 3(PCV3)was firstly discovered in the United States in 2016.Since then,PCV3 has been reported in China,South Korea,Poland,Italy,the United Kingdom,Brazil,Thailand,Denmark,Germany and Spain.Diseases associated with PCV3included porcine dermatitis and nephrotic syndrome,reproductive failure,cardiac and multi-systemic inflammation and piglet diarrhea,which were extremely similar to the clinical symptoms of Porcine circovirus 2(PCV2)associated diseases that brings difficulty to the clinical diagnosis of PCV3.As a new emerging pathogeon,the impact on the pig industry of PCV3 is difficult to estimate.In this study,PCV3 was firstly identified from piglets with congenital tremor(CT).A total of 403 samples including 203 from piglets with CT in 7 farms and 200 from sows with other clinical symptoms in 8 farms were collected from Guangdong and Guangxi Provinces between 2016 and 2017.Through the the traditional PCR,PCV3 was detected positively at the rate of 17.87%in all samples,26.60%in piglets and 9.00%in sows.Epidemiological investigations of 7 PCV3-positive farms with CT indicated that the farrowing rate of CT piglets in primiparous sows was 20-50%and the incidence of CT in neonatal pigs and malformed piglets was 2.00-6.05%and 48.58-79.10%,respectively.Several interactive pathogens such as Atypical porcine pestivirus(APPV),Porcine deltacoronavirus(PDCoV),Porcine epidemic diarrhoea virus(PEDV),Porcine kobuvirus(PKV),Porcine reproductive and respiratory syndrome Virus(PRRSV),Porcine pseudorabies virus(PRV)and Porcine sapelovirus(PSV)were detected in CT-piglets.Moreover,we found that only PCV3 were examined in the farm named GDLC1.15 PCV3 genomes including 7 from CT-piglets and 8 from sows were acquired in our study.Phylogenetic analysis demonstrated that the PCV3 genome was highly conserved since the similarities between 93 PCV3 genomes were 97.3-100%and divided into three evolutionary branches.Compared with other Circoviruses,the evolutionary branch of KJ641716 strain which belongs to Bat circovirus(BtCV)was the closest branch to PCV3.The similarities between all of the nucleotide sequences of PCV3 capsid protein(Cap)and replication protein(Rep)were 98.1-100%and 98.9-100%,respectively.The predicted signal peptide sequence of PCV3 Cap was 1-36 amino acids.A pair of specific primers aimed the conserved regions of the PCV3 Rep gene was designed to develope the SYBR Green-based real-time qPCR assay.The positive standard of PCV3 was prepared by gene cloning and a standard curve with good linearity was obtained.The assay was developed for the rapid and reliable diagnosis of PCV3 infection in pigs and had a good specificity with no cross-amplification reactions between common pathogens such as Foot-and-mouth disease virus(FMDV),Swine vesicular disease virus(SVDV),Senecavirus A(SVA),Porcine bocavirus(PBoV),Classical swine fever virus(CSFV),Porcine parvovirus(PPV),Swine influenza virus(SIV),PEDV,PKV,PSV,PDCoV,PRV,PRRSV and PCV2.The method had a good sensitivity with the limit of detection of PCV3 was 1.73×10~2 copies/?L and also possessed a good repeatability with both coefficients of variations(CVs)of intragroup and intergroup were less than 2.27%.The detection rate of PCV3 with this real-time quantitative PCR was 86.7%(176/203),much higher than that 26.6%(54/203)of the traditional PCR.According to the prediction of signal peptides of PCV3 Cap,a pair of primers was also designed to amplify the truncated sequence of PCV3 Cap gene that was inserted into the pET32a.The recombinant plasmid named pET32a-PCV3 Cap was transformed into E.coli BL21(DE3).The PCV3 Cap fusion protein with a molecular weight about 38 kDa was induced successfully.SDS-PAGE analysis testified that the optimal time of induction of PCV3 Cap fusion protein which expressed as insoluble inclusion body was 4 h.The PCV3Cap fusion protein was purified by using the Ni-sepharose purification and the purity was used as the immunogen to immunize mice.Eight hybridoma cell lines that secreted the antibody of PCV3 Cap protein were obtained.The potencies of the positive hybridoma cell lines named A8F and C11C were both greater than 1:64,000.The results of Western Blot confirmed that PCV3 Cap specifically reacts with the monoclonal antibody which indicated that the recombinant protein had good antigenicity.This study provides a good technical support and theoretical basis for future controls of PCV3 that has a significant meaning for pig productions.