Molecula Epidemiology Investigation Of Porcine Circovirus Type 3 And Establishment Of TaqMan Fluorescent Quantitative PCR Detection Method

Porcine Circovirus Type 3(PCV3)belongs to Circovirus family,Circovirus genus,which can cause Porcine reproductive disorders,dermatitis nephropathy syndrome and heart polysystem inflammation.In 2015,Palinski et al.of Kansas state university detected a new porcine circovirus(porcine circovirus type 3)in an outbreak of PDNS(dermatitis and nephrotic syndrome)in north Carolina.PCV3 gene group leader was about 2.0kb,which was divided into two gene subsets,PCV3 a and PCV3 b,with three open reading frames:ORF1,ORF2 and ORF3,in which ORF1 and ORF2 encode virus replicase protein(Rep)and capsid protein(Cap protein)respectively.The result of Genetic evolution analysis indicated that PCV3 and PCV1,PCV2,circovirus and bat circovirus belong to the same evolutionary branch.At present,PCV3 is reported from time to time in some provinces and cities in China.Therefore,the prevalence and genetic variation of PCV3 in China remain to be studied.In this study,According to the PCV3-Hebei-LY＿2015 strain of porcine circovirus type3(Genebank login number:MF318451.1)the specific identification primers were designed,and the detection method of PCV3 conventional PCR was established.The results showed that the method was of high specificity,and swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV2),pig pseudo rabies virus(PRV),etc.No cross reaction,and the method has good repeatability.DNA extracted with 3 positive samples was repeated for 3 times and 9 times,and the results were all positive The results showed that this method can be applied to the clinical detection and diagnosis of PCV3.Using this method,454 suspected infectious agents from 64 large pigfarms in 15 provinces and cities including Henan,Shanxi,Jiangxi,Guangxi,Sichuan and Chongqing were investigated.The results showed that PCV3 was detected in samples from nine provinces and cities,with a positive rate of 31.7% and a positive rate of 28.1% in pig farms.Among them,the detection rates in Sichuan,Guangdong,Henan and other major pig breeding provinces were higher than 35%.In addition,PCV3 can be detected in the tissues and organs of pigs,in which the positive rate of PCV3 in the heart,lung,kidney and brain tissues is more than 30%.Therefore,PCV3 can proliferate in various organs of the pig body.In this study,16 Cap genes of positive pathogens from different provinces were sequenced.Molecular genetic evolution analysis and amino acid analysis of Cap proteins were performed with Cap genes of circoviruses at home and abroad.The results showed that 16 Cap genes of PCV3 had 98.0%～100% homology,respectively 97.6%～99.8%homology with other Cap protein genes of domestic PCV3,and 97.5%～99.8% homology with Cap gene sequences of foreign PCV3.The homology of Cap gene and circovirus was38.1%～38.3%.The homology of Cap gene and PCV1 was 32.8%～33.5%.The homology of Cap gene and PCV2 was 31.4%～32.1%,and the homology of Cap gene and bat circovirus was 40.7%～41.5%.The result indicated that PCV3 and bat circovirus were more closely related.The results of molecular genetic evolution analysis showed that 15 of 16 PCV3 belonged to PCV3a1 and 1 to PCV3 b,indicating that China’s PCV3 is mainly dominated by PCV3a1,accompanied by PCV3a2 and PCV3 b,which is basically consistent with the prevalence at home and abroad.Amino acid sequence analysis showed that the amino acid site mutation in Cap gene of PCV3 was mainly at 9～28,45～66,139～144 points.Basing on the PCV3-Hebei-LY＿2015 strain of porcine circovirus type 3(Genebank accession number: MF318451.1),we designed specific primers and probes,and established PCV3 Taq Man fluorescence quantitative PCR detection method.The experimental results showed that the method had high sensitivity and specificity,and had no cross reaction with CSFV,PRRSV,PCV2 and PRV.The results of clinical positive samples showed that the method had high sensitivity and it could be used for PCV3 clinical detection and its standard indicators of incidence.Through this study,we have grasped the epidemic situation and genetic variation ofPCV3 in China,and laid a solid theoretical foundation for the prevention and control of the epidemic and spread of PCV3 in China.Through the establishment of Taq Man fluorescent quantitative PCR detection method,we can provide a reliable instrument for the further development of PCV3 vaccine and pathogenesis research.