| Porcine circovirus type 3(PCV3)is a single-stranded and non-encapsulated circular DNA virus,which was discovered and reported by American scholars in 2016.PCV3 was firstly prevalent in the United States,and the virus is susceptible to various stages of of pigs,especially for piglets.Since its first report in 2016,it has appeared worldwide.In 2017,Prof.He Qigai from Huazhong Agricultural University of China reported the first case of PCV3 infection in China.The clinical symptoms of PCV3 were extremely similar to those of Porcine circovirus type 2(PCV2),which was characterized by Porcine dermatitis and nephrotic syndrome(PDNS).There are no commercially available vaccines and diagnostic kits on the market,so establishing a fast and accurate differential diagnosis method is essential for the prevention and control of the disease.A pair of specific primers were designed based on the conserved sequences of PCV2 and PCV3,and a dual PCR detection method for detecting both PCV2 and PCV3 was established.By optimizing the reaction conditions and reaction system,the optimal annealing temperature was 56.0℃,and the optimal primer concentration was 0.2 μmol/L.This experiment successfully established a dual PCR method for detecting PCV2 and PCV3,which provided a good method for clinical diagnosis of porcine circovirus and lays a foundation for the development of diagnostic kits.The epidemiological investigations of PCV3 was conducted by PCR,and the samples of suspected PCV3 in different regions of China(Guangdong,Guangxi,Hainan,Jiangxi,Jiangsu,Hunan,Hubei,Henan,Shanxi,Heilongjiang)were collected from 2016 to 2018.A total of 1189 samples of different stages and types were collected from 86 pig farms.The test results showed that the positive rate of mixed samples of oral,nasal and faecal swabs46.19%(291/630),the seroprevalence rate was 6.48%(16/247),and the positive rate of tissues and organs was 27.24%(85/312).The results of fecal and oral secretions from pigs from three PCV3 positive farms showed that the positive rate of suckling pigs(1-20 days old)was 59.17%(71/120);nursery pigs(21-60 days old)was 56.67%(51/90);growth and fattening pigs was 32.22%(29/90);gilts was 23.33%(21/90);pregnant sows was 54.44%(49/90);lactating sows was 60%(54/90);and boars was 26.67%(16/60).A total of 7 full-length sequences of PCV3 were screened and obtained in this study.The homology analysis showed the following results: The nucleotide similarity between the7 strains and Korean strains,as well as other Chinese strains were as high as 99.7%,but there was large difference in homology with sequences of PCV2.According to the result of genetic evolutionary analysis: these isolates were closely related to other Chinese PCV3 isolates discovered and identified in recent years,and they belonged to the same branch.A pair of specific primers were designed based on the conserved sequence of the PCV3/GD/08/16 isolate,and the Bam HI and HInd III restriction sites were added to the upstream and downstream primers.The successfully identified Cap gene was ligated into the p MAL-c5 x expression vector,thus the recombinant expression vector PCV3-Cap-p MAL was successfully constructed and then induced to express.The size of the recombinant fusion protein,which existed in the form of inclusion bodies,was determined by SDS-PAGE electrophoresis to be about 70 k D.Furthermore,IPTG was used to used to determin the induce concentration,concentration,temperature and time,the optimal IPTG concentration was 0.5 m M,the optimal induction temperature was 37℃,and the optimal induction time was 6 hours.The recombinant protein was subjected to Western blot using the optimal induction conditions determined above.The results showed that the PCV3-Cap-p MAL expression vector was successfully constructed,which would lay a foundation for the construction of ELISA method in the next step. |
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