Development Of A Real-time PCR Assay For Detection Of Porcine Circovirus 4 And Epidemiological Investigation Of Porcine Circovirus Disease In Some Areas Of Hebei

Porcine circovirus(PCV)is a circular,single-stranded,non-enveloped DNA virus that can be separated into four main types.PCV1 is generally considered non-pathogenic,and PCV2 and PCV3 are associated with multiple system failure syndromes in weaned piglets.,Porcine dermatitis is related to a variety of diseases such as nephrotic syndrome.In 2019,PCV4 was discovered in a diseased pig herd in Hunan Province,China.In order to understand the prevalence of PCV in pig herds in Hebei Province,this study established a PCV4 real-time fluorescent PCR method,carried out molecular epidemiological investigations on PCV in pig herds in Hebei Province,and carried out genetic evolution of the PCV4 epidemic strain analysis.1.One pair of specific primers and Taq Man probe were designed based on the conserved sequence of ORF2 gene of PCV4 available in Gen Bank.Through the optimization of the reaction system,a real-time PCR assay for detection of PCV4 was successfully developed.The results showed that the Ct values remained a good linear relationship with the standard plasmids within the range of 1.53×10~7～1.53×10~3copies,R2 was 0.999.The developed real-time PCR demonstrated good specificity,in which only PCV4 could be amplified and no amplifications were observed with PCV1,PCV2,PCV3 and other common swine pathogens used in this study.The sensitivity was high with the limit of detection of 1.53×10~1copies/reaction.The assay also showed good repeatability with the coefficient variations of intra-assay and inter-assay less than 2%.2.The 960 samples from 11 regions of Hebei Province from 2013 to 2020 were tested for PCV by real-time PCR.The results showed that the positive rates of PCV2,PCV3 and PCV4 were 85.52%,25.52%and 1.15%,respectively,and there were mixed infections.The positive rate of PCV is different at different times.The positive rate of PCV2 decreased year by year from 2013 to 2020;the detection rate of PCV3in 2017 was as high as 86.79%;PCV4 was only detected in 2019,with a positive rate of 2.83%.The positive rates of PCV in different regions are different.The positive rates of PCV2 in all regions are above 70%.PCV4 was only detected in samples from Hengshui and Shijiazhuang,and the positive rates were 6.67%and 1.33%,respectively.The positive rates of PCV in different types of samples are different.PCV2 and PCV3 have the highest positive rates in lymph node/spleen/lung samples,99.58%and 80.33%,respectively;PCV4 has the highest positive rate in lymph nodes,which is 2.13%.3.Select part of the PCV4 positive disease materials,amplify the PCV4 ORF2gene and the whole genome sequence,and perform genetic evolution analysis after sequencing.A total of 3 PCV4 ORF2 gene sequences(Hebei-AP15-2019,Hebei-AP67-2019 and Hebei-LS1-2019)and 1 Hebei-AP-67-2019 genome sequence were obtained and uploaded to Gen Bank(accession numbers are MW439193,MT995847,MW439192 and MW084633).The Hebei strain shares a small branch with PCV4 strains from other provinces in my country and is closely related;the nucleotide and deduced amino acid homology of the ORF2 gene sequence to the 11domestic representative strains in Gen Bank is 98.5%～99.6%and 98.7%～100%respectively;the nucleotide homology between the whole genome sequence and the7 representative strains is 98.8%～99.5%,which is closely related to the representative strains.