Establishment Of An ELISA For Detecting PCV3 Antibodies And Preliminary Epidemiological Investigation Of PCV3 In Hunan Province

Porcine circovirus(PCV3)is a new type of circovirus.Nowadays,PCV3 was found in the US,China,the UK,and other countries.Clinically,PCV3 is considered to associate with Porcine Dermatitis Nephropathy Syndrome(PDNS),congenital tremors of piglet,sow reproduction disorder,and multi-system inflammation.It poses potential threats to the pig-breeding industry and needs to be highly valued.To comprehensively understand the PCV3 infection in pig herds in Hunan Province and help pig farms to develop comprehensive prevention and control measures against the background of African swine fever,it is necessary to establish a simple,rapid,and easy-to-high-throughput ELISA method to detect PCV3 antibody.At the same time,the PCV3 epidemiological investigation was carried out in Hunan Province.In the study,the gene of Cap protein without nuclear localization sequence(NLS)of PCV3 was cloned into p ET28-a(+)and expressed in Escherichia coli BL21(DE3).The recombinant d Cap protein was purified by Ni-IDA affinity chromatography and used as a coating antigen to establish an indirect ELISA for detection of antibodies to PCV3.To investigate the antibody status of PCV3 in pigs,1175 clinical serum samples collected from 11 cities in Hunan province were tested.The pathogen was detected by q PCR,and the whole genome sequences of PCV3 in 11 prefecture-level cities in Hunan Province were isolated by PCR,and the sequences were analyzed.The results showed that the optimal working conditions for ELISA were 0.2μg/m L of the recombinant d Cap protein diluted in the carbonate buffer with p H=10.83,combined with 1: 8,000 HRP-conjugated goat anti-swine Ig G and 1:50 dilution of serum sample.The threshold of negative-positive cutoff S/P value for defining positive samples was set to be 0.270.The difference between interassay and intraassay was less than 10%.The method had no cross-reaction with antibodies to PCV2,CSFV,PRV,and PRRSV.The results of seroepidemiological investigation of PCV3 in Hunan Province showed that the positive rate of pig farms was 100%(29/29)and the positive rate of serum samples was 58.64%(689/1175).PCV3 infection is common in fattening pigs.The S / P values of serum antibodies in pigs ranged from 0.270 to 2.0,but more than 70 % of the samples were below 0.4,and the antibody levels were not high.In this study,the complete genome sequences of a total of 11 strains of PCV3 were obtained.The homology ranges of the whole genome sequence,Rep protein and Cap protein amino acid sequences of the strains in Hunan Province were 98.6 % ～100.0 %,98.7 % ～ 100.0 % and 96.3 % ～ 100.0 %,and the homology ranges of the strains with domestic and foreign viruses were 97.9 % ～ 99.7 %,98.3 % ～ 100.0 %and 95.3 % ～ 100.0 %.The results showed that the indirect ELISA method established in this study had good sensitivity and specificity.It can be used for PCV3 antibody detection.Through serum and molecular epidemiological investigation,PCV3 infection was common in pigs in Hunan Province,and the homology of PCV3 strains in various regions was high.This study provides a scientific basis for the epidemiological study of PCV3 in Hunan Province.