Investigation Of PCV3 Infection In Small And Medium-sized Aquaculture Farms In Some Areas Of Sichuan,Chongqing,and Guizhou From 2020 To 2022 And Establishment Of An IELISA Antibody Detection Method

Porcine circovirus 3（PCV3）is a circular envelope free DNA virus with a genome of 2000 nt.Since its first discovery in 2016,its prevalence and harmfulness have been of great concern.Currently,it has been found and confirmed in several countries and regions for a long time.In order to understand the prevalence of PCV3in southwest China,especially in small and medium-sized farms,this experiment first established a dual PCR differential diagnosis method for PCV3 and PCV2,detected855 clinical samples from some small and medium-sized farms in Sichuan,Guizhou,and Chongqing from 2020-2022,analyzed their infection status,and further revealed the molecular genetic evolution characteristics by determining the entire genome of PCV3 in positive samples;Then,the main structure and immunogenic protein（Cap）of PCV3 virus were obtained by prokaryotic expression,and an indirect ELISA method was established to detect its antibodies.Serological methods were initially used to further understand the situation of PCV3 infection in three provinces（cities）.The main results of this test are as follows:1.Establishment of PCV3 and PCV2 dual differential PCR method and detecti on and analysis of clinical samples.The clinical symptoms caused by PCV3 and PCV2 infection are similar and prone to mixed infection.In order to facilitate differentiation,this experiment first established a rapid,accurate,and highly specific dual PCR detection method to achieve the differential diagnosis of PCV3 and PCV2.The sensitivity of the established dual methods was,respectively 3.39×10³copies/μL、6.63×10⁴copies/μ.It has no cross reaction with other common pig pathogens such as classical swine fever,and can be used for clinical detection.In order to understand the prevalence of PCV3infection in small and medium-sized pig farms in southwest China,this experiment used the established dual PCR method to detect clinical samples collected from small and medium-sized pig farms in parts of Sichuan,Chongqing,and Guizhou from2020-2022.It was found that the positive rate in Sichuan Province was 1.15%（3/260）,Chongqing City 2.65%（8/302）,Guizhou Province 0.34%（1/293）,and the total positive rate in the three provinces（cities）was 1.4%（12/855）.The analysis found that the prevalence of PCV3 in clinical samples from three provinces（cities）was higher in summer,and most of them were co infected with PCV2.2.Determination and analysis of the whole genome of PCV3In order to further understand the genetic evolution of PCV3 in clinical samples from three regions,the entire genome of 12 positive PCV3 samples was amplified and sequenced.The complete sequences of 12 strains were spliced and typed.At the same time,DNA Star and MEGA software were used to construct a homology matrix and phylogenetic evolution tree using the neighbor method.The results showed that the main type of PCV3 obtained was 3a（7 strains）,followed by 3b（3 strains）and 3c（2strains）.Homologous evolution analysis showed that the nucleotide sequence of the whole genome and bat circovirus had high homology,low genetic variation with PCV3 from pigs in various provinces in China,and high homology with PCV3 from other animals;Both Rep protein and Cap protein have high similarity with waterfowl circovirus,and have high sequence homology with domestic reference strains and other animal derived Rep and Cap proteins.3.Prokaryotic expression and identification of PCV3 Cap proteinAs a structural protein of circovirus,Cap protein has good immunogenicity.Therefore,this experiment carried out prokaryotic expression of the obtained Cap protein of PCV3.First,through the bioinformatics analysis of PCV3 Cap,the optimal fragment was selected and the recombinant plasmid was constructed using p ET32a vector.Then,p ET32a-Cap was prokaryotically expressed in Rosetta host bacteria,which can enhance the expression of rare codons.The optimal expression condition was to induce IPTG with a final concentration of 0.5m M/L at 37℃for 4h.After Western blot verification and nickel column purification,it was confirmed that the recombinant protein p ET32a-Cap was successfully expressed and purified,and could react with clinically positive serum.4.Establishment and preliminary application of an indirect ELISA method for detecting antibodies based on PCV3 Cap proteinPurified and refolded p ET32a-Cap protein was used as an antigen,and an indirect ELISA method for detecting serum PCV3 antibody was established by optimizing the conditions.The optimal antigen concentration was 1μg/m L,the blocking solution is 5%skimmed milk powder,the serum to be tested is diluted 10times,the enzyme labeled second antibody sheep anti pig Ig G-HRP is diluted 1:5000,and the TMB color reaction is used.The judgment standard is that when the OD_450nmabsorbance value S/P value is greater than 0.224,it is positive.The coefficient of variation of this method is less than 10%,and there is no cross reaction with antibody sera such as CSFV,PCV2,PRRSV,and PRV.The specificity of this method is good.Preliminary application found that the positive rate of PCV3 antibody in three farms in Sichuan Province was 72.1%（158/219）;81.9%（149/182）of the three farms in Chongqing;57.1%（97/170）of the five aquaculture farms in Guizhou Province,and71.4%（404/571）of the total positive rate in the three provinces（cities）.In summary,this experiment has established a dual differential PCR detection method for the differential detection of PCV3 and PCV2,which has enriched the PCV3 infection data of small and medium-sized farms in Sichuan,Guizhou,and Chongqing in recent years;An indirect ELISA method for PCV3 antibody detection was established using PCV3 recombinant Cap protein to further understand the infection status of PCV3 from a serological perspective,providing technical support for the prevention and control of PCV3.