Molecular Epidemiology And Serological Analysis Of Porcine Circovirus Type 2 In Henan Province From 2017 To 2021

Porcine circovirus（PCV）,a member of the genus Circovirus within the family Circoviridae,is a small,non-enveloped,hemagglutination-inactive,single-stranded circular DNA virus.PCV is divided into four types: porcine circovirus type 1（PCV1）,porcine circovirus type 2（PCV2）,porcine circovirus type 3（PCV3）,and porcine circovirus type 4（PCV4）.PCV1 was originally discovered in the contaminant of the porcine kidney cell line（PK-15）in 1974,which is not pathogenic.Unlike PCV1,PCV2 can cause various clinical symptoms,such as multisystem wasting syndrome（PMWS）in weaned piglets,porcine dermatitis and nephrotic syndrome（PDNS）,porcine respiratory disease syndrome（PRDC）,congenital tremor（CT）in piglets and porcine reproductive failure,and often co-infects with other pathogens to cause immunosuppression.It is recognized worldwide as an important immunosuppressive pathogen endangering the pig industry.At present,pig herds in many countries and regions in the world are still deeply affected by PCV2,with a high infection rate and incidence.Henan is a large animal husbandry province,and its pig breeding industry plays an important role in animal husbandry.The persistent infection of porcine circovirus seriously restricts the healthy development of pig industry.There are few epidemiological analyses on PCV2 in Henan Province.In this study,the etiology and serology of PCV2 in Henan Province from 2017 to 2021 were analyzed,and the Cap protein of PCV2 was expressed in prokaryotic cells,laying a foundation for the study of ELISA antibody kits in the future.1.Etiological analysis of porcine circovirus type 2 in Henan ProvinceIn order to understand the molecular epidemiology and evolutionary characteristics of porcine circovirus type 2（PCV2）in Henan from 2017 to 2020,a total of 401 clinical samples were collected from different large-scale pig farms in Henan for PCR detection,and the complete genome was amplified from the positive samples for homology and genetic evolution analysis of the sequences.The results showed that among 401 clinical samples,the positive rate of PCV2 was 39.90%（160/401）,of which the positive rates from2017 to 2020 were 58.65%（61/104）,49.48%（48/97）,28.57%（36/126）and 20.27%（15/74）,respectively.Sequence analysis of the 24 positive samples showed that the similarity of the tested strains ranged from 95.90% to 99.80%,and the similarity with the reference sequence ranged from 95.10% to 99.80%.According to the genetic evolution analysis,24 isolates were in two clades,PCV2b（8/24）and PCV2d（16/24）,indicating that PCV2 d gradually replaced PCV2 b as a new dominant genotype in Henan.This topic reveals the prevalence of PCV2 in Henan,providing some reference value for the selection and breeding of PCV2 vaccine strains and vaccine research and development in Henan.2.Serological analysis of porcine circovirus type 2 in Henan ProvinceIn order to understand the PCV2 antibody level in large-scale pig farms in Henan Province,1677 pig serum samples were collected from 2019 to 2021,and serological detection was performed by indirect ELISA.The detection results showed that 1268 of1677 serum samples submitted were positive for PCV2 antibody,with an average antibody positive rate of 75.61%,which exceeded the minimum 70% immunization qualification standard specified by the National Ministry of Agriculture and Rural Areas,indicating that the overall immune effect of pig herds in Henan was good.In 2019,the antibody positive rate was 83.20%（322/387）;in 2020,the antibody positive rate was 86.91%（459/526）;in2021,the antibody positive rate was 63.74%（487/764）.The antibody level may be affected by swine price,new coronavirus,African swine fever and other macroscopic factors.The number of immunizations should be increased or the vaccine with better immune effect should be replaced.The serum samples were divided into sows,nursery pigs and fattening pigs according to the herd category.The antibody positive rates were 78.67%（332/422）,63.23%（375/593）and 84.74%（561/662）,respectively.The nursery stage was the high incidence stage of PCV2.It was necessary to improve the immunization procedure and timely supplement the immunization to ensure that the PCV2 antibody of nursery pigs was maintained at an effective protective level.This study provides some reference for immunization prevention and control of PCV2 in Henan Province.3.Prokaryotic expression of porcine circovirus type 2 Cap proteinIn order to obtain PCV2 Cap protein,Cap protein-specific primers were designed and amplified by PCR.The amplified Cap gene was cloned into p MD18-T vector,digested by genetic engineering,and cloned into prokaryotic expression p GEX-6P-1 vector.The plasmid was extracted and amplified by PCR.The alignment was verified to be correct by double digestion and sequencing,and transformed into BL21（DE3）E.coli expression system.The conditions affecting the induction of expression,namely temperature,time,and IPTG concentration,were optimized.The expressed PCV2 Cap protein was verified by polyacrylamide gel electrophoresis（SDS-PAGE）and immunoblotting（Western blotting）tests.The validation results showed that the prokaryotic expression plasmid of PCV2 Cap protein was successfully constructed,and the expressed target protein could be detected at about 54 KDa by SDS-PAGE electrophoresis after 4 hours of induction with 0.5 mmol/L IPTG at 37℃ under the optimal induction expression conditions.The expressed protein was detected by Western-blotting with porcine serum as the primary antibody and（rabbit anti-pig）Ig G-HRP as the secondary antibody by immunoblotting,and the results showed that the target protein could specifically bind and develop color.This test lays a foundation for the study of ELISA kit after PCV2.