Molecular Pathogenic Monitoring Of Porcine Circovirus Type 3 And Pathogenicity Analyses

Porcine circovirus type 3（PCV3）is a new type of porcine circovirus firstly reported in the United States in 2016,whose etiological characteristics and pathogenic mechanisms are still unknown.In order to explore the epidemic characteristics,genetic evolution and pathogenicity of PCV3 in China,this research had established and optimized a conventional PCR method against PCV3,a differential diagnosis PCR method against PCV2 and PCV3,and a Taq Man fluorescence quantitative PCR methods against PCV3.The established PCR methods were used in the investigation of 11169 clinical samples from 2016 to 2021,and the molecular epidemiology was analyzed by gene sequencing.Virus isolation and piglet infection experiment were carried out to verify the pathogenicity of PCV3.The results come as follows:1.Establishment of a PCR method for detection of porcine circovirus type 3.In this study,three PCR detection methods for PCV3 were established,among which the conventional PCR method and Taq Man fluorescence quantitative PCR method targeted its ORF2 gene.The constructed p MD-18T-PCV3-ORF2 recombinant plasmid was used to verify the lowest detection limits of conventional PCR and Taq Man fluorescence quantita-tive PCR,which were 6×10³copies/μL and 4.13×10¹copies/μL,respectively.In order to distinguish the mixed infection of PCV2 and PCV3,the differential diagnosis PCR method was established.By analyzing the whole gene sequence of PCV2 and PCV3,degenerate primers were designed for their conservative and overlapping regions,so that PCV2 and PCV3 could be dectected by the only pair of primers at the same time.The detection limit of this method for PCV2 and PCV3 was 2.65×10³copies/μL and 2.45×10³copies/μL,re-spectively.Through optimization and comparison,it was found that the three PCR methods had good specificity and sensitivity and could be used for pathogen detection of PCV3under different circumstances.2.Etiological investigation of PCV in 2016-2021.11169 clinical samples from 967 farms in the link of pig breeding-sale-slaughtering from 2016 to 2021 were tested by the established PCV3 PCR method to investigate porcine circovirus infection.In 2016,77 samples were PCV3 nucleic acid positive from 24 farms of all 35 pig farms with reproductive disorders.The positive rates of PCV3 infection in farms and pigs were68.6%and 34.7%,respectively.PCV2 tests showed that 138 samples were PCV2 nucleic acid positive,35 samples were PCV2 and PCV3 nucleic acid positive,and the mixed infection rate of both was 15.8%.The above etiological investigation results showed that PCV3 exists in China and has a mixed infection with PCV2.The differential PCR method of PCV2 and PCV3 was used to detect 2894 clinical samples suspected of porcine circovirus infection in 537 pig farms from 2016 to 2021.The results showed that PCV2 and PCV3 infection in pig farms was 45.62%（245/537）and33.52%（180/537）respectively,and the positive rate of co-infection in pig farms was 24.95%（116/537）.The positive rates of pigs infected with PCV2 and PCV3 were 34.45%（997/2894）and 24.95%（722/2894）,respectively,and the positive rate of pigs co-infected with both pathogens was 12.72%（368/2894）.It was demostrated that porcine circovirus could be detected in all feeding stages,and the highest detection rate of PCV2 and PCV3was 47.36%（206/435）in fattening pigs and 37.24%（264/709）in breeding pigs,respectively.The detection rate of PCV2 in stillbirths was the lowest,11.78%（35/297）,while the detection rate of PCV3 in nursery pigs was the lowest,16.01%（105/297）.PCV3 infection in pigs transported in Hubei Province in 2019 and fattening pigs slaughtered in Hubei,Hunan,Sichuan and Chongqing from 2020 to 2021 were investigated by Taq Man fluorescence quantitative PCR（FQ-PCR）.6943 anticoagulant samples from pigs transported in Hubei Province in 2019 were collected from 274 pig farms in 13 cities in Hubei Province.The results showed that the positive rates of PCV3 infection in farms and pigs in Hubei Province were 22.26%（61/274）and 4.55%（316/6943）,respectively.According to the statistical test results of the feeding stage of pigs at the source of the sample,it was found that the positive rates of conservation pigs and fattening pigs were5.33%（94/1764）and 4.28%（222/5179）,respectively.According to the results of seasonal statistics,it was found that the detection rate of PCV3 were much higher in autumn and winter,which were 7.86%（99/1259）and 12.02%（63/524）,respectively.The corresponding samples of different types（anticoagulant,spleen and inguinal lymph nodes）of 370 slaughtered fattening pigs collected from 50 slaughterhouses in Hubei,Hunan,Sichuan and Chongqing from 2020 to 2021 were tested for PCV3.It was found that the positive rates of PCV3 infection in farms and pigs were 52.07%and 35.68%,respectively.The detection rates of anticoagulant,spleen and inguinal lymph nodes were 12.70%（47/370）,20.27%（75/370）and 23.78%（88/370）,respectively.From 2016 to 2021,ORF2 Gene sequences of 295 strains of PCV3 were determined,among which the proportion of PCV3a gene subtypes was 30.95%（13/32）,13.04%（6/46）,21.74%（10/46）,31.82%（7/22）,52.27%（23/44）and 40.95%（43/105）,respectively.The proportion of PCV3c gene subtypes was 46.88%（15/32）,67.39%（31/46）,23.91%（11/46）,59.09%（13/22）,13.64%（6/44）and 45.71%（48/105）,respectively.It was found that PCV3c was the current dominant gene subtype.3.Pathogenicity study of PCV3.In this study,a strain of PCV3 was successfully isolated and named PCV3/CH/HB/XY/2018 whose whole genome sequencing analysis showed that the strain belonged to the PCV3a subtype.Weaned piglets were infected with PCV3 positive material homogenate and 8^th generation virus cell culture medium.5 piglets in each group were infected by nasal drip and intramuscular injection separately,and a blank control group was set up.The results of infection test of piglets were evaluated in terms of clinical manifestation,body temperature,weight gain,viremia,tissue distribution and pathological changes of virus.It was found that PCV3 positive material homogenate and virus cell culture medium infected weaned piglets could show body temperature rise,weight loss,dermatitis and slow-growth,and viremia lasted for more than 14 days,so blood samples can be used as clinical monitoring samples.The results of tissue distribution of the virus showed that PCV3 mainly infected tonsil,lymph nodes and other important immune organs,and the pathogen content of PCV3 was the highest in spleen,kidney and tonsil,causing pathological changes such as interstitial pneumonia,lymphoid nodule hyperplasia and eosinophil increase in lung.To sum up,in this study,a PCV3 conventional PCR method a PCV2 and PCV3differential diagnosis PCR Method and a Taq Man fluorescence quantitative PCR3detection method were established,and these methods were used to complete the investigation of PCV3 infection in reproductive disorders,porcine circovirus etiology in2016-2021,PCV3 infection in transporting pigs in Hubei Province in 2019 and slaughtering and fattening pigs in 2020-2021.One strain of PCV3a subtype was successfully isolated,and 28-day-old weaned piglets were infected with PCV3 positive material homogenate and virus cell culture medium.In the infection test,all pigs from experimental group showed clinical symptoms such as emaciation,rough coat and dermatitis,which proved that the virus strain was pathogenic to weaned piglets,which laid a foundation for the diagnosis and pathogenicity study of PCV3.