Epidemiological Investigation Of Porcine Circovirus Types 2,3 And 4 In Yunnan Province

PCV2 can cause a variety of swine disease syndromes caused by immune system suppression,and is one of the important pathogens threatening the health of Chinese pigs.There are currently no effective commercial vaccines for PCV3 and PCV4.In recent years,3 virus infections have been reported from different provinces.In this study,a triple PCR method of PCV2,PCV3 and PCV4 was established to carry out an epidemiological investigation of PCVs in Yunnan Province and clarify the distribution of porcine circovirus.It is of great significance to analyze the genetic evolution of the positive samples and understand the homology and variation of nucleotide and amino acid sequences for revealing the epidemic and pathogenic ecology of porcine circovirus in our province.1.Establishment of PCV2,PCV3 and PCV4 triple PCR methodIn this study,Gene sequences of PCV2,PCV3 and PCV4 were searched in NCBI Gene Bank database,and three pairs of primers were designed to amplify conserved regions of PCV2,PCV3 and PCV4,respectively.The recombinant plasmid was constructed as the positive plasmid standard of this method,and the reaction system and conditions were adjusted to conduct sensitivity and specificity tests.The results showed that the sensitivity and specificity of triple PCR were good.2.Epidemiological investigation of PCV2,PCV3 and PCV4 in Yunnan ProvinceA total of 3406 samples(spleen,lung,lymph node)from livestock farms,slaughterhouses,free-range households and markets in 16 prefectures and cities of Yunnan Province during 2018-2022 were collected for triple PCR detection,and the distribution and prevalence of PCVs in different years,seasons,regions and sites were analyzed.Between 2018 and 2022,the total positive detection rate of PCVs was 62.24%.The individual infection rates of PCV2 and PCV3 were the highest in autumn,with 41.2% and26.64% respectively.The lowest values were 35.17% and 20.59% in winter.The highest(27.65%)infection was found in summer and the lowest(24.57%)was found in winter.The spatial distribution showed that there were separate and mixed infections of PCV2 and PCV3 in all cities and cities.The highest positive detection rates of PCV2 and PCV3 were in Dehong(49.15% and 31.19%),while the lowest were in Kunming(28.66%)and Zhaotong(17.68%).Qujing had the highest rate of mixed infection(38.33%),while Lincang had the lowest rate(15.04%).The positive rates of PCV2 and PCV3 in free-range households were 87.23% and 69.33%,respectively.In the scale field and market,the lowest,respectively 66.67%,23.95%.The mixed infection of PCV2 and PCV3 was the highest in free-range households(30.92%)and the lowest in large-scale farms(8%).3.Sequencing and genetic evolution analysis of PCV2 and PCV3 in Yunnan ProvinceFourteen PCV2 positive samples were selected for the whole genome,Cap gene and25 PCV3 positive samples for Cap gene sequencing analysis,multiple sequence alignment analysis of nucleotide and amino acid sequence homology and variation sites,to construct the genetic evolution tree.The nucleotide homology between the whole gene sequence and the reference sequence of the 14 PCV2 sequenced strains was 93.6% to 99.4%.The analysis of nucleotide variation sites of Cap gene showed that there was base insertion and replacement in this region,but no deletion phenomenon.Genetic evolution analysis showed that 1 strain of 2a subtype,5 strains of 2b subtype,8 strains of 2d subtype.PCV3 typing analysis showed that 4 strains were 3a,5 strains were 3b and 16 strains were 3c.The nucleotide homology of Cap gene was 94%～99.5% compared with the reference strain.Analysis of amino acid mutation sites showed that there were 13 amino acid substitution sites without insertion or deletion.Conclusions: The established PCV2,PCV3,PCV4 triple PCR method has stable detection effect.In Yunnan Province,the infection rate of PCVs was high,including individual and mixed infection of PCV2 and PCV3.The nucleotide sequence homology of PCV2 gene was high,the nucleotide variation of Cap gene was small,and PCV2 d was the main prevalent gene subtype.PCV3 had high nucleotide homology of Cap gene,and the amino acid difference of Cap protein of sequenced strains was small.The main subtype of circulating strain was PCV3 c.