| Porcine Circovirus(PCV),belonging to the genus Circovirus of the family Circoviridae,is the smallest DNA virus known to date.PCV1,PCV2,PCV3 and PCV4 are four genotypes currently discovered,among which PCV2 is the main pathogen causing Porcine Circovirus Associated Diseases(PCVAD)and is widely prevalent in clinical.Consequently,this study focuses on PCV2.In order to understand the epidemic characteristics,genetic evolution and pathogenicity of PCV2 in China,this study investigated the PCV2 infection in 1233 clinical samples from December 2021 to November 2022 in some parts of the country by qPCR,and analyzed its epidemic.The isolation and identification of viruses and piglet infection experiments were carried out to compare the pathogenicity of isolated strains.The main research contents and results are as follows:1.Molecular epidemiological study of porcine circovirus type 2 in some parts of the country from December 2021 to November 2022With the intention of understanding the epidemic status and genetic characteristics of Porcine Circovirus in some parts of China,this study collected 1233 clinical samples from pig farmhoggery in some parts of the country from December 2021 to November 2022,and used qPCR method for PCV2 detection,and a total of 458 positive samples were detected,with a total positive rate of 37.15%(458/1233),indicating that PCV2 was widespread throughout the country.Aiming at understanding the dynamic changes of PCV2 subtypes in the investigation area,458 positive clinical samples were tested for PCV2 a,2b and 2d subtypes by multiplex quantitative PCR.Among them,PCV2 a,PCV2b and PCV2 d subtypes mixed infection was particularly serious.The results showed that PCV2 crossrecombination and self-recombination occurred during natural infection.To further understand the genetic characteristics of PCV2,458 positive samples were amplified and sequenced PCV2 ORF2 and whole genome by PCR,resulting in a total of197 ORF2 gene sequences and 34 whole genome sequences.The phylogenetic analysis of197 ORF2 gene sequences showed that PCV2 a subtype accounted for 17.26%(34/197),PCV2 b subtype accounted for 12.69%(25/197),and PCV2 d subtype accounted for 70.05%(138/197).The results indicated that PCV2 d was the dominant genotype.Nucleic acid homology analysis of 197 PCV2 ORF2 gene sequences showed that the nucleotide homology among the PCV2 ORF2 sequenced strains was 88.0%-100%,and was 85.6%-99.8% among the selected PCV2 reference strains of different gene subtypes.The analysis of amino acid sequence mutation of Cap protein revealed that 29 amino acid mutation sites coexisted,most of which were located in the epitope of Cap protein.Phylogenetic analysis showed that 34 strains belonged to PCV2 a,PCV2b and PCV2 d subtypes.Among them,1strain belongs to PCV2 a,7 strains belong to PCV2 b,and 26 strains belong to PCV2 d,indicating that PCV2 d was the main endemic strain in the investigated area.Nucleotide homology analysis showed that the nucleotide homology of 34 strains was 94.9%-100%among them and was 89.5%-99.8% among the selected PCV2 reference strains of different gene subtypes at home and abroad.2.Isolation,identification and pathogenicity of the virusThis study set out to deeply explore the pathogenicity and differences of different subtypes of PCV2.PCV2 b and PCV2 d strains in the investigated area were isolated and identified,and the pathogenicity was compared by animal regression test.In this study,one strain of PCV2 b was named ZJ0263 and one strain of PCV2 d was named SD0027,both of which had certain pathogenicity to pigs.The infected piglets showed loss of appetite and messy body hair,but there was no significant difference between the two groups(P>0.05).In addition,the weight of the two challenge groups was lower than that of the control group,and the weight of SD0027(PCV2d)was more significant(P<0.05),which had a more obvious effect on the growth rate of pigs.According to the analysis of antibody levels,it was found that after the two isolates were challenged,the host’s immune system could quickly produce specific antibodies,and the immune response of SD0027(PCV2d)attack group was earlier and faster.Viral load measurement showed that viremia appeared on the7 th day after ZJ0263(PCV2b)challenge,and then the viral load continued to rise and peaked at the 14 th day,and then showed a downward trend.Viremia appeared on the 3rd day after the challenge in the SD0027(PCV2d)group,and then the viral load also continued to rise and reached a peak on the 21 st day,which was higher than that in the ZJ0263(PCV2b)challenge group,and then gradually decreased.After 28 days of dissection,the heart,liver,lungs,spleen,kidney,and lymph nodes were tested and found that the viral load of the lungs and lymph nodes was high.After sectional observation,the thickening of the alveolar wall and inflammatory cell infiltration were found,and the inflammatory cell infiltration of the lungs was more serious in the SD0027(PCV2d)group.The two attack groups of lymph nodes showed mild mononuclear macrophage infiltration and lymphopenia.The above results show that the PCV2 b and PCV2 d strains isolated in this study were pathogenic.Comparing the two attack groups,the isolated SD0027(PCV2d)strain after challenge led to earlier appearance of viremia in the host,faster immune response,more obvious lung lesions,and higher pathogenicity. |
