Isolation and identification of porcine circovirus subtype 2d from guizhou strain and mouse infection test
In order to identify porcine circovirus type 2(PCV2)suspected to be suffering from porcine dermatitis nephrotic syndrome(PDNS)in a pig farm in Guizhou province,The serum of diseased pig,which was tested positive for PCV2 by qPCR,was collected and inoculated onto PK-15 cells for PCV2 isolation.Indirect immunofluorescence(IFA),PCR and whole genome clone sequencing were used to identify PCV2 virus.Relevant information software was used to analyze genetic evolution of the PCV2 and analyze its prolife-ration on PK-15 cells and determine TCID50.BALB/c mice were artificially infected with isolated strains to study infection traits.Results showed that PK-15 cells inoculated with F9 virus had specific green fluorescence.PCR results of the virus was positive for PCV2.Whole-genome clone sequencing showed that the full length of the PCV2 was 1 767 bp,which was named PCV-2 GZGD2022.The isolated strains and PCV2d reference strains were in the same branch of the evolutionary tree.The nucleotide similarity between F9 and F1 viruses reached 99.7％,and 94.1％-98.7％with reference strains.Compared with the F1 genera-tion,F9 had 6 nucleotide mutations(G155A,G669A,T1 474C,T1 500C,A1 518G and C1 572T),3 mutations in Cap protein(F77L,L227P and Y233C),and 1 mutation in Rep protein(Y195H).PCV2d can proliferate in PK-15 cells and TCID50 can reach 10-4.75/0.1 mL.The isolated strain had strong invasion to the lung and lymph nodes of mice.In this study,one PCV2d strain with stable proliferation was successfully isolated by PK-15 cells,and the results laid a certain foundation for the subsequent etiology study of PCV2 and provided materials for the screening of PCV2 vaccine strains.
porcine circovirus type 2porcine dermatitis nephrotic syndromeseparation identi-ficationmouse infection assay
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