Isolation And Identification Of PSV And Analysis Of Its Biological Characteristics

This study aims to understand the pathogenicity and genetic evolution rules of the porcine sapelovirus(PSV)by isolating and culturing of PSV epidemic strain.Furthermore,polyclonal antibody were prepared and an indirect ELISA method for detecting PSV Ig G antibody was established by using recombinant PSV VP1 protein expressed in vitro as antigen,which provide a scientific basis for in-depth understanding the characteristics of PSV and developing serological diagnostic techniques.1.Isolation,identification,genome sequencing and phylogenetic analysis of PSVA PSV strain was isolated by PK-15 cells from faecal samples of diarrheic piglets,and named SHCM2019.The full-length genome of this isolate was 7,567 bp(Genbank ID:MN685785).Phylogenetic analysis based on ORF fragment showed that the SHCM2019 strain was most similar with the YC2011 strain with 90.8%nucleotide identity and 98%amino acid identity,thus forming a subgroup with other PSV strains,which were clustered in the China group.The strain might be the recombinant strain of He B04 and ISU-SHIC,and there was a potential recombinant break point upstream of the 3D region.The analysis of amino acid mutation sites of VP1 protein showed that there were amino acid mutations in VP1 region.2.Pathogenicity of the SHCM2019 isolate to pigletsSix 5-day-old piglets were randomly divided into 3 groups with 2 piglets in each group.After 3 days of observation,experiment group 1 and 2 were inoculated with 5m L and 15 m L 10^7.41TCID₅₀SHCM2019,repectively.Experiment group 3 was inoculated with DMEM as control group.Observation the disease of piglets,pathological observation and nucleic acid quantitative detection were carried out on the tissue of infected piglets.Anal swabs were collected daily to detect viral load by q PCR.The results showed that one piglet in experimental group 1 developed watery diarrhea on the 5th day after inoculation,and one piglet in experimental group 2developed watery diarrhea on the 2nd day after inoculation.At the 6th day,necropsy of all piglets showed that the lesions mainly occurred in the intestine and lungs.The pathological manifestations were:intestinal thinning,intestinal contents diluted,intestinal villi atrophy and rupture,intestinal submucosa edema;lung swelling,local bleeding,alveolar septum thickening,serous exudation and inflammatory cell infiltration.PSV was mainly colonized in the intestine and lungs,with a small amount of distribution in the brain and intestinal lymph,but no virus was detected in the heart,liver and kidney.Infected animals can detoxify through feces,and the viral load is the highest in 3?6 days after challenge.3.Prokaryotic expression of PSV VP1 protein,preparation of polyclonal antibody and establishment of indirect ELISA method based on this proteinThe VP1 segment amplified from the PSV isolate,was cloned into p ET28a vector to perform prokaryotic expression.SDS-PAGE showed that the VP1recombinant protein was about 40 k Da and exists as inclusion bodies.Mice were immunized with purified VP1 protein to prepare murine anti-PSV polyclonal antibody.ELISA confirmed that the antibody had high potency,Western blot confirmed that it had good specificity,and immunofluorescence assay showed that it had good reactivity.An indirect ELISA was established for the detection of PSV Ig G antibody using VP1 protein as antigen.The optimal coating concentration of the method was 2?g/m L,and the optimal serum dilution was 1:400.The method was only specific to PSV positive serum,and the coefficients of variation of the intra-assay and inter-assay were less than 10%.