Isolation And Identification Of SVV And A Study Of The Pathogenicity

Senecavirus A, formerly known as Seneca Valley virus(SVV), a non-enveloped, single-stranded RNA virus, is the member of the family Piconaviridae, species Senecavirus Ain the genus Senecavirus.SVV was characterized by vesicular fluid on snout and hoof, sometimes accompanied with fever and anorexia in sows.In this study, four SVV strains designated SVV CH-01-2015、SVV CH-02-2015、SVV CH-03-2015 and SVV CH-04-2015 respectivelywere obtained from the tissues of infected sowsand the complete genome were sequenced.Based on the conserved regions of SVV 3C gene,a pair of primers and a probe were respectively designed,the standard recombinant plasmids for 3C gene was constructed and usd for Taq Man Real-Time PCR.The SVV CH-02-2015 strain was used to infected healthy piglets of 4-day age to observe the changes ofclinical symptoms and pathology,and detected virus titers in infected tissues.The isolation and identification of the SVV results indicated that the CPE could be seen on 1th genration in 24 hrs and were harvested in 48 hrs. CPE was shorter and intensified with increased passages, and the virus can steadilypropagated in PK-15 cells.Whole genome sequence alignment results show that SVV CH-01-2015 complete genome sequences shared a 97.6% nucleotide identity to SVV CH-02-2015、CH-03-2015 and SVV CH-04-2015; SVV CH-02-2015 complete genome sequences shared a 99.9% nucleotide identity to SVV CH-03-2015 and SVV CH-04-2015;SVV CH-03-2015 complete genome sequences shared a 100% nucleotide identity to SVV CH-04-2015. The 4 SVV strains in China had 94.3%-94.4% nucleotide identityto the Americanstrains SVV-001 in 2008; SVV CH-01-2015 complete genome sequences shared a 96.8% nucleotide identity with two new Americanstrains(USA/IA46008/2015 and USA/IA40380/2015); and shared a 97.1% nucleotide identity with three Brazilian strains(SVV/BRA/GO3/2015 、SVV/BRA/MG1/2015、SVV/BRA/MG2/2015); SVV CH-02-2015、CH-03-2015 and SVV CH-04-2015 complete genome sequences shared a 98.0% nucleotide identity with two new Americanstrains(USA/IA46008/2015 and USA/IA40380/2015); and shared a 98.2% nucleotide identity with three Brazilian strains(SVV/BRA/GO3/2015 、SVV/BRA/MG1/2015、SVV/BRA/MG2/2015); SVV CH-01-2015、SVV CH-03-2015 and SVV CH-04-2015 shared a 96.7% nucleotide identity with Canadianstrain(11-55910-3) while SVV CH-02-2015 shared a 96.6% nucleotide identity with it.The phylogenetic tree indicated that the 4 SVV strains were clustered together with Senecavirus A,revealed that the 4 SVV strains shared high homology with Senecavirus A.Result of Taq Man Real-Time PCR confirmed that the correlation coefficient of standard curve(R2) was0.999 and 1 × 102copies/μLof standard plasmids could be detected,moreover the positive fluorescent signal only was detected in the reaction which template is SVV c DNA and the cross-reaction of other virus or cells was not detected.The coefficient of variation between intra-assay and inter-assay were less than 0.96 percent. It is evident that the method based on PCR assay is highly specific and rather sensitive and stable for the detection of SVV.The pathogenic trial in piglets showed that:(1) the piglets inoculated with live SVV in different routes of infection show a mild clinical sign of disease in our experiment;(2) it was possible that SVV is a contagious disease.(3)SVV may have astrong affinity for tissues, including lung, spleen, tonsil, brain and heart. This conclusion was supported by thefact that the viral titers in the above tissues were higher and further demonstrates thatthesuccessful replication of SVV in the pig.