Isolation And Identification And Biological Characteristics Analysis Of Porcine Senecavirus A(SVA)HN16

Senecavirus A?SVA?is a porcine pandemic virus that has only been diagnosed in recent years.It can cause blisters,ulcers,and ulcerative wounds in pigs'nose,mouth,and hoof.In China,the SVA infection occurred in pigs in March 2015,and the first strain of SVA was isolated and identified in 2016.Today,SVA has been reported in Guangzhou,Hubei,Heilongjiang,and other places in China,meaning that the epidemic has obviously expanded.Therefore,It is of great significance to isolate and identify newly discove red strains,and to study the biological characteristics of SVA.In this study,the SVA was detected in samples from a pig farm in South China,and the main contents are as follows:A pair of specific primers,designed according to the published SVA gene,was used to detect the SVA in the pig viscera and blisters from pigs with blisters-like appearances at a pig farm in southern China.The isolate was identified as SVA by sequencing and named SVA HN16.Sensitive cell lines were studied on the detected SVA HN16 strain.The virus fluid was inoculated into PK-15 and BHK cell lines and subcultured respectively.SVA showed pathological changes in both cells and could be transmitted to 30 generations stably.RT-PCR was used to identify the virus fluid harvested every 3 generations.The SVA HN16strain was successfully isolated and stably propagated.Viral titers were measured in both cells and the results showed that PK-15 cells were passed to 30th generation,and the TCID₅₀ was 10^5.81/mL,while the TCID₅₀ of BHK cells was 10^5.12/mL.The SVA HN16 was more sensitive to the PK-15 cells.After transmitted to 50th generation in the PK-15 cells,the TCID₅₀ of the SVA HN16 strain was detected.Results showed that the TCID₅₀ of the5th generation was 10^5.59/mL,the 30th generation was 10^5.81/mL,while the 50th generation was 10^6.12/mL,indicating that the time of CPE appearance shorter and shorter,while the degree of CPE more and more serious during virus transmission.The Virus titer of the SVA was significantly increased by the 50th generation.The whole genome of SVA HN16 strain was cloned and sequenced by segmented amplification method,and the sequencing results were analyzed by using biological software such as splicing,coding region analysis,nucleotide homology a nalysis and genetic evolution analysis.The sequencing results suggested that isolate SVA HN16 consisted of7280 bp,in which the 5'Untranslated region?UTR?contained 668 bp and the 3'UTR,excluding the poly A tail,contained 66 bp.Moreover,an open reading frame contained6546 bp,encoding a polyprotein with 2181 amino acids.The results of nucleotide homology analysis showed that the most homologous to SVA HN16 were Chinese isolates CH-02-2015 and CH-03-2015,were 99.1%,while the least homologous to SVA HN16 was the American isolate ATCC_PTA-5343,which was 93.7%.In addition,the homology of SVA HN16 with other isolates from China,the United States,and Thailand was 95.5%to 98.9%.The results of genetic evolution analysis showed that the genetic relationship between the SVA HN16 strain and the Chinese isolates CH-02-2015 and CH-03-015 was the closest.They were classified into pedigrees III with other isolates from China,the United States,Canada,and Thailand reported in recent years.The American strains ATCC_PTA-5343 and SVV-001 were classified into pedigrees I,and American strains 88-23626?89-47752?90-10324?92-48963 were classified into pedigrees?.The SVA HN16 is a new isolate in China,belonging to the Senecavirus,and it is closely related to other Guangdong strains.The SVA HN16 strain was used for pig infection regression experiments.The SVA pathogens were tested on infected swine serum,anal swabs,and nasal swabs.The results showed that the virus was weakly positive at 10 dpi,but no virus was detected at 5 dpi and15 dpi.Anal swabs were positive for virus detection at 3-6 dpi and negative for 9-15 dpi,but no virus was detected in nasal swabs collected.Serum neutralizing antibody titer was 1:22 at 10 dpi.In this study,SVA HN16 strain was used to infect New Zealand white rabbits and the dynamic changes of antibodies were studied.The results showed that neutralizing antibodies?1:8?appeared at 6 dpi,and the neutralizing titer of serum antibodies gradually increased at 12-18 dpi over time.It reached a maximum?1:27?at 25 dpi,after which the antibody titer began to decrease.In conclusion,this study was based on the basic research on the isolation,identification,sequence analysis,phylogenetic analysis,animal regression experiment s,and dynamic changes of neutralizing antibodies against newly discovered strain SVA HN16 in southern China.It provides a theoretical basis for the follow-up study of the pathogenic mechanism and prevention and control of the disease.