Isolation And Identification Of Senecavirus A And Construction Of Its Infectious Clone

Senecavirus A(SVA)is a newly discovered pathogen of swine vesicular disease in re-cent years and belongs to the sole species in the genus Senecavirus of family Picornaviridae Clinical signs included vesicles on the snouts and/or coronary bands,sometimes accompa-nied with lameness,anorexia,lethargy,and transient fever,and the mortality rate of newborn piglets was as high.The first occurrence of SVA in China was reported in pig herds in Guangdong province in 2015.Since then,cases of SVA have been reported from eight prov-inces including Hubei,Heilongjiang,Henan,Fujian,Guangxi,Shandong,Sichuan,and Guangdong.It has caused serious economic losses to the pig industry.However,the emer-gence and pathogenesis of SVA infection are unclear.There are no drugs or vaccines that have been approved to treat the disease caused by SVA on the market currently.Here,we isolated five novel SVA strains by cell culture and sequencing.Furthermore,the growth characteristic of the five SVA strains in vitro was also investigated.Then,GD05/2107 strain was selected to construct SVA infectious clones using reverse genetics technology.The for-eign genes were inserted into the genome of SVA infectious clone virus,and SVA expressing the reporter gene was rescued and characterized.1.Isolation and identification of Senecavirus AA number of cases of vesicular disease occurred in pig farms in Guangdong during 2017-2018.The infected pigs showed lethargy,claudication,and increased body temperature,resulting in severe increase in mortality in neonatal piglets.In order to determine the etiolo-gy of the disease,twenty samples were collected from some of the infected herds and five of them were tested positive for SVA by RT-PCR.The positive samples were processed for vi-rus isolation by ST-R cells.The isolated strains were verified by RT-PCR,IFA,western blot and named as GD01/2017,GD03/2017,GD05/2017,GD06/2017,GD-S1/2018.The result of sequence analysis showed that five novel SVA strains were?7.2 kb in length,respectively.It contains a single open reading frame(ORF)encoding a 2181 aa polypeptide,flanked by a 5 untranslated region(UTR)and a 3' UTR followed by a poly(A)tail.In addition,phyloge-netic analysis based on the ORF and VP1 genes showed that the Chinese SVA strains can be mainly grouped into nine genetic branches.The current study reported that five novel SVA strains are within clade-?,?,?,?,? respectively.It also suggests the SVA strains currently circulating in China may have different origins2.Construction of an infectious clone of a SVA GD05/2017 strainIn order to construct SVA infectious clone using reverse genetics,we chose GD05/2017 strains with a relatively stable growth to construct a full-length infectious clone.The DNA fragments covering complete virus genome were strategical cloned into pEGFP-C3 vector by utilizing enzymatic digestion and homologous recombination.Finally,a full-length cDNA clone of GD05/2017 strain,named as pC3-SVA-GD05,was obtained.The virus infectious clone plasmid was transfected into BHK-21 cells.Then,the cell supernatants were collected after 60-72 h incubation.ST-R cells were further infected with the harvested supernatants,and apparent CPE was observed after incubation.The harvested virus was identified by RT-PCR,IFA and WB.The results indicated that the SVA named as V-GD05-clone was res-cued,suggesting that a SVA virus infectious clone system has been established successfully.3.Construction of recombinant SVA viruses expressing foreign genesIn order to detect the replication of the virus in the cells quickly and intuitively,three foreign genes of different sizes were separately inserted into the genome of SVA infectious clone virus.Recombinant SVA viruses expressing exogenous genes were rescued,named as V-GD05-iLOV,V-GD05-RFP,V-GD05-Nluc.Genetic stability testing verified that V-GD05-iLOV can maintain the highest level of stable passage on the cell.However,insertion of the foreign gene RFP or Nluc into SVA was deleted in the course of cell culture three to five passage.It indicates that exogenous genes are unstable in the viral genome.Ad-ditionally,flow cytometry and IFA test showed that V-GD05-RFP were detected high inten-sity fluorescence signals and V-GD05-iLOV detected medium-intensity fluorescent signals.And the luciferase activity assay showed that V-GD05-Nluc could quantitatively detect the level of virus replication.Furthermore,in this study,we infected ST-R ANTXR1 KO cell with three SVA reporter viruses,which initially proved that ANTXR1 is a receptor of SVA invasion of pig-derived cells.So,it has laid a good foundation for the subsequent research on the interaction between virus and receptor.