| Senecavirus A(SVA)is the cause of Seneca disease in pig herds,which shares the similar clinical symptoms with other vesicular disease and lead economic losses of the pig industry.SVA had attracted much attention in recent years,however there is no commercial vaccine at present.The aim of this study was to analyze the distribution of SVA and the evolution of SVA strains in some provinces(municipalities directly under the central government and autonomous regions)of China by epidemiological investigation from 2019 to 2020.Meanwhile,the reverse genetic platform of SVA was also constructed to pave a way for SVA infection mechanism research and marker vaccine construction.Firstly,a total of 3 995 mixed tissue samples and serum samples were collected from some provinces(municipalities directly under the central government and autonomous regions)of China from 2019 to 2020.Real-time RT-PCR and competitive ELISA were used for the sample testing,and the SVA nucleic acid positive samples were then inoculated to cells for further virus isolation and genome sequencing.In 2019,1 310 mixed tissue samples were collected from 92 pig herds in 13 provinces(municipalities and autonomous regions),in which the individual positive rate(IP)was 6.72% and the herd positive rate(HP)was 23.91%.Besides,828 serum samples were also collected from 21 pig herdsin 5 provinces(municipality directly under the Central Government and autonomous region),and the IP,HP were 13.04%,85.71% respectively.In 2020,1 397 mixed tissue samples were collected from 101 pig herds in 13 provinces(municipalities directly under the central government and autonomous regions),and the IP,HP were 8.66%,36.63% respectively.Meanwhile,a total of 460 serum samples were collected from 13 pig herds in 5 provinces(municipalities and autonomous regions),and the IP,HP were7.61%,46.15% respectively.Finally,10 SVA strains were successfully isolated,the strain SVA-GXF011-2019,SVA-GXF053-2019,SVA-FJ036-2019,SVA-GX011-2019,SVA-FJ039-2019 and SVA-Hu N032-2019 shared 99.20%-99.30% similarity with the previously uploaded Chinese strains(SVA/GX/CH/2018),SVA-FJ018-2019,SVA-SD069-2019,SVA-SD072-2019 and SVA-SD074-2019 shared 98.30% similarity with USA/In Purdue 36998/2016 strain.Secondly,the CMV promoter,the whole SVA genome of the SVA GXT91 strain,the Poly A tail and HDV ribozyme sequence(HDVr)were amplified successively,and the three fragments were integrated into the low-copy vector pWSK-29 by homologous recombination technology.The results showed that the transfection efficiency of the constructed plasmid p WSK-29-SVA was about 70%.After 3 generation of blind-passages,cytopathic effect(CPE)was appeared,and the sequencing results of the rescued SVA virus(rSVA)at the fifth generation was correct.The r SVA was the inoculated to the cells and harvested at different time points,and the titer of the harvested virus was measured and the growth curve was plotted,which showed similar replication ability and proliferation characteristics to the parent strain(GXT91).The rSVA was also identified by indirect immunofluorescence detection,and the size and morphology of the plaque formed by rSVA infection was basically the same as the parent GXT91 strain.In conclusion,the infection rate of SVA in China is still high and the contamination area is still large in 2019-2020.The main epidemic strains in China are in Clade ?.However,the isolates SVA-FJ018-2019,SVA-SD069-2019,SVA-SD072-2019 and SVA-SD074-2019 made up of a new Clade(Clade ?)and got a high similarity with the US strain,so their biological characteristics need to be further studied.At the same time,the SVA reverse genetic platform plasmid(pWSK-29-SVA)was successfully constructed in this experiment and the rSVA was successfully rescued,which paved a way for the subsequent research on SVA. |
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