| Senecavirus A(SVA),the only member of the Senecavirus genus within the Picornaviridae family,can cause severe vesicular disease and epidemic transient neonatal losses in swine,resulting in significant economic loss to the breeding industry.Vesicular disease caused by SVA infection is very similar to those that caused by foot-and-mouth disease(FMD),swine vesicular disease(SVD),porcine herpes of swing(VES)and vesicular stomatitis(VS).In 2015,the first Chinese SVA strain was isolated from in Guangdong province,and gradually spread into Fujian,Hunan,Henan,Heilongjiang and other provinces.Currently,majority of the provinces had been reported to be affected by SVA infection in China.Given that the rapid transmission ability of SVA,establishment of efficient and accurate diagnostic methods and the development of SVA vaccine are the key to the prevention and control of the disease.Based on this,in this study has carried out the following research:1.SVA isolation and identification.The suspected samples collected from pig farms were detected by PCR,and the virus was isolated by PK-15 cells.The cell cultures were identified by cytopathic effect,whole genome amplification,electron microscope observation and phylogenetic tree.The results showed that SVA(SVACH-HNZK-2017)was successfully isolated.Genetic analysis revealed that the highest nucleotide sequence identity was 98.6% between CH-HNZK-2017 and US-15-40381 IA,while the lowest minimum genome identity was 93.7% between CHHNZK-2017 and SVV-001.Phylogenetic analysis based on the complete genome sequence suggested that SVA-CH-HNZK-2017 is located in the genus Senecavirus with closely related to US-15-40381 IA.2.Establishment of RPA-LFD rapid diagnosis method for SVA.Primers and probes were designed based on the conserved region of VP1 gene of SVA using Recombinase polymerase amplification(RPA)and lateral flow dipstick(LFD).The screening,optimization of reaction conditions,sensitivity and specificity assay were carried out to establish the RPA-LFD method.Moreover,the RPA-LFD method was further applied to test clinical samples.The optimum reaction conditions of RPALFD were performed at 35℃ for 25 minutes,and the results were visualized directly on the dipstrip.The specificity assay showed no cross-reactivity with other tested viruses,and the sensitivity test showed that the minimum detection limit was 15copies/μL.Moreover,the RPA-LFD method was successfully applied to test clinical samples,with no significant difference being observed between RPA-LFD and q RTPCR.3.Immunogenicity study based on VP2 of Senecavirus A.The VP2 gene was amplified by PCR,and cloned into the recombinant plasmid p ET-30a-VP2 using the c DNA of SVA HNZK isolate as the template.After identification,the recombinant plasmid was transformed into E.coli for prokaryotic expression.The purified recombinant protein was emulsified with 206 adjuvant to prepare a vaccine,and the vaccine efficacy was evaluated in immunized pigs.The results showed that VP2 protein was successfully expressed,mainly in soluble form.Western blot showed that the recombinant protein VP2 had good reactivity.The SVA subunit vaccine based on VP2 protein was successfully prepared,which can significantly improve the specific antibody,IL-4 and IFN-γ(P < 0.01).The challenge protection test showed that the group immunized VP2 subunit vaccine provided 4/5 protection against SVA challenge,while adjuvant group and PBS control group had no protection.In summary,in this study,a SVA strain was successfully isolated,which laid a foundation for the further study of the virus.The established RPA-LFD assay could be used as a potential optional rapid,reliable,sensitive and low-cost method for field diagnosis of SVA,especially in resource-limited regions.The VP2 subunit vaccine developed in this study has a good immune protective effect,providing a new direction for the development of novel SVA vaccine. |
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