Development Of Colloidal Gold Strip Based On Monoclonal Antibody For Porcine Senecavirus A(SVA)detection And Study On Immunogenicity Of Inactivated Vaccine Against SVA

Porcine Senecavirus A（SVA）,an emerging picornavirus and the only member of the genus Senecavirus in the family Picornaviridae,can cause vesicular lesions in pigs and epidemic transient neonatal losses（ETNL）in newborn piglets,which seriously restricts the healthy development of pig industry in major pig breeding countries（USA,Brazil,Canada,China,etc.）.In 2015,a pig farm in Guangdong Province of China reported the SVA for the first time,and then spread to more than a dozen provinces such as Hunan,Henan,Guangxi,Gansu,and Heilongjiang.Vesicular lesions caused by SVA infection are very similar to those that the clinical symptoms caused by Swine vesicular disease（SVD）,Foot and mouth disease（FMD）,Vesicular stomatitis（VS）and Vesicular exanthema of swine（VES）,caused great obstacles in the differential diagnosis of SVA.In addition,SVA is highly transmissible with the potential risk of pandemic.At present,there is no commercial vaccine available,and the domestic pig population lacks the immune barrier of SVA.Therefore,it is urgent to develop effective vaccines and differential diagnosis methods for SVA.Based on this,this paper carried out the following studies:1.Isolation and biological identification of SVAThe virus was isolated and cultured using BHK-21 cells after the collected specimen were detected by RT-PCR,and the isolated virus were biologically identified by the cytopathic observation,virus titer determination,indirect immunofluorescence test,transmission electron microscope observation,whole genome amplification and sequencing and genetic analyses.The results showed that a SVA strain named as CH-GXYL-2019 was successfully isolated from BHK-21 cells.After 24 hours of culture,the virus titer reached 1×10^7.23TCID₅₀/0.1 m L,and the full length of the genome was 7286 nucleotides（nt）.The genetic analysis showed that CH-GXYL-2019 and SVA/BRA/GO3/2015 had the highest nucleotide sequence homology（98.9%）,the nucleotide sequence homology with domestic strains is 98.5%～98.7%,while the genome homology between CH-GXYL-2019 and SVV-001was the lowest（92.4%）.The phylogenetic tree was constructed based on the whole SVA genome sequence,indicating that CH-GXYL-2019 belonged to the Senecavirus genus within Picornaviridae family.2.Preparation of monoclonal antibody against SVA VP1 proteinThe recombinant expression plasmid pET-28a-SVA VP1 was constructed by gene cloning.After the identification,the recombinant expression plasmid was transformed into E.coli for prokaryotic expression and purification.BALB/c mice were immunized with purified SVA VP1 protein as antigen.Two monoclonal antibodies（McAbs）that stably secreted anti-SVA VP1 were successfully obtained,named 2E10 and 4E3 respectively.According to the monoclonal antibody subtype identification kit,2E10 was Ig G2b,κchain,3E4 was IgG2a,κchain.The number of chromosomes was 102 in 2E10 and 88 in 3E4.Western blot and indirect immunofluorescence assay（IFA）results showed that the two McAbs specifically bound to SVA and recognized different epitopes on SVA VP1 protein.3.Preparation of colloidal gold test strips for SVAUsing the prepared two strains of McAbs（2E10,4E3）,2E10 McAbs was used as the gold-labeled antibody,and goat anti-mouse Ig G and 3E4 McAbs were used as the quality control line（C line）and detection line（T line）,respectively,to prepare SVA colloidal gold test strips by colloidal gold immunochromatography.After repeated tests,it was determined that the optimal marker amount of 2E10 McAbs was 38.5μg/m L,and the optimal pH was 8.0.The prepared colloidal gold test strips were specifically bound to the SVA,but had no cross reaction with Foot and mouth disease virus（FMDV）,Porcine reproductive and respiratory syndrome virus（PRRSV）,Classical swine fever virus（CSFV）and Porcine circovirus type 2（PCV2）.The minimum detection limit is 5×10^1.23TCID₅₀/0.1 m L.The coincidence rate between established method and the q RT-PCR method for clinical samples was 96.55%.4.Immunogenicity study of inactivated vaccine based on SVA isolateThe inactivated vaccine was prepared by expanding the culture of SVA isolate CH-GXYL-2019 and harvesting the virus liquid.After BEI inactivation,the inactivated vaccine was emulsified with ISA 201 VG adjuvant.The character test,immune effect evaluation and challenge test were carried out on the inactivated vaccine.The character test,immune effect evaluation and challenge protection test were carried out of the trial-produced inactivated vaccine.The results showed that the trial inactivated vaccine met the characteristics and safety inspection standards of veterinary biological products,and immunized pigs could induce higher levels of specific IgG antibody,neutralizing antibody and IFN-γ（P<0.01）.The challenge protection test showed that the vaccine group could provide complete protection（5/5）,while the ISA 201 adjuvant group and the normal saline group had no protective effect.In summary,a SVA strain was successfully isolated in this study,which laid a material foundation for the subsequent etiology research and vaccine development of SVA.SVA VP1 protein,expressed and purified by prokaryotic expression system,was used as immunogen to immunize BALB/c mice,successfully obtaining two strains of monoclonal antibodies（2E10,3E4）against SVA VP1 protein.The colloidal gold strip was successfully prepared for SVA detection based on two monoclonal antibodies above,providing more options for grassroots epidemic prevention personnel to timely carry out point-of-care testing of SVA.The trial production of inactivated vaccine based on SVA isolates has a good protective effect,providing a new robust tool for SVA prevention,control and eradication in China.