Establishment Of Indirect ELISA Method Of ASFV And Identification Of B Cell Epitopes In Extracellular Domain Of CD2v Protein

African swine fever（ASF）is a potent infectious disease with detrimental effects on the global swine industry and no vaccine available so far.The CD2 v protein is a glycoprotein on the outer envelope of African swine fever virus（ASFV）,which mediates the transmission of virus in the body,playing an important role in ASFV vaccine development and disease prevention.The CD2v-deleted mutants of ASFV exhibit characteristics of non-hemadsorption（non-HAD）,low-virulence,recessive and chronic infection.The prevalence of the mutants bring greater challenge to the prevention and control of ASF.In this study,we aimed to establish an antibody detection method based on CD2 v protein to identify wild-type and CD2v-deleted strains infection of ASFV.The CD2 v protein extracellular domain sequence（CD2v-ex,1 ～ 588 bp）of the highly pathogenic strain China/2018/Anhui XCGQ was integrated into the genome of suspension culture-adapted Chinese hamster ovary-S（CHO-S）cells using lentivirus vectors（LVs）.By subcloning and screening,a monoclonal CHO-S cell line（CHO-CD2v）that stably expressed secretory CD2v-ex protein was identified.And the protein was purified through affinity chromatography.Then the purified CD2v-ex protein was used as the detection antigen to establish an indirect ELISA method（CD2v-i ELISA）for identification of the ASFV wild-type and CD2v-deleted（CD2v^-）strains.Furthermore,we generated two specific monoclonal antibodies（m Abs）,6C11 and 8F12（subtype Ig G1/kappa-type）.Peptide scanning technology was used to identify the epitopes recognized by m Abs 6C11 and 8F12.The results showed that the CHO-CD2 v cell line established in this study was able to stably secrete and express CD2v-ex protein with glycosylation modification.Genome level validation results showed that the target gene was successfully integrated into the CHO-S cell genome and transcribed into m RNA.The CD2v-i ELISA method we established in this study showed excellent specificity with no cross-reaction with serum samples infected with ASFV（CD2v^-）,porcine reproductive and respiratory syndrome virus（PRRSV）,classical swine fever virus（CSFV）,porcine circovirus（PCV）,porcine pseudorabies virus（PRV）,swine foot and mouth disease virus（FMDV）and porcine epidemic diarrhea virus（PEDV）.Furthermore,this method showed high sensitivity,allowing identification of ASFV-infected clinical serum samples up to a dilution of 1:2,560.The coefficient of variation both in and between batches was <10% with good reproducibility and a high compliance rate of 99.4%.And in this study,two conservative B cell epitopes,³⁸DINGVSWN⁴⁵ and ¹³⁴GTNTNIY¹⁴⁰,were defined.Amino acid sequence alignment showed that the defined epitopes were conserved in all eight referenced ASFV strains from various regions of China including the highly pathogenic,epidemic strain,Georgia 2007/1（NC＿044959.2）,with the same noted substitutions compared to the four foreign ASFV wild-type strains.This CD2v-i ELISA method developed here can be used to distinguish infection by ASFV wild-type strains（HAD）and CD2v-deleted strains（non-HAD）and might be of great importance in epidemiological investigation and normalized monitoring of ASFV.The identified antigenic epitopes provide new targets for the design and development of ASF vaccines,and the prepared monoclonal antibodies can also serve as biological materials for studying the action mechanism of CD2 v protein.