Screening And Identification Of Monoclonal Antibodies Differentiating Attenuated Vaccine C Strain From Field Isolates Of Classical Swine Fever Virus

Classical swine fever?CSF?caused by classical swine fever virus?CSFV?with single stranded RNA as viral genome about 12.3 knt is a hightly contagious disease that caused serious damage to global swine industry.According to the phylogenetic analysis based on the nucleotide sequence of E2 gene,CSFV isolates can be grouped into three genotypes?genotypes 1,2 and 3?,which were further divided into about 3 or4 subgenotypes?1.1¹.4,2.1².3 and 3.1³.4?.Domestic pigs and wild boars are the single host of CSFV leading to urgent,continuously febrile disease with higher mortality.However,atypical CSF with lower mortality increased and becomes chronic and persistent recently,leading to a difficult diagnosis.The pigs of atypical CSF or persistent infection of CSFV can continuously carry and secret viral particles,which were important sources of CSFV and threat to pig health seriously.Accurate identification of persistent infection of CSFV in healthy pigs is the key and technical problem for CSF purification.The infection of CSFV wild isolates and vaccination with attenuated vaccine C strain will induce neutralizating antibody.Due to the extensive application of attenuated vaccine C strain in China,the infection of CSFV field isolates can not be rapidly identified by the conventional antibody detection method.And there is no rapid serological detection kit to differentiate the antibody induced by infection of CSFV field isolates and that induced by attenuated vaccine C strain.Therefore,development of the rapid serological diagnosis method to identify persistent infection or recessive infection of CSFV is essential to the radication of CSF in China.In the present study,we aim to analyze antigenic differentiation between CSFV field isolates from China and attenuated vaccine C strain,which is critical for establishing the rapid-serological diagnosis method to distinguish persistent infection or recessive infection of CSFV from vaccination.First,four monoclonal antibodies?mAbs?against CSFV were used to react withC strain,Shimen strain,108 CSFV field isolates and 3 strains of BVDV-1 by using IFA,and reaction between mAbs and viral E^rns or E2 protein expressed by the baculovirus expression system,which were originated from vaccine C strain,Shimen,8 subgenotypes?2.2,2.3,1.2¹.4,3.1,3.2 and 3.4?,and 7 sub-subgenotypes of 2.1?2.1a,2.1b,2.1c,2.1g,2.1h,2.1i and 2.1j?was also tested.To reveal the type of antigenic epitopes recognized by mAbs,eukaryotic and prokaryotic E^rns and E2protein were treated with SDS loading buffer containing?-mercaptoethanol or not,and then subjected to Western blot.As a result,No.1 mAb cannot react with vaccine C strain,but can react with shimen strain,sub-subgenotypes 2.1a,2.2g,2.2i and 2.1j isolates,and most isolates belonging to sub-subgenotypes 2.1b,2.1c,2.2 and 2.3.No.2 mAb can strongly react with vaccine C strain and weekly with 2 CSFV isolates of subgenotypes 1.1,but not with Shimen strain and other field isolates.No.3 mAb can react with C strain,Shimen strain and other isolates belonging to subgenotypes 1.1,1out of 18 isolates grouped into 2.1c,and 6 out of 23 isolates belonging to subgenotypes 2.2.No.4 mAb can react with C strain,Shimen strain and other isolates belonging to subgenotypes 1.1,2.1i and 2.1j,most isolates belonging to 2.1b,2.2,and2.3,and few isolates grouped into sub-subgenotypes 2.1c,2.1g,and 2.1h.The above results were further confirmed by Western blot of mAb with viral proteins.No.1 mAb can react with E2 protein of Shimen and isolates belonging to subgenotypes 1.2¹.4and 3.1,but not with C strain,subgenotypes 3.2 and 3.4;No.2 only reacts with E^rnsns protein of C strain,and No.3 mAb cannot react with E2 protein of isolates belonging to subgenotypes 1.2¹.4,3.1 3.2 and 3.4,but with E2 proteins of C strain and Shimen strain;and No.4 mAb can react with E2 protein of C strain,Shimen and isolates belonging to subgenotypes 1.2¹.4,3.1,3.2 and 3.4,but not with E2 proteins of most isolates belonging to 2.1c,2.1g,2.1a,and 2.1h.Furthermore,these mAbs were identified not to react with BVDV-1.To identify the type of the antigenic epitopes recognized by those mAbs,E2protein of Shimen and E^rns protein of vaccine C strain,which were expressed by baculovirus expression system and E.coli expression system,were treated with SDS Loading buffer with or without?-Mercaptoethanol,the chemical that can reduce the disulfide bonds.Then the treated protein and native protein were reacted by western blot with the analyzed mAbs.Results showed that these mAbs only react with the native proteins expressed by baculovirus expression system,indicating that the analyzed mAbs can recognize conformational epitopes.Overall,reaction between four mAbs and C strain or CSFV field isolates or related viral proteins reveal the antigenic differentiation between these CSFV isolates,and two mAbs differentiating vaccine C strain from current CSFV field isolates were obtained,which were not cross-reacted with isolates belonging to BVDV-1.Thus,the obtained results and differentiating mAbs will improve our understanding of the virological properties of CSFV field isolates and provide a solid basis for the development of serological diagnosis for differentiation of vaccination from infection with CSFV field isolates,thus eradication of CSV in China will be accelerated.