Differentiation Of Bovine Enterovirus Species By Double PCR And Preparation Of Monoclonal Antibody Against HY12 Enterovirus-encoded 3A Protein

Bovine enterovirus(BEV)infection is a new infectious disease characterized by gastrointestinal and respiratory disorder reported in China in recent years.As the causative agent,BEV belongs to the genus of enterovirus within the the family of Picornaviridae.Currently,BEV is divided into EV-E and EV-F enterovirus species.Since these two species have a varied nucleotide sequences and belong to different serotypes/genotypes,it is difficult to distinguish them by conventional serological methods.Additionally,there is no diagnostic method to distinguish EV-E from EV-F infection.Enterovirus nonstructural protein 3A is a membrane-bound protein composed of89 amino acids.It is composed of soluble N-terminal(58 residues,symmetrical dimer),hydrophobic region containing 22 residues(amino acids 59-81)and C-terminal containing 7 amino acid residues.Early study on 3A protein structure found that there is a strong hydrophobic region at the C-terminal,which is divided into two parts(I:amino acids 64-72;II:amino acids 73-80).Part II may be involved in mediating the binding of 3A and 3B to the membrane during the initiation of RNA synthesis.It is reported that the change of amino acids in this hydrophobic region can lead to virus replication defects and death.Since there is no method to differentiate EV-E from EV-F enterovirus infection and no relevant detection reagents or methods used to investigate the function of 3A protein encoded by BEV,two pairs of primers based on the genome sequences of EV-E and EV-F enterovirus published by Gen Bank were designed,synthesized,and used to establish a double PCR method to differentiate EV-E from EV-F.The specificity,sensitivity and repeatability of this method were determined.At the same time,the method was applied to detect the samples suspected of bovine enterovirus infection in Changchun.The results showed that the established double PCR method had good specificity and only detected EV-E or EV-F.Sensitivity assay showed that the minimum detectable amounts of EV-E and EV-F were 3.67×10~2copies/?L and 5.21×10~3copies/?L,respectively.Detection of the suspicious specimens showed that the positive rates for EV-E and EV-F viruses were 28.13%and 34.38%respectively,with the mixed infection rate of 15.63%.In addition,the 3A gene sequence of BEV-HY12 virus was amplified by RT-PCR and cloned into prokaryotic expression vector to construct prokaryotic expression plasmid p EGX-4T-3A.After induction by IPTG,the expressed 3A protein was purified and used as immunogen to immunize mice.The obtained immune splenocytes were fused with myeloma cells by PEG.The monoclonal antibody against 3A protein were screened by indirect ELISA.Then,the subtypes of 20 monoclonal antibodies were identified.The reactivity of five monoclonal antibodies to HY12 virus was identified by immunoblotting and immunofluorescence.The antigenic epitopes of two monoclonal antibodies were screened by segmented antigen detection method.The key region was in ³¹NEEVREYCRSKGWIV⁴⁵.The amino acids in this region are relatively conserved among E enteroviruses through sequence alignment analysis,but there are great differences among F enteroviruses.Immunofluorescence assay demonstrated that Mc Abs 3A6 and 3B2 only recognized EV-E,but failed to recognize EV-F,bovine parvovirus,and bovine viral diarrhea virus.In summary,in this study,we established a method for differentiating EV-E from EV-F enterovirus species.This method has strong specificity,high sensitivity,and advantages for detecting EV-E and EV-F in one step at the same time.The monoclonal antibody against HY12 virus 3Aprotein was prepared,and the key epitope regions of Mc Abs 3A6 and 3B2 were mapped.The Mc Abs 3A6 and 3B2 only recognize EV-E with a high specificity.The above results provide a technical means for species differentiation and epidemiological investigation on bovine enterovirus infection,and lay a foundation for further studying the function of 3A protein of HY12 strain and developing relevant detection methods.