Characterization Of A Monoclonal Antibody Against Glycoprotein E2 Of Classical Swine Fever Virus Isolate SM And Identification Of Its Recognition Sites

Classical swine fever (CSF) is a highly contagious disease of swine and wild boars, causing significant economical losses in a wide range of the world. The causative agent of this disease is classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. CSFV is an enveloped RNA virus with the whole genome length being approximately 12.3 kb. The typical pathological change caused by CSFV is degeneration of vascular wall, which may result in hemorrhage, infarction and necrosis of internal organs in addition to fever. CSF is contracted mainly through direct contact. Pigs carring CSFV often act as sources of transmission, and can spread the virus to the environmennts via faeces before any clinical syndroms can be observed. Recent research indicates that the prevalent genotype of CSFV has changed to group 2 from group 1 to which the vaccine C-strain belongs, suggesting that current vaccine could not provide efficient protection against infections by field CSFV.The present study was aimed to:(1) probe the molecular epidemiology of CSFV in Zhejiang, (2) characterize monoclonal antibodies against E2 protein of CSFV strain SM, (3) identify the recognition sites of monoclonal antibodies on the E2 protein. Results thus obtained may provide the foundation for further analysis of antigenic diversity of classical swine fever virus.1. Detection and molecular epidemiology of CSFVTwo pairs of primers were designed for nest RT-PCR based on the conserved regions upstream and downstream of E2 gene. This PCR reaction exhibited good specificity and could detect as low as 1400 copies of CSFV genome. An RFLP approach was developed to differentiate vaccine strain and wild strains based on the distinct distribution of Mspl enzyme restriction sites in their E2 genes. This method was applied to screen CSFV in 59 sapmles from swines in Zhejiang Province. The results revealed that 9 samples were recognized by PCR, with 4 belonging to vaccine strains,4 to wild strains, and the remaining representing the mixture of vaccine strains and wild strains. Phylogenetic analysis of the 190bp variable region of E2 gene indicates that the field viruse isolates recovered in Zhejiang and neibourghing areas belonged to genotype 2.1b, which were divergent from the group 1 isolates including the vaccine C-strain.2. Characterization of E2 monoclonal antibodies from CSFV strain SMTo investigate the effects of some key amino acids involved in the antibody recognition within highly variable region of E2 protein on the antigen structures, we vaccinated BALB/c mice with E2 protein from CSFV classical virulent strain SM, and obtained one hybridoma cells (2B10) that could secrete antibodies against the E protein via hybridoma technique and IFA screening. Subtyping indicated that the monoclonal antibody was IgG2a isotype.2B10 could react with strain SM in IFA assay. These reaults demonstrated that the recognition sites by 2B10 only existed in strain SM.3. Identification of recognition sites of the monoclonal antibody 2B10Monoclonal antibody 2B10 had strong reaction with recombinant protein E2-AD, and also reacts with truncated E2-BC by Western blot and indirect ELISA, indicating that the linear epitopes were located within the 1-125 amino acid resdues of E2. The diversity of E2 protein from prevalent strains was analyzed using two monoclonal antibodies.2B10 could recognize SM strain belonging to group 1, but failed to recognize vaccine strain of the same group and wild strains of group 2. Given that the vaccine strain or wild strains could not react with 2B10, the recognition sites were present only in strain SM. Some amino acids differences were found through comparison of E2-BC sequences between wild strains and the vaccine strain. These amino acids were site-mutated, and mutant peptides were expressed. These peptides were subsequently subjected to reaction with 2B10. The results demonstrated that the recongnition site for 2B10 was P709 in E2 of CSFV strain SM.In summary, this study has provided good foundation for continuing research on the relationship between variations of CSFV E2 gene and antigenic divergence as well as on the development of more effective anti-CSF vaccine.