Identification of a Novel Conserved B Cell Epitope in the N Protein of Equine Arteritis Virus (Bucyrus Strain).

This research examines the most conserved protein, the nucleocapsid or N protein, of the equine arteritis virus (EAV) and identifies the smallest shared B cell epitope on it. This was achieved by immunizing mice with the N protein, generating monoclonal antibodies and using a variety of tests to identify the minimal conserved epitope.

Methods

• The purified N protein from EAV was used to immunize mice to generate monoclonal antibodies (mAbs).
• The reactivity of the produced mAbs was evaluated using Western blot and immunofluorescence assay. This step aimed to confirm that the antibodies were able to detect and interact with the N protein.
• To further understand and localize the epitope (area of the antigen recognized by the antibody) within the N protein, the researchers designed 11 overlapping peptides, named MBP-N1 to MBPB-N11.
• Indirect enzyme-linked immunosorbent assay (ELISA) and Western blot were used to identify the peptides recognized by the generated mAbs.

Results

• The minimal conserved B cell epitope on the EAV N protein, or the smallest shared protein sequence that is recognized by the B cells of the immune system, was identified successfully.
• An EAV N-reactive mAb, designated as 1C11, was selected out of those produced. Indirect ELISA results showed that the mAb 1C11 recognized the overlapping domain between MBP-N10 and MBP-N11.
• Further studies using indirect ELISA and Western blot revealed that the minimal linear epitope of the EAV N protein was (101)QRKVAP(106), a sequence of six amino acids.
• The researchers compared this epitope with other EAV strains and found it was conserved, present in all of them except one strain known as ARVAC.

Conclusion

• The researchers were successful in creating an EAV N-specific mAb (1C11) and identified a minimal linear peptide epitope ((101)QRKVAP(106)) present within the N protein.
• This discovery is significant, as it can help in designing diagnostic tests for EAV based on this specific antibody interaction, or potentially in treatment strategies targeting this conserved epitope.