Genomic characterization of neutralization-resistant monoclonal antibody escape variants of aquatic birnaviruses

To identify regions on the major capsid protein (VP2) of aquatic birnaviruses associated with antibody neutralization and replication, four neutralizing monoclonal antibodies (MAbs AS-1, W3, W4 and E3) which recognize different epitopes on VP2 were used to select neutralization-resistant variants from the West Buxton (WB) and European eel virus (EEV) strains. All variants were fully refractory to neutralization by the selecting MAb. Nucleotide and deduced amino acid sequences of the VP2 coding region were determined for each variant and compared with the wild type virus sequence. Amino acid substitutions were found at one or two positions in VP2 of the variant viruses. These amino acid substitutions occurred in a relatively few regions of VP2. All three variants selected with MAb AS-1 exhibited an amino acid substitution at position 73 at the N-terminal, highly conserved end of VP2. Amino acid changes in VP2 of all variants selected with other MAbs occurred in the central variable region at amino acid positions 245–251, 268, 288, 317, and 321. In some cases, specific amino acid substitutions resulted in loss of ability to react with the selecting MAb in immunodot assays, indicating the possibility that this region of VP2 falls within the epitope recognized by the MAb. In other cases, the escape variant still bound the selecting MAb, suggesting that the amino acid changes in VP2 of these viruses likely were not in regions forming the structure of the specific epitope. In addition to conferring resistance to MAb neutralization, the changes in one or two amino acids of VP2 of most variants also resulted in changes in ability of the variants to replicate in various fish cell cultures.; This investigation provides the first information about the locations of VP2 forming specific neutralizing epitopes or affecting the specificity of specific neutralizing epitopes. This information will facilitate the development of genetically engineered vaccines and/or contribute to our understanding of the mechanisms of antibody neutralization of this group of viruses. Furthermore, these variants may serve as useful in vitro models for further investigations host range, tissue tropism and virulence observed among aquatic birnaviruses.