Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence changes in GL associated with neutralization resistance.

The research discusses the identification and characterization of three murine monoclonal antibodies known for neutralizing equine arteritis virus. It examines the sequence changes that render the virus immune to neutralization and how these changes can’t be used to differentiate viruses from acutely and persistently infected horses.

Identified and Characterized Monoclonal Antibodies

• The study successfully identified three monoclonal antibodies, named 93B, 74D(B), and 38F, that neutralize the equine arteritis virus (EAV) infectivity.
• The antibodies were confirmed to recognize the major envelope glycoprotein (GL), which is encoded by open reading frame (ORF) 5. This was established through immunoblots and immunoprecipitation methods.
• These antibodies were then used to compare various EAV isolates such as the Bucyrus isolate, MAb neutralization-resistant escape mutants, the vaccine virus, and 19 independent field isolates.

Neutralization Resistance and Nucleotide Sequence Changes

• The research found different capabilities of the antibodies to neutralize the virus isolates, which indicated that the antibodies recognize non-identical epitopes or parts of the virus.
• Neutralization susceptibility was not found to be effective in distinguishing viruses from acutely and persistively infected horses.
• Amino acid sequence alterations at positions 99 and 100 were found in neutralization-resistant and neutralization-sensitive virus isolates. These changes correlated with the neutralization-resistant phenotype.
• Other unique sequence changes at positions 96 and 113 might also contribute to the resistance to neutralization by these antibodies.

Glycosylation Sites and Sequence Changes

• The Bucyrus-derived virus isolates were found to contain one N-glycosylation site, which is a biochemical process necessary for the functioning of proteins. In contrast, the field isolates DL8 and DL11 contained two such sites, both of which were utilized.
• Most of the non-conservative amino acid sequence alterations were located within the second half of the N-terminal hydrophilic domain, suggesting that significant changes occur in this region.
• On the other hand, the sequence changes in the first half of the N-terminal ectodomain, the predicted transmembrane domain, and the C-terminal hydrophilic domain mostly led to silent base substitutions or resulted in conservative amino acid substitutions. This indicates that these regions of the glycoprotein are functionally conserved and maintain their structure and function despite slight changes in the sequence.