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Comparison of equine arteritis virus isolates using neutralizing monoclonal antibodies and identification of sequence
A L Glaser1, A A de Vries, E J Dubovi
1Diagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca 14852-5786, USA.
Abstract:
Three murine monoclonal antibodies (MAbs) that neutralize equine arteritis virus (EAV) infectivity were identified and characterized. The antibodies, 93B, 74D(B) and 38F, recognized the major envelope glycoprotein (GL) encoded by open reading frame (ORF) 5 in immunoblots and by immunoprecipitation. All three MAbs were used to compare the Bucyrus isolate of EAV and MAb neutralization-resistant (NR) escape mutants with the vaccine virus and 19 independent field isolates of EAV by virus neutralization. The different abilities of the MAbs to neutralize virus isolates indicated that they recognize non-identical epitopes. Susceptibility to virus neutralization could not be used to distinguish viruses from acutely and persistently infected horses. Comparison of the ORF 5 nucleotide and deduced amino acid sequence from NR and neutralization-sensitive virus isolates revealed amino acid sequence changes at positions 99 and 100 which correlate with the NR phenotype. Additional unique changes in the amino acid sequence of MAb NR viruses at positions 96 and 113 may also contribute to neutralization resistance. The sequence data further showed that the Bucyrus-derived viruses contain one N-glycosylation site, whereas the field isolates DL8 and DL11 possess two sites, both of which are used. Most of the non-conservative amino acid sequence changes were located within the second half of the N-terminal hydrophilic domain. Sequence changes within the first half of the N-terminal ectodomain, the predicted transmembrane domain and the C-terminal hydrophilic domain were mainly silent base substitutions or resulted in conservative amino acid substitutions, suggesting that these regions of the protein are functionally conserved.
Insights
Three monoclonal antibodies neutralize equine arteritis virus (EAV) by targeting its envelope glycoprotein (GL). Amino acid changes in ORF 5 correlate with neutralization resistance, aiding in understanding EAV evolution and infection.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Equine arteritis virus (EAV) causes significant disease in horses.
- Understanding EAV neutralization is crucial for vaccine development and disease control.
- The EAV envelope glycoprotein (GL) encoded by ORF 5 is a key target for neutralizing antibodies.
Purpose of the Study:
- To characterize murine monoclonal antibodies (MAbs) that neutralize EAV.
- To identify the epitopes recognized by these MAbs on the EAV GL.
- To investigate the genetic basis of MAb neutralization resistance in EAV isolates.
Main Methods:
- Production and characterization of three neutralizing MAbs (93B, 74D(B), 38F).
- Immunoblotting and immunoprecipitation to identify MAb targets.
- Virus neutralization assays comparing EAV isolates and escape mutants.
- Nucleotide and amino acid sequencing of EAV ORF 5.
Main Results:
- MAbs 93B, 74D(B), and 38F recognize the EAV GL (ORF 5).
- These MAbs bind to distinct epitopes, as indicated by differential neutralization of EAV isolates.
- Amino acid substitutions at positions 99 and 100 in GL correlate with MAb neutralization resistance.
- Additional changes at positions 96 and 113 may also contribute to resistance.
- Sequence analysis revealed variations in N-glycosylation sites between different EAV strains.
Conclusions:
- Neutralizing MAbs target specific epitopes on the EAV GL.
- Amino acid changes in ORF 5 are critical for EAV's resistance to antibody neutralization.
- These findings provide insights into EAV evolution and host-pathogen interactions.