Neutralization determinants of laboratory strains and field isolates of equine arteritis virus

Identification of four neutralization sites in the amino-terminal ectodomain of the G(L) envelope glycoprotein

Udeni B R Balasuriya, John F. Patton, Paul V. Rossitto, Peter J. Timoney, William H. McCollum, Nigel J Maclachlan

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Abstract

The N-terminal hydrophilic ectodomain of the G(L) envelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding G(L) envelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 1084, indicating that they also recognize epitopes on the G(L) protein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified- live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the G(L) protein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1 variable region (amino acids 61-121) within the second half of the N-terminal hydrophilic ectodomain of the G(L) protein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.

Original languageEnglish (US)
Pages (from-to)114-128
Number of pages15
JournalVirology
Volume232
Issue number1
DOIs
StatePublished - May 26 1997

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Equine Arteritis Virus
Glycoproteins
Monoclonal Antibodies
Viruses
Epitopes
Amino Acids
Proteins
Viral Proteins
Amino Acid Substitution
North America
Neutralizing Antibodies
Immunoblotting
Horses
Immune Sera
Amino Acid Sequence
Vaccines
Western Blotting
Rabbits
Phenotype

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Neutralization determinants of laboratory strains and field isolates of equine arteritis virus : Identification of four neutralization sites in the amino-terminal ectodomain of the G(L) envelope glycoprotein. / Balasuriya, Udeni B R; Patton, John F.; Rossitto, Paul V.; Timoney, Peter J.; McCollum, William H.; Maclachlan, Nigel J.

In: Virology, Vol. 232, No. 1, 26.05.1997, p. 114-128.

Research output: Contribution to journalArticle

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abstract = "The N-terminal hydrophilic ectodomain of the G(L) envelope glycoprotein of equine arteritis virus (EAV) contains neutralization determinants of the virus. We developed a panel of 17 neutralizing murine monoclonal antibodies (MAbs) to further characterize the neutralization determinants of EAV. Included were 6 MAbs previously raised against a laboratory strain (EAVUCD) of the original Bucyrus strain of EAV, as well as 11 additional MAbs that were raised against a neutralization-resistant variant [escape mutant (EM)] virus (EM6D10) that was derived from EAVUCD. All MAbs raised against EAVUCD and 4 of the MAbs raised against EM6D10 (2B3, 5F8, 8D4, and 10B4) reacted with the corresponding G(L) envelope glycoprotein in a Western immunoblotting assay, whereas the remaining 7 MAbs raised against EM6D10 did not react with any viral protein in the immunoblotting assay but competitively inhibited the binding of MAbs 2B3, 5F8, 8D4, and 1084, indicating that they also recognize epitopes on the G(L) protein. A panel of 18 EM viruses raised to the MAb panel, 19 field isolates of EAV from North America and Europe, the modified- live virus vaccine (ARVAC), and 3 other laboratory strains of EAV were characterized by microneutralization assay with the panel of neutralizing MAbs and polyclonal rabbit and horse antisera. Comparative analysis of the nucleotide sequences of ORF5 and the deduced amino acid sequences of the G(L) protein of individual EM viruses and field isolates of EAV identified four distinct neutralization sites. These sites include amino acids 49 (site A), 61 (site B), 67 through 90 (site C), and 99 through 106 (site D). With the notable exception of site A, the sites were all located in the V1 variable region (amino acids 61-121) within the second half of the N-terminal hydrophilic ectodomain of the G(L) protein. Site D includes several overlapping linear epitopes which appear to interact with amino acids in the other three sites to form conformationally dependent epitopes. Amino acid substitutions within any of these four sites can alter the neutralization phenotype of individual strains of EAV.",
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