Neutralization Determinants of Laboratory Strains and Field Isolates of Equine Arteritis Virus: Identification of Four Neutralization Sites in the Amino-Terminal Ectodomain of the GLEnvelope Glycoprotein

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 10B4, 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.

Serologic Response of Horses to the Structural Proteins of Equine Arteritis Virus

“…Furthermore, a number of sera that had significant titers of neutralizing antibody as determined by SN test did not bind the G L protein in the immunoblotting assay despite the fact that the G L protein expresses the known neutralization determinants of EAV. [1][2][3]8,17,21 However, the immunoblotting assay described here is very useful in confirming the protein specificity of antibodies in horse serum, and the data strongly suggest that the M protein should be targeted for future development of an improved diagnostic ELISA for serologic detection of EAV infection of horses. Furthermore, the immunoblotting assay has potential diagnostic utility.…”

“…g The authenticity of the ORF in each recombinant plasmid then was confirmed by sequence analysis of both strands using internal primers. Sequencing was done as previously described 2,23 with AmpliTaq DNA polymerase, FS, and an automatic sequencer. h Appropriate clones were selected for transfection, and large quantities of each plasmid were purified using a commercial kit.…”

“…Eight of the 13 sera from horses inoculated with field strains of EAV and 3 of 4 sera from vaccinated horses recognized the N protein, although 2 of these sera recognized only the baculovirus-expressed N protein antigen and not the N protein in the EAV virion preparation. The G L protein expresses the known neutralization determinants of EAV, [1][2][3]8,17,21 but despite the fact that all 17 sera from the vaccinated and experimentally inoculated horses were positive by SN test, only 8 of these sera bound the G L protein and 3 bound only the G L protein in EAV virions and not the G L protein expressed from baculovirus. None of the sera from experimentally infected horses reacted with the G S protein.…”

“…13 The G L protein expresses the known neutralization determinants of EAV. [1][2][3]8,17,21 The genome of EAV includes at least 8 open reading frames (ORFs). 11,16 ORFs 1a and 1b encode the viral replicase, whereas ORFs 2-7 are nested and located at the 3Ј end of the genome.…”

Sero-epidemiologic survey of the equine arteritis in the region of Khenchela (Algeria)

“…They were immediately frozen and stored at-20ºC until analysed by the technique of sero-neutralization in microplate on vero cells in monolayer. This is a reference method widely used for the diagnosis of equine viral arteritis (Balasuriya and al., 1997;Senne and al., 1985;Timoney, 2000b).…”