A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies

Balasuriya, Udeni B. R.; Rossitto, Paul V.; DeMaula, Christopher D.; Maclachlan, Nigel J

doi:10.1099/0022-1317-74-11-2525

Cited by 58 publications

(61 citation statements)

References 20 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…The production and characterization of MAbs to the G L (MAb 6D10) and N (MAb 3E2) proteins of EAV has been previously described. 1,3,35 Polyclonal rabbit antisera to the G S and M proteins of EAV were prepared by immunization of rabbits with synthetic peptides specific for the amino acid sequence of the extreme carboxy terminus of each protein (G S : NH 2 -Cys-Pro-Ser-Arg-Arg-Thr-Ser-Ser-Gly-Thr-LeuPro-Arg-Arg-Lys-Ile-Leu-COOH; M: NH 2 -Tyr-Ala-GlyArg-Leu-Phe-Ser-Lys-Arg-Thr-Ala-Ala-Thr-Ala-Tyr-LysLeu-Gln-COOH). 13 Rabbits initially were immunized by subcutaneous inoculation of 10 mg of peptide conjugated to keyhole limpet hemocyanin in adjuvant j and subsequently boosted 3 times at 3-wk intervals by subcutaneous inoculation of the peptide in Freund's complete or incomplete adjuvant.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Characterization Of Baculovirus-expressed Eav Proteinsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

et al. 1998

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Abstract. Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, an apparently emerging disease of equids. In this study, the antibody response of horses to the structural proteins of EAV was evaluated using gradient-purified EAV virions and baculovirus-expressed recombinant EAV structural proteins (G L , G S , M, N) as antigens in a Western immunoblotting assay. Thirty-three sera from horses that previously had been naturally or experimentally infected with EAV were evaluated, including samples from mares, geldings, and both persistently and nonpersistently infected stallions. Sera also were evaluated from 4 horses that had been vaccinated with the commercial modified live EAV vaccine. The data suggest that the serologic response of individual horses to EAV may vary with the infecting virus strain and duration of infection. The M protein was most consistently recognized by the various serum samples, whereas the response to the N and G L proteins was variable and the G S protein was bound by only 1 serum sample. The immunoblotting assay definitively established the protein specificity of the humoral response of horses to EAV; however, it clearly is less sensitive than the standard serum neutralization (SN) test-2 of the 37 sera that were seropositive by the SN test failed to react in the immunoblot assay with any EAV structural protein. Furthermore, the G L protein expresses the known neutralization determinants of EAV, yet only 22 of the 37 sera that had SN antibodies bound the G L protein in the immunoblotting assay. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection of horses.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a disease of horses that may manifest as interstitial pneumonia in very young foals, influenza-like illness of adult horses, and abortion of pregnant mares. 6,10,18,19,22,31,43,45,51 Persistently infected stallions that shed virus in their semen are a critical natural reservoir of EAV because venereal infection of mares frequently occurs after mating with such stallions. 26,34,40,[44][45][46][47]50 Horizontal spread of the virus occurs via aerosol exposure of susceptible horses to acutely infected animals. 38,44,45 EAV infection is especially prevalent in Standardbred horses, but since the mid-1980s there has been a disturbing increase in the incidence of EVA in a variety of horse breeds. 5,20,24,25,39,44,45,48,49 EAV is an enveloped, positive-stranded RNA virus 27,36,37,41 that recently was taxonomically placed in the genus Arterivirus in the order Nidovirales, along with porcine reproductive and respiratory syndrome virus, lactate dehydrogenase-elevating virus, and simian hemorrhagic fever virus. 7 The EAV virion has a diameter of approximately 55 nm and consists of a

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Characterization Of Baculovirus-expressed Eav Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 1998

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“…A live attenuated vaccine (Doll 0001-3134 © 1995 SGM E. D. Chirnside and others et al, 1968;Harry & McCollum, 1981;Timoney et al, 1988) and inactivated whole virus vaccines (McCollum, 1986;Fukunaga et al, 1990Fukunaga et al, , 1992 induce VN antibody responses in horses. Western blotting and competition ELISA data (Balasuriya et al, 1993) indicate that strongly neutralizing monoclonal antibodies recognize a single epitope on a 29 kDa EAV protein which is likely to correspond to G L. Recently Deregt et al (1994) demonstrated that from a panel of monoclonal antibodies to the EAV N and GT, proteins all of the neutralizing antibodies recognized G~. Competitive binding assays revealed that they all bound to the same or to closely adjacent sites on the GL protein.…”

Section: Introductionmentioning

confidence: 99%

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL

Chirnside

Vries

Mumford

et al. 1995

Journal of General Virology

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Complementary DNAs encoding ORFs 2 to 7 of equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immunodominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-Specific synthetic peptide (residues 75 through 97) induced EAV-nentralizing antibody in vaccinated horses. The defined antigenic region of G L is likely to be exposed on the surface of the native EAV virion and consequently may be useful in the development of diagnostic tests and vaccines.

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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

et al. 1997

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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.

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A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies

Cited by 58 publications

References 20 publications

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

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

Equine arteritis virus-neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein GL

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

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