Identification of the Major Epitope in the GL Protein of Equine Arteritis Virus (EAV) Recognized by Antibody in EAV-infected Horses Using Synthetic Peptides.

Kondo, Takashi; Sugita, Shigeo; Fukunaga, Yoshio; Imagawa, Hiroshi

doi:10.1294/jes.9.19

JES

1998

DOI: 10.1294/jes.9.19

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Identification of the Major Epitope in the GL Protein of Equine Arteritis Virus (EAV) Recognized by Antibody in EAV-infected Horses Using Synthetic Peptides.

Takashi Kondo¹,

Shigeo Sugita

Yoshio Fukunaga

et al.

Abstract: A panel of overlapping peptides covering the ectodomain of the GL protein of equine arteritis virus (EAV) was synthesized to identify the major epitope recognized by the EAV-seropositive horse sera by using enzyme-linked immunosorbent assay (ELISA). Three consecutive peptides, G15 (amino acid residues 75-90), G16 (79-94) and G17 (83-98), strongly reacted with 3 experimentally EAV-infected horse sera. G16 peptide was highly antigenic with all 8 EAVinfected horse sera tested. The ELISA absorbance values and the … Show more

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Cited by 5 publications

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References 18 publications

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“…The humoral immune response of horses to EAV is mainly directed against the three major protein components of the virus (7,8,9,24,28,31). By pepscan analysis, an immunodominant epitope was localized between amino acids 70 and 99 of the EAV G L protein (30). Immunization of mice with EAV particles yielded both VN-positive (VN ϩ ) and VN-negative (VN Ϫ ) monoclonal antibodies (MAbs).…”

mentioning

confidence: 99%

Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

Weiland¹,

Bolz²,

Weiland³

et al. 2000

J Clin Microbiol

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We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G L ). Two of the EAV G L -specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G L MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G L -specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G L MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G L MAbs.on July 5, 2020 by guest http://jcm.asm.org/ Downloaded from FIG. 4. Immunoelectron microscopy of EAV particles. Sucrose gradient-purified virions were first incubated with MAb E7/d5-c1 (A), E3/c16 (B), or E1/A19 (C) and were then labeled with gold-conjugated secondary antibodies. Bar, 100 nm. 2072WEILAND ET AL.

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mentioning

confidence: 99%

Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

Weiland¹,

Bolz²,

Weiland³

et al. 2000

J Clin Microbiol

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“…In this 23 work, no positive sera from horses infected with the American strain were used. As 24 suggested by Kondo et al (1998), the reactivity to the peptide is highly specific to theA c c e p t e d M a n u s c r i p t 7 heterologous strain (not American) does not react with the peptide by an ELISA designed 1 over the Bucyrus strain. Other authors have been able to discriminate between serological 2 responses to European-genotype vaccines and European-genotype field strains of porcine 3 reproductive and respiratory syndrome virus, by using an ORF 4 peptide-based ELISA 4 (Oleksiewicz et al, 2005).…”

mentioning

confidence: 79%

Development of a peptide ELISA for the diagnosis of Equine arteritis virus

Metz

Lorenzón

Serena

et al. 2014

Journal of Virological Methods

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19A peptide-based indirect ELISA was developed to detect antibodies against Equine 20 arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein 21 M were designed, synthesized, purified and used as antigens either alone or combined. 22Ninety-two serum samples obtained from the 2010 equine viral arteritis outbreak, analyzed 23 previously by virus neutralization, were evaluated by the ELISA here developed. The best 24 resolution was obtained using peptide GP5. The analysis of the inter-and intraplate 25 variability showed that the assay was robust. The results allow concluding that this 26Page 2 of 16 A c c e p t e d M a n u s c r i p t 2 peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test 1 because it can be standardized between laboratories, can serve as rapid screening, can 2 improve the speed of diagnosis of EAV-negative horses and can be particularly useful for 3 routine surveillance in large populations. 4 KEY WORDS synthetic peptides-ELISA-Equine arteritis virus 5 6 Equine arteritis virus (EAV) belongs to the order Nidovirales, family Arteriviridae, 7 genus Arterivirus (Snijder and Meulenberg, 1998). The most relevant feature of EAV 8 infection is that it produces subclinical infection. However, the most important clinical signs 9 of the disease are abortions, respiratory disease in adult animals and pneumonia in foals. 10In Argentina, although serological evidence was first documented in 1984 (Nosetto et al., 11 1984), EAV was first isolated in 2001(Echeverría et al., 2003. Following the EAV 12 outbreak in 2010, the number of samples sent to the laboratory for EAV analysis was 13 significantly higher than the annual average of the previous years, reaching almost 5000 14 samples analyzed over a period of seven months. This increase highlighted the need for 15 an alternative technique to replace the virus neutralization test, which is, to date, the test 16 for international trade prescribed by the OIE (OIE, 2012). The virus neutralization test 17 detects antibodies against to EAV GP5 protein but is complex and high cost and requires 18 72 h to yield a result. Other difficulties include the considerable interlaboratory variation 19 and the contamination or nonspecific cellular cytotoxicity in sera from vaccinated horses 20 (Newton et al., 2004). Although several ELISAs have been developed, none have been 21 validated as extensively as the virus neutralization test. Some, however, offer comparable 22 specificity and almost equivalent sensitivity. The aim of this work was to design an ELISA 23 as a screening assay for EAV, using synthetic peptides. Indirect ELISA using peptides 24 containing GP5 neutralization epitopes may provide a simpler and more cost-effective 25 method to quantify EAV antibodies than the virus neutralization test. Another benefit of an 26Page 3 of 16 A c c e p t e d M a n u s c r i p t 3 EAV ELISA is that it can provide a same-day test result compared with the 72 h needed for 1 the virus neutralization test....

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Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (GL) of equine arteritis virus

Nugent

Sinclair

DeVries

et al. 2000

Journal of Virological Methods

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Identification of the Major Epitope in the GL Protein of Equine Arteritis Virus (EAV) Recognized by Antibody in EAV-infected Horses Using Synthetic Peptides.

Cited by 5 publications

References 18 publications

Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

Development of a peptide ELISA for the diagnosis of Equine arteritis virus

Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (GL) of equine arteritis virus

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