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

Abstract

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....

Synthetic Peptides Derived from Ribosomal Proteins of Leishmania spp. in Mucocutaneous Leishmaniasis: Diagnostic Usefulness

“…Epitope mapping is a methodology used for identifying each linear sequence acting as epitope in order to make detecting antibodies more specific. In that sense, intracellular protein-derived peptides have been studied, in which immunogenic determinants have been identified, so using epitope mapping methods, these immunodominant peptides have shown to be useful for diagnosing leishmaniasis in humans and canines, [19,20] as well as in Chagas' disease [21,22] and diseases caused by other infectious agents (such technique having up to 100% sensitivity and specificity) [23,24].…”

The Use of Peptides in Veterinary Serodiagnosis of Infectious Diseases: A Review

“…Another disease, the equine arteritis virus is responsible for vascular lesions, respiratory disease in adults and foals, and abortion; the pELISA was designed with two peptides of different proteins but in the end, one of them showed better discrimination capacity between positive and negative serum, additionally, the samples were characterized previously with a virus neutralization test. The study revealed a sensitivity of 95.65% and a specificity of 80.43% [ 59 ]. This pELISA and the other described in the previous paragraph were developed in Argentina.…”