Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Shan Zhao,Nicola Pusterla,Samantha Barnum,Berend-Jan Bosch,Nancy Schuurman,Herman Egberink,Constance Smits,Frank Van Kuppeveld,Kees Van Maanen

doi:10.3390/v11121109

Abstract

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Highlights

Coronaviruses (CoVs) are enveloped, positive single-stranded RNA viruses that belong to the subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales
To identify equine sera containing Equine coronavirus (ECoV)-neutralizing antibodies, we screened a subset of 231 equine sera, composed of randomly selected serum samples from panels A–C, and all samples from panel D were screened in the virus neutralization (VN) assay
Results assess the correlation between the values obtained with the two Results indicate that optical densities (OD) values obtained with both enzyme-linked immunosorbent assay (ELISA) show a high degree of correlation, with correlation indicate that OD

Summary

Introduction

Coronaviruses (CoVs) are enveloped, positive single-stranded RNA viruses that belong to the subfamily Orthocoronavirinae in the family Coronaviridae of the order Nidovirales. They are classified into four genera (alpha-, beta-, gamma- and deltacoronavirus) and infect both mammalian and avian hosts [1,2]. Equine coronavirus (ECoV) belongs to Betacoronavirus 1 species, within the Embecovirus subgenus of the Betacoronavirus genus, as does human coronavirus OC43, HKU1 and bovine coronavirus [3]. Since 2010, several cases of ECoV infections have been reported in adult horses from the United States, Europe and Japan [5,6,7,8,9].

Objectives

Methods

Results