Development of a peptide ELISA for discrimination between serological responses to equine herpesvirus type 1 and 4 (EHV-1 and EHV-4)

Abstract

Infections with equine herpesviruses type 1 (EHV-1) and 4 (EHV-4) can result in important economic losses for the horse industryworldwide. Both viruses are common causes of acute respiratory disease, while EHV-1 frequently induces abortion but also perinatal foal mortality and devastating neurological disease. EHV-1 and -4 are closely related alphaherpesviruses. Detection of EHV antibodies in serum is a common tool to determine whether a horse has been exposed to EHV. Despite the differences in their biological characteristics and genomic sequences, both viruses show strong antigenic cross-reactivity in any immunological assay that uses polyclonal serum as the source of antibodies. An effective serological tool capable of discriminating between antibodies responses to EHV-1 or EHV-4 in horses is of importance in disease control. In the present study, we describe the development and application of a type-specific EHV-1/EHV-4 ELISA based on peptide antigens. Seven and four peptides for EHV-1 and EHV-4, respectively, were initially studied with respect to their discriminatory potential in an ELISA setup using sera from experimentally infected foals. In our optimized protocol, plates were coated overnight at 4 C with 100 ml/well streptavidin at 1 mg/ml in 50 mM carbonate/bicarbonate buffer (pH 9.6), washed 3 times with washing buffer (PBS containing 0.1% Tween 20) and coated with 100 ml/well of respective biotinylated peptide at a concentration of 2 mg/ ml in carbonate/bicarbonate buffer. After incubation for 2 h at 37 C and extensive washing, wells were blocked with 100 ul/well of blocking buffer (1% goat serum in washing buffer) for 1 h at 37 C. After washing, 100 ml of horse serum diluted 1/400 or 1/1200 in washing buffer was added in triplicate and incubated for 1 h at 37 C. After 3 further washes, plates were incubated with 100 ml/well of HRP goat anti-horse immunoglobulin diluted 1/20,000 in blocking buffer for 1 h at 37 C. Following a final wash, the color reaction was developed for 10 min at room temperature by adding 100 ml/well of a chromogen/substrate mixture of TMB (240 mg/mL 3,3’,5,5’ tetramethylbenzidine) in Gallati buffer (42 mg/mL citric acid, pH 3.95/0.01% H2O2). The reaction was stopped with 100 ml/well of 1 M sulfuric acid and the optical density measured at 450 nm. Results were expressed as absorbance OD values and sera tested in triplicate against the EHV-1 and EHV-4 antigen. The most promising pair of peptides, EHV-1 glycoprotein E and EHV4 glycoprotein G peptides, was further evaluated using acute and convalescent sera from experimentally and naturally infected horses (Figure 1) as well as a panel of field sera. The results show that our peptide ELISA clearly identifies horses that have been infected with EHV-1 or EHV-4 using acute and convalescent sera and, when applied to a large number of field samples, revealed to be a robust assay for determining the EHV-1 and EHV-4 antibody status. With further validation, the developed EHV-1/EHV-4 peptide ELISA could serve as an effective and cheaper alternative to other current tools for EHV-1 and -4 serodiagnosis.