Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Antibody to Epizootic Hemorrhagic Disease of Deer Virus

Abstract

Abstract. An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.Epizootic hemorrhagic disease of deer viruses (EHDVs) are members of the orbivirus genus of the family Reoviridae. These viruses cause an often fatal hemorrhagic disease of white-tailed deer and can cause a bluetongue-like illness in cattle. 8 Two serotypes of EHDV, designated EHDV-1 (New Jersey) and EHDV-2 (Alberta), are enzootic in the US. The agar gel immunodiffusion (AGID) test is the primary groupspecific test used for detection of antibodies to either bluetongue viruses (BTVs) or EHDVs; however, serological cross-reactivity between these two groups of viruses can result in false-positive results. Serum neutralization is used for serotype determination. 13 Enzyme-linked immunosorbent assay (ELISA) procedures that are highly sensitive and specific have been developed for the detection of antibody to BTV. 10 Because of their disease potential, the development of improved diagnostic tests for EHDV is needed. ELISA procedures showing various degrees of specificity and sensitivity with both polyclonal and monoclonal antibody-based formats have been described for the detection of antibody to EHDV. 1,3,7,[14][15][16] This paper describes the development of a monoclonal antibody-based ELISA for the detection of antibody to EHDV.Hybridoma cultures were prepared from the spleens of adult female BALB/c mice that had been immunized by a series of intraperitoneal and intravenous inoculations with EHDV-2 (Alberta). Monoclonal antibody production in the hybridoma cultures was determined in an indirect binding ELISA with antigen produced from EHDV-2-infected cell cultures 6 and anti-mouse IgG conjugated to horseradish peroxidase. The specificity of positive hybridoma culture fluids

Antigen Capture Competitive Enzyme-Linked Immunosorbent Assays Using Baculovirus-Expressed Antigens for Diagnosis of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus

“…Optical density (OD) measurement of the color change following addition of OPD allowed calculation of the percent inhibition (PI) of binding of the MAb to the captured antigen and a relative measure of the concentration of specific antibody in the test serum sample. Percent inhibition was calculated as PI ϭ 100 Ϫ (OD 492 of test serum/ OD 492 of negative serum) ϫ 100 (12). Optimal reagent concentrations for the Ag Cap c-ELISA were determined by cris-cross serial dilutions (6).…”

“…Samples from baculovirus-infected Sf9 cell cultures were electrophoresed using the discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system of Laemmli (7) and transferred by electroblotting to nitrocellulose membranes. Recombinant viral proteins were detected by chemiluminescence with the West Femto kit (Pierce), using either a rabbit polyclonal antibody to BTV-11 or a mouse MAb (4F4.H1) (12) to EHDV-2, VP7.…”

“…Diagnosis of infections caused by these two viruses is often confounded because of their antigenic similarity (15). To overcome this problem, competitive enzyme-linked immunosorbent assay (c-ELISA) procedures have been developed for the serologic diagnosis of infections caused by these two groups of viruses (1,2,12,18). Because of their sensitivity and specificity, c-ELISAs have become the assays of choice for serologic monitoring of infections caused by these viruses (3, 16).…”

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups

“…26 The need for a differential diagnostic assay is emphasized by the serologic evidence of widespread EHDV infection in U.S. cattle. 22 An understanding of the molecular genetics of these viruses is needed for the development of rapid and sensitive molecular diagnostic assays. The protein coding assignments for BTV and EHDV are identical, consisting of 10 genome segments that encode 7 structural and 4 nonstructural proteins.…”

Detection of All Eight Serotypes ofEpizootic Hemorrhagic Disease Virusby Real-Time Reverse Transcription Polymerase Chain Reaction

“…1 There is also serologic evidence of infection in and isolation of EHDV from U.S. cattle and sheep. 14,23 Therefore, rapid diagnosis and differentiation of BTV and EHDV are needed for both livestock and wildlife.…”