Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

Emmanuel Bréard,Aurore Fablet,Damien Vitour,Bernd Hoffmann,Grégory Caignard,Corinne Sailleau,Cyril Viarouge,Lydie Postic,Stéphan Zientara,Martin Beer,Georges Beaud,Mathilde Turpaud

doi:10.3390/v13091741

Abstract

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

Highlights

enzyme-linked immunosorbent assay (ELISA) results obtained on sheep and 55 cattle from the field (Table 1), which were BTV-4 seroneutralization testing (SNT)-positive because they were naturally infected, were all ELISA-positive with optical density (OD) values between 0.3 and 2
The overall results show that the double antigen ELISA developed can be used to screen for BTV-4-specific antibodies from susceptible animals reared in Europe and the Mediterranean basin
The specificity of the test is excellent for the specific detection of BTV-4 vaccinated or infected animals; the test can yield positive results with sera from animals infected with serotypes 10, 12, 17 and 20, which have never been detected in Europe and the Mediterranean basin [4]

Summary

Introduction

Since the beginning of the 20th century, of the 24 conventional serotypes (1, 2, 3, 4, 6, 8, 9, 11 and 16) have been detected in Europe [4,5,6,7], as well as serotypes 25, 27 and 32 [8,9]. These three BTV strains belong to the class of BTV serotypes found in asymptomatic small ruminants [4,8]. In the European Union, animals infected with these non-virulent BTV strains are not reportable and do not lead to any restrictions on animal trade. 35 confirmed BTV serotypes have been identified worldwide [8]

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Conclusion