Recombinant VP6-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Eyach virus (genus Coltivirus)

Background: Eyach virus (EYAV) is a tick-borne virus belonging to genus Coltivirus, family Reoviridae. It was isolated in Germany and France and has been suspected to be responsible for neurological diseases in humans. To date, there has been no relatively rapid and relatively specific serological assay for EYAV Objectives: To develop an ELISA for EYAV, suitable for epidemiological and/or diagnostic purposes. This ELISA should allow to distinguish between infections with EYAV and the related Colorado tick fever virus (CTFV). Study Design: VP6, VP7 and VP12 of Eyach virus (the three proteins most divergent between EYAV and CTFV) were expressed in bacteria using the pGEX-4T-2 vector. A partial sequence of VP6 (designated pVP6) was chosen to develop an ELISA for detecting anti-EYAV IgG antibodies in serum. This choice was based on two observations: (i) the homologous VP7 protein of CTFV was successfully used as a target for detecting antibodies to CTFV (the VP7 showed the highest reactivity to an anti-CTFV antibody among all CTFV expressed proteins); (ii) to distinguish infection with EYAV from a CTFV infection: the expressed sequence was chosen within a region which is highly divergent (49% of amino acid identity) from the homologous VP7 sequence of CTFV Results and conclusions: pVP6 was shown to be the most reactive among the three expressed proteins. The elaborated pVP6 ELISA was evaluated with 340 sera of French blood donors, and found to exhibit a specificity of 100% (no false positives). Furthermore, no cross reaction was detected with antibody to CTFV thus permitting us to distinguish between infections by either virus. The use of this recombinant protein for serological assays is a good alternative to the use of native EYAV antigen due to the extremely low productivity of the virus in cell culture, and the requirement for suckling mice. This ELISA will be useful to clarify the epidemiological status and the suspected pathogenicity of the virus. (C) 2004 Elsevier B.V. All rights reserved.