Identification of African Horse Sickness Virus in Cell Culture using a Digoxigenin-Labeled RNA Probe

A digoxigenin-la beled RNA probe was synthesized from a plasmid containing a portion of the African horse sickness virus (AHSV) serotype 4 genome segment coding for nonstructural protein 1. In an in situ hybridization procedure, this probe hybridized successfully to Vero cells infected with each of the 9 serotypes of AHSV. There was no hybridization with noninfected cell cultures or cell cultures infected with bluetongue virus. African horse sickness (AHS) is a viral disease of horses characterized by an acute, severe clinical disease with high mortality. 11 This disease is spread primarily by biting flies of the genus Culicoides. Because of the nonspecific nature of many of the clinical signs, rec- ognition of the syndrome can be problematic. 2 African horse sickness currently is confined to the African con- tinent, but within the last decade it has spread to other areas (Spain, Portugal) and caused severe epizootics. Currently, all diagnostic tests rely on immunologic procedures. Definitive diagnosis rests on virus isola- tion, with the identity of the virus and serotyping con- firmed by virus neutralization. Numerous serologic tests have been devised, 6 and sandwich enzyme-linked im- munosorbent assays for the detection of antigen in infected animal tissues recently have been devel- oped. 5,10 The aim of this study was to determine if a nucleic acid-based technology could be used to successfully detect AHS virus (AHSV) in cell culture monolayers, specifically to determine if a digoxigenin-labeled RNA probe could hybridize with all 9 serotypes of AHSV in cell culture monolayers.