In-situ hybridization for demonstration of equine herpesvirus type 1 DNA in paraffin wax-embedded tissues and its use in horses with disseminated necrotizing myeloencephalitis

Abstract

The detection of equine herpesvirus type 1 (EHV-1) in infected cell cultures, and in tissues taken at necropsy, by the in-situ hybridization technique is described. A 4.9 kb Bam HI fragment of EHV-1 vaccine strain RacH was used as a probe after labelling with [alpha-32P] thymidine 5'-triphosphate ([32P]TTP) or digoxigenin-deoxyuridine 5'-triphosphate (dUTP). Both probes specifically detected EHV-1 DNA in either cytospin or paraffin wax-embedded preparations of infected cells. The digoxigenin-labelled probe was further used to examine tissue sections of equine fetuses which had been aborted due to EHV-1 infection. In all cases positive hybridization signals were mainly associated with the nuclei. Positive results were confirmed by immunostaining of EHV-1 antigen in adjacent sections. However, both methods failed to detect EHV-1 in spinal cord sections of six horses suffering from disseminated necrotizing myeloencephalitis (DNM). These results support the hypothesis that DNM is not caused by a productive viral infection of parenchyma of the nervous system but is immunologically mediated.