Detection of chelonid herpesvirus DNA by nonradioactive in situ hybridization in tissues from tortoises suffering from stomatitis-rhinitis complex inEurope and North America

Chelonid herpesvirus (ChHV) infection in tortoises associated with stomatit is-rhinitis complex is a severe, mostly epizootic disease characterized by proliferative and diphtheroid-necrotizing glossitis, pharyngitis, rhinitis, and tracheitis, often occurring with pneumonia and encephalitis. The UL5 g ene from a German ChNV isolate was used to generate a digoxigenin-labeled 3 07-base-pair DNA probe by polymerase chain reaction (PCR). ChHV DNA was det ected in paraffin-embedded tissues of five naturally infected tortoises (tw o Afghan tortoises [Testudo horsefieldii], USA; two Hermann's tortoises [Te studo hermanni], Switzerland; one T. hermanni, Germany) by means of in situ hybridization (ISH) and PCR. Distribution of ChHV DNA exhibits many charac teristics of alphaherpesvirus but also some characteristics of betaherpesvi rus infections. The amino acid sequence of a portion of the ChHV UL5 homolo g exhibited more than 50% similarity to alphaherpesvirus UL5 proteins. Nucl ear hybridization signals were detected in epithelial cells of the lingual mucosa and glands. Furthermore, ChHV DNA was observed in tracheal epitheliu m, pneumocytes, hepatocytes, the renal tubular epithelium, cerebral glia ce lls and neurons, and intramural intestinal ganglia. ChHV DNA in endothelial cells of many organs underlines the systemic character of the disease. Imp ortantly, ChHV DNA was detected by ISH in multiple tissues of tortoises ori ginating from different geographic provenances. This indicates a high degre e of conservation of the UL5 gene fragment among viruses prevalent in torto ises on different continents. With the described ISH, a molecular biologica l tool is available for rapid and specific diagnosis of ChHV infections and , Inert: importantly, comparative pathogenetic studies of ChHV isolates fro m geographically unrelated regions.