DETECTION OF RABBIT HEMORRHAGIC-DISEASE VIRUS (RHDV) BY IN-SITU HYBRIDIZATION WITH A DIGOXIGENIN-LABELED RNA PROBE

An in-situ hybridisation (ISH) technique for the detection of rabbit h aemorrhagic disease virus (RHDV) was developed. Thirteen seronegative adult rabbits were infected ore-nasally using the BS89 RHDV strain. Li ver and spleen samples were collected from 4 h post infection (p.i.) a nd repeated every 4 h till 44 h p.i. Each sample was tested immunohist ochemically, by sandwich ELISA and by ISH. A 2.482-kb RNA probe, match ing the genomic fragment coding for the VP60 structural protein of RHD V, was arranged. Two RNA probes (sense and antisense) were transcribed in vitro and UTP-digoxigenin-labelled. The antisense probe clearly de tected positivity in the cytoplasm of the hepatocytes at 8 h p.i. Labe lled hepatocytes were scattered throughout the sections until 24 h p.i . followed by a more diffuse perilobular positive reaction. A much wea ker signal of similar distribution was detected up to 24 h p.i. using the sense RNA probe. All spleen samples tested negative for both probe s. Liver samples were positive at 32 h p.i. using both ELISA and the i mmunoperoxidase test. Spleen samples were positive using only the ELIS A at 32 h p,i. This study showed that RHDV replication occurred almost immediately after inoculation and that the liver appears to be the ma in site of replication. (C) 1998 Elsevier Science B.V. All rights rese rved.