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Detection of rabbit haemorrhagic disease virus (RHDV) by in situ hybridisation with a digoxigenin labelled RNA probe

An in-situ hybridisation (ISH) technique for the detection of rabbit haemorrhagic disease virus (RHDV) was developed. Thirteen seronegative adult rabbits were infected oro-nasally using the BS89 RHDV strain. Liver and spleen samples were collected from 4 h post infection (p.i.) and repeated every 4...

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Detalles Bibliográficos
Autores principales: Gelmetti, Daniela, Grieco, Valeria, Rossi, Cesare, Capucci, Lorenzo, Lavazza, Antonio
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier Science B.V. 1998
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7120613/
https://www.ncbi.nlm.nih.gov/pubmed/9694329
http://dx.doi.org/10.1016/S0166-0934(98)00030-5
Descripción
Sumario:An in-situ hybridisation (ISH) technique for the detection of rabbit haemorrhagic disease virus (RHDV) was developed. Thirteen seronegative adult rabbits were infected oro-nasally using the BS89 RHDV strain. Liver and spleen samples were collected from 4 h post infection (p.i.) and repeated every 4 h till 44 h p.i. Each sample was tested immunohistochemically, by sandwich ELISA and by ISH. A 2.482-kb RNA probe, matching the genomic fragment coding for the VP60 structural protein of RHDV, was arranged. Two RNA probes (sense and antisense) were transcribed in vitro and UTP-digoxigenin-labelled. The antisense probe clearly detected positivity in the cytoplasm of the hepatocytes at 8 h p.i. Labelled hepatocytes were scattered throughout the sections until 24 h p.i. followed by a more diffuse perilobular positive reaction. A much weaker signal of similar distribution was detected up to 24 h p.i. using the sense RNA probe. All spleen samples tested negative for both probes. Liver samples were positive at 32 h p.i. using both ELISA and the immunoperoxidase test. Spleen samples were positive using only the ELISA at 32 h p.i. This study showed that RHDV replication occurred almost immediately after inoculation and that the liver appears to be the main site of replication.