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 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.
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, Italy.
D
D
Gelmetti
V
V
Grieco
C
C
Rossi
L
L
Capucci
A
A
Lavazza
D000818:Animals; D004076:Digoxigenin; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D017929:Hemorrhagic Disease Virus, Rabbit / Q000235:genetics / Q000302:isolation & purification*; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods / Q000662:veterinary*; D008099:Liver / Q000821:virology; D015336:Molecular Probe Techniques / Q000662:veterinary; D015347:RNA Probes / Q000235:genetics; D011817:Rabbits; D013154:Spleen / Q000821:virology; D013997:Time Factors; D015678:Viral Structural Proteins / Q000235:genetics
D015347:RNA Probes; D015678:Viral Structural Proteins; C081149:viral protein 60, rabbit hemorrhagic disease virus; D004076:Digoxigenin