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

Abstract

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.

Fig. 1

Streptavidin-biotin immunoperoxidase, liver, 32 h p.i. Small, brownish, vacuolated and granular structures are evident in the cytoplasm of hepatocytes (1000×).

Fig. 2

ISH, anti-sense probe, liver, positive control rabbit. Blue and granular positive signal was confined to the cytoplasm of the hepatocytes (1000×).

Fig. 3

ISH, anti-sense probe, liver, 28 h p.i. Labelled hepatocytes were scattered throughout the section (600×).

Fig. 4

ISH, anti-sense probe, liver, 44 h p.i. Labelled hepatocytes with perilobular distribution (150×).