Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures.
An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613 bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 10(3) TCID50/ml, whilst the labelling appears at 24h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent.
Alonso
M C
MC
Department of Microbiology, Faculty of Sciences, University of Malaga, Campus Teatinos, 29071 Malaga, Spain.
Cano
I
I
Castro
D
D
Perez-Prieto
S I
SI
Borrego
J J
JJ
D000818:Animals; D019201:Aquabirnavirus / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D004625:Embryo, Nonmammalian; D005399:Fishes / Q000821:virology*; D005412:Flatfishes / Q000821:virology; D017403:In Situ Hybridization / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012474:Salmon / Q000196:embryology / Q000821:virology
D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary