title: Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures.
abstract: 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.
authors.affiliation: Department of Microbiology, Faculty of Sciences, University of Malaga, Campus Teatinos, 29071 Malaga, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Alonso
authors.forename: I
authors.initials: I
authors.lastname: Cano
authors.forename: D
authors.initials: D
authors.lastname: Castro
authors.forename: S I
authors.initials: SI
authors.lastname: Perez-Prieto
authors.forename: J J
authors.initials: JJ
authors.lastname: Borrego
mesh_terms: 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
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary