title: Quantitation of ranaviruses in cell culture and tissue samples.
abstract: A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.
authors.affiliation: Finnish Food Safety Authority Evira, Veterinary Virology Research Unit, Helsinki, Finland. riikka.holopainen@evira.fi
authors.forename: Riikka
authors.initials: R
authors.lastname: Holopainen
authors.forename: Jarno
authors.initials: J
authors.lastname: Honkanen
authors.forename: Britt Bang
authors.initials: BB
authors.lastname: Jensen
authors.forename: Ellen
authors.initials: E
authors.lastname: Ariel
authors.forename: Hannele
authors.initials: H
authors.lastname: Tapiovaara
mesh_terms: D000818:Animals; D000835:Animals, Wild; D018929:Cell Culture Techniques; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D007558:Italy; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017995:Ranavirus / Q000235:genetics / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA; D019562:Viral Load / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase