title: Enhanced detection of infectious salmon anaemia virus using a low-speed centrifugation technique in three fish cell lines.
abstract: Infectious salmon anaemia (ISA), caused by ISA virus (ISAV), is a serious disease of farmed Atlantic salmon, Salmo salar L. Recently, molecular- and immunofluorescent-based techniques have become powerful diagnostic tools for ISAV detection, but culture-based techniques remain the gold standard. A disadvantage of ISAV culture is that the incubation time required before cytopathic effect (CPE) is observed in cell monolayers. To decrease time until CPE is observed, a low-speed centrifugation technique was applied to existing standard operating procedures for ISAV culture in three fish cell lines. Time until CPE observation was compared in CHSE, SHK and ASK cells, treated or not treated with low-speed centrifugation after inoculation with ISAV. Low-speed centrifugation treatment significantly enhanced observable cell infection. Compared to control cells, the length of time until ISAV CPE observation decreased in centrifuged ASK and CHSE cells. Low-speed centrifugation was also incorporated into a modified clinical shell vial assay. At 48 h post-inoculation with approximately 20 viral particles, ISAV was detected by an immunofluorescence antibody test in treated ASK and SHK1 cells but not in control cells. Finally, this enhanced viral adsorption assay performed in ASK cells demonstrated higher sensitivity than a real-time RT-PCR assay performed on RNA isolated from ISAV-spiked salmon kidney homogenates.
authors.affiliation: Department of Molecular and Biomedical Sciences, Aquaculture Research Institute, University of Maine, Orono, ME, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Molloy
authors.forename: E
authors.initials: E
authors.lastname: Thomas
authors.forename: K
authors.initials: K
authors.lastname: Hoyt
authors.forename: D A
authors.initials: DA
authors.lastname: Bouchard
mesh_terms: D000818:Animals; D002460:Cell Line; D002498:Centrifugation; D005398:Fisheries / Q000379:methods*; D046829:Isavirus / Q000502:physiology*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction; D012474:Salmon / Q000821:virology; D012680:Sensitivity and Specificity