A validated quantitative polymerase chain reaction assay for the detection of ranaviruses (Family Iridoviridae) in fish tissue and cell cultures, using EHNV as a model

Abstract

Viruses in the genus Ranavirus (Family Iridoviridae) are important pathogens of fish, amphibians and reptiles and likely have a global distribution. Certification of freedom from infection, prevalence surveys, surveillance and routine diagnosis are hampered by lack of a validated rapid molecular test. In this study, a quantitative PCR (qPCR) assay for the detection of the major capsid protein gene of ranaviruses was developed and validated using Epizootic Haematopoietic Necrosis Virus (EHNV) as a model ranavirus. The assay had 100-fold greater analytical sensitivity compared to virus isolation in Bluegill fry (BF-2) cells, detected all viruses in a panel of 20 ranaviruses from Australia, America, Europe and the United Kingdom, did not detect two disputed members of the genus Ranavirus (doctorfish virus and guppy virus 6) and did not detect a megalocytivirus or two cyprinid herpesviruses. When applied to the detection of EHNV in fish tissue homogenates, 94.3% of samples which caused cytopathic effect (CPE) in BF-2 cells were positive in the qPCR, together with many others which did not display CPE but that arose from fish exposed to EHNV. Similarly, 98% of cell culture supernatants from cell monolayers with CPE were positive in the qPCR, together with other cultures of tissues from EHNV-exposed fish which did not display CPE. Overall a greater number of samples from exposed fish tested positive in qPCR compared to culture in BF-2 cells. The diagnostic specificity of the qPCR assay was 100% based on tests of tissue homogenates from 100 fish which were not exposed to EHNV. The assay appears to be suitable for a range of applications including surveillance and diagnosis, and for confirming the cause of CPE in BF-2 cells.