DAO 106:103-115 (2013)  -  doi:10.3354/dao02644

Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

Maureen K. Purcell1,*, Rachel L. Thompson1, Kyle A. Garver2, Laura M. Hawley2, William N. Batts1, Laura Sprague3, Corie Sampson3, James R. Winton

1Western Fisheries Research Center, US Geological Survey, 6505 NE 65th St., Seattle, Washington 98115, USA
2Pacific Biological Station, Fisheries and Oceans Canada, Aquatic Animal Health, 3190 Hammond Bay Road, Nanaimo, British Columbia V9T 6N7, Canada
3Idaho Fish Health Center, US Fish and Wildlife Service, Orofino, Idaho 83544, USA

ABSTRACT: Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.


KEY WORDS: Infectious hematopoietic necrosis virus · HNV · Real-time PCR · Diagnostic validation · Field validation · Steelhead trout · Reproducibility · Genogroup


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Cite this article as: Purcell MK, Thompson RL, Garver KA, Hawley LM and others (2013) Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV). Dis Aquat Org 106:103-115

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