A New Multiplex RT-qPCR For The Rapid Differentiation Of ISAV-HPR0 From ISAV HPR-delete

Authors: ROUNSVILLE, THOMAS - University Of Maine; TURNER, SARAH - University Of Maine; Pietrak, Michael; Peterson, Brian; BOUCHARD, DEBORAH - University Of Maine; Polinski, Mark

Submitted to: Annual Eastern Fish Health Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 3/1/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: ISAV is an internationally regulated virus that infects salmon. There are two genetic forms of the virus - one form that causes serious disease and one that is benign - and each are regulated differently. The methods used to distinguish these two genetic forms have been genome sequencing, which is costly and typically takes at least a week to get results. Here we describe a new rapid (one day) method for identifying and distinguishing both genetic types of ISAV. We further validate and define the parameters of this test for anticipated future use in regulatory diagnostics.

Technical Abstract: Infectious salmon anemia (ISA) is an economically important disease of Atlantic salmon (Salmo salar L.) caused by infectious salmon anemia virus (ISAV). ISA outbreaks have resulted in significant losses of farmed salmon globally. Due to the ability of ISAV to spread rapidly and cause significant mortality, the detection of ISA or ISAV is notifiable to the World Organization of Animal Health (WOAH). Many governments of salmon producing countries also have independent requirements for reporting and surveillance monitoring of ISAV. However, two different phenotypically distinct variants of ISAV exist with divergent disease outcomes, associated regulations, and control measures. ISAV-HPR', also known as ISAV-HPR deleted, is responsible for ISA outbreaks, while ISAV-HPR0, is avirulent and is not known to cause fish mortality. The primary genetic difference between the two phenotypes is the length of the hemagglutinin-esterase gene in the hypervariable region (HPR) and current diagnostic methodology requires genetic sequencing to differentiate phenotypes. To increase the speed of phenotypic determinations of ISAV, we developed a new, rapid multiplex RT-qPCR method and validated the analytical specificity of the assay on more than 30 genetically diverse ISAV isolates collected from North America and Europe, followed by determining analytical sensitivity. In addition, we evaluated this assay in three different laboratories and determined this new assay has equivalent sensitivity to current RT-qPCR methods used to detect ISAV without differentiating phenotypes.