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Title: Accurate detection and quantification of the fish viral hemorrhagic septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay

Author
item PIERCE, LINDSEY - University Of Toledo
item WILLEY, JAMES - University Of Toledo
item PALSULE, VRUSHALEE - University Of Toledo
item YEO, JIYOUN - University Of Toledo
item Shepherd, Brian
item CRAWFORD, ERIN - University Of Toledo
item STEPIEN, CAROL - University Of Toledo

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/5/2013
Publication Date: 8/20/2013
Citation: Pierce, L.R., Willey, J.C., Palsule, V.V., Yeo, J., Shepherd, B.S., Crawford, E.L., Stepien, C.A. 2013. Accurate detection and quantification of the fish viral hemorrhagic septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay. PLoS One. 8(8): e71851.

Interpretive Summary: Viral Hemorrhagic Septicemia virus (VHSv) is one of the world’s most challenging finfish diseases, killing more than 70 wild and cultured species across Eurasia. In addition, a new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Diagnostic tests for VHSv have been inaccurate and prohibitively long and costly; a cell culture that takes weeks is the sole approved veterinary diagnostic standard. Recently, a new assay for VHSv detection and quantification, called Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR), was developed. Compared with other testing methods, the StaRT-PCR assay is faster and demonstrated higher accuracy, wider detection range, high pathogen specificity and also controlled for false negative results. However, the StaRT-PCR assay required the use of equipment that is not commonly used in disease diagnostic laboratories. To make this test more accessible and streamlined, we adapted the StaRT-PCR procedure to a standard real-time PCR platform using a 2-color detection system that identifies the pathogen and control genes. This new assay is rapid and inexpensive compared with other tests. More importantly, because it shares the higher accuracy and specificity seen with the StaRT-PCR testing assay, it does not have the drawbacks seen with other real-time PCR tests and traditional cell culture diagnostics. For fish farms, hatcheries, and baitfish operators, a month-long holding period for viral detection (that has a high false-negative rate) could lead to industry loss and possible viral spread. Consequently, this new VHSv testing platform will improve accuracy and reduce the time needed to reliably, and cost-effectively, detect the VHSv pathogen in economically- and ecologically- important finfish species.

Technical Abstract: Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting > 80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive reverse transcription real-time quantitative PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00 ± 0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6 x 10^6 molecules. Infected fishes are found to harbor levels of virus that range to 1.21 x 10^6 VHSv molecules/10^6 actb1 molecules, with ~ 1.0 x 10^3 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published real-time PCR tests and traditional cell culture diagnostics.