|PIERCE, LINDSEY - University Of Toledo|
|WILLEY, JAMES - University Of Toledo|
|CRAWFORD, ERIN - University Of Toledo|
|PALSULE, VRUSHALEE - University Of Toledo|
|LEAMAN, DOUGLAS - University Of Toledo|
|FAISAL, MOHAMED - Michigan State University|
|KIM, ROBERT - Michigan State University|
|STANOSZEK, LAUREN - University Of Toledo|
|STEPIEN, CAROL - University Of Toledo|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/2013
Publication Date: 4/1/2013
Citation: Pierce, L.R., Willey, J.C., Crawford, E.L., Palsule, V.V., Leaman, D.A., Faisal, M., Kim, R.K., Shepherd, B.S., Stanoszek, L.M., Stepien, C.A. 2013. A new StaRT-PCR approach to detect and quantify fish viral hemorrhagic septicemia virus (VHSv): enhanced quality control with internal standards. Journal of Virological Methods. 189:129-142.
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 2005, 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. A new assay for VHSv detection and quantification, called Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR), has been developed. Compared with cell culture and other polymerase chain reaction (PCR)-based methods, the StaRT-PCR assay demonstrated higher accuracy and wider detection range and also controlled for false negative results. This assay detected the VHSv pathogen in a wide range of sample types including two Great Lakes fish species and a fish cell line, and it was found to be specific to the VHSv pathogen. 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, the StaRT-PCR assay will improve accuracy and reduce the time needed to reliably detect the VHSv pathogen in economically- and ecologically-important finfish species.
Technical Abstract: Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world’s most important finfish diseases, killing >80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR® green real time qRT-PCR, and cell culture are compared, as well as effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14-47% in SYBR® green real time qRT-PCR tests, and 47-70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0 x 10^0-1.2 x 10^5 VHSv/10^6 actb1 molecules in wild caught fishes and 1.0 x 10^0-8.4 x 10^5 in laboratory challenged specimens. In the latter experiments, muskellunge with lesions had significantly more viral molecules (mean = 1.9 x 10^4) than those without (1.1 x 10^3) (P < 0.04). VHS v infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test detects accurately and reliably and quantifies VHSv.