Accurate Detection and Quantitation of the Fish Viral Hemorrhagic Septicemia Virus (VHSv) With a New Real-Time PCR Assay

Viral Hemorrhagic Septicemia virus (VHSv) is one of the most serious fish pathogens, killing >80 marine, freshwater, and estuarine fish species from Europe and North America. A novel and especially virulent strain -IVb- first appeared in the Great Lakes in 2003, shutting down interstate transport of bait fish. Cell culture is the method approved by the USDA-APHIS, which takes a month or longer to obtain results, lacks sensitivity, and does not quantify the amount of virus. We thus developed a novel, easy, rapid, and highly sensitive diagnostic 2-color fluorometric (2CF) real-time quantitative PCR assay, which incorporates an internal standard for quality control to cirumvent false negative results and uses the housekeeping gene actb1 as a reference. This new test modifies our recently-published Standardized Reverse-Transcriptase PCR (StaRT-PCR) assay, which is based on Agilent electrophoretic equipment that is prohibitively expensive for many diagnostic laboratories. Both methods show 100% true accuracy down to five viral molecules. Results demonstrate that our 2CF test has high signal-to-analyte response (slope=1.00±0.02), high precision (0–6.86% error), and a linear dynamic range that spans eight orders of magnitude (R²=0.99), ranging from 0.6x10⁰ to 6x10⁷ molecules. Infected fishes harbor levels of virus that range from 1x10⁰ to 1.21x10⁶ VHSv molecules/10⁶ actb1 molecules, with ~1.0x10³ being a rough cut-off for clinical signs of disease. Our diagnostic tests are free of false negatives, and thus are superior to other published real-time PCR tests and traditional cell culture.