Unique RT-PCR test for replicating VHS virus, with internal controls

An outbreak of a unique and especially virulent strain (IVb) of viral hemorrhagic septicemia (VHS) occurred in 2005 in the Great Lakes, killing several economically prominent freshwater fishes; including yellow perch (Perca flavescens) and muskellunge (Esox masquinongy); with additional outbreaks in subsequent years. This virus poses extensive risk to the aquaculture, baitfish industries, and native fisheries. Despite efforts to reduce detection time with DNA diagnostics, cell culture, which is a weeks-long laborious process, is the only currently USDA-APHIS approved diagnostic method. Our laboratories have developed a new molecular based assay, Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR), to detect VHSv, which speeds detection time (to hours), lowers detection threshold, uniquely identifies among VHSv strains, determines whether the virus is actively replicating (transmissible), and provides intrinsic quality control via a standardized mixture of internal standards (SMIS). We present evaluations of our StaRT-PCR test using infected fish cell lines, immersion, and injection-challenged experiments. Here we compare the ability of our StaRT-PCR assay to accurately detect VHSv infection with results from current cell culture/plaque assay tests and traditional qRT-PCR. Additionally we compared quantitative results from (A) plaque assay, (B) qRT-PCR, versus (C) StaRT-PCR to determine accuracy and detection threshold at varying infection levels.