Evaluation of ViroCyt® Virus Counter for Rapid Filovirus Quantitation

Abstract

Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM) for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR). The ViroCyt® Virus Counter (VC) 2100 (ViroCyt, Boulder, CO, USA) is a flow-based instrument capable of quantifying virus particles in solution. Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.

Introducing EbolaCheck: potential for point-of-need infectious disease diagnosis

“…USAMRIID have demonstrated performance across five logs of viral RNA genome equivalents (GE) with 100% analytical specificity against 65 other pathogens and analytical sensitivities of 0.001 (NP) and 0.0001 (GP) plaque-forming units per reaction [9]. The roughly 4000 GE/plaque-forming units ratio observed under biosafety level 4 (BSL4) experimentation [21], suggests a lower limit of detection (LLOD) of 10 GE/reaction, or 10 4 GE/ml of WBS. Given typical time-to-presentation in autumn 2014 was >3 days post symptom onset, a LLOD goal of 10 4 GE/ml WBS was set for the Trombley+ assays on EbolaCheck.…”

Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

“…Diagnostic methods based on the direct quantification and identification of intact virus particles, such as flow cytometry (3,4) have been used to quantify a variety of viruses with differing morphologies (reviewed in 5). These techniques make use of fluorescent dyes that bind to the viral genome (6) or proteins (7), all of which require a minimal 30 minute incubation period for efficient virus labelling prior to virus quantification. Negative stain transmission electron microscopy (TEM) images have been used since the 1940s (8) as an important tool to image individual virus particles and quantitatively determine virus concentrations, but due to the high costs and amount of space required for a TEM instrument, this is still only available in certain facilities.…”

An optimized method for counting viral particles using electron microscopy

“…To count the amount of viral particles several methods can be used, including plaque-forming cells; quantitative RT-PCR; immunofluorescence microscopy; analytical flow cytometry; electron microscopy, etc. (Ferris et al, 2002;Reid et al, 2003;Malenovska, 2013;Heider, Metzner, 2014;Rossi et al, 2015).…”