Evaluation of ViroCyt® Virus Counter for rapid filovirus quantitation

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 micro...

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Bibliographic Details
Published in:Viruses 2015-02, Vol.7 (3), p.857-872
Main Authors: Rossi, Cynthia A, Kearney, Brian J, Olschner, Scott P, Williams, Priscilla L, Robinson, Camenzind G, Heinrich, Megan L, Zovanyi, Ashley M, Ingram, Michael F, Norwood, David A, Schoepp, Randal J
Format: Article
Language:English
Subjects:
Animals
Ebolavirus - isolation & purification
filovirus
Humans
Infectious diseases
Medical research
Pathogens
plaque assay
Proteins
qRT-PCR
quantitation
Staining and Labeling - methods
Stains & staining
TEM
Time Factors
Transmission electron microscopy
Vaccines
Viral Load - methods
ViroCyt® Virus Counter
Viruses
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Summary: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.
ISSN:1999-4915
1999-4915
DOI:10.3390/v7030857