title: 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.
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. cynthia.a.rossi.civ@mail.mil.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Rossi
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. brian.j.kearney.civ@mail.mil.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Kearney
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. scott.p.olschner.civ@mail.mil.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Olschner
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. priscilla.l.williams4.ctr@mail.mil.
authors.forename: Priscilla L
authors.initials: PL
authors.lastname: Williams
authors.affiliation: Pathology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. robinsonc@janelia.hhmi.org.
authors.forename: Camenzind G
authors.initials: CG
authors.lastname: Robinson
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. mheinrich7295@gmail.com.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Heinrich
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. azovanyi@gmail.com.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Zovanyi
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. michael.f.ingram.mil@mail.mil.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Ingram
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. david.a.norwood.civ@mail.mil.
authors.forename: David A
authors.initials: DA
authors.lastname: Norwood
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. randal.j.schoepp.civ@mail.mil.
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Schoepp
mesh_terms: D000818:Animals; D029043:Ebolavirus / Q000302:isolation & purification*; D006801:Humans; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D019562:Viral Load / Q000379:methods*