title: Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles.
abstract: Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9%. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus had greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed.
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, United States.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Bousse
authors.forename: David A
authors.initials: DA
authors.lastname: Shore
authors.forename: Cynthia S
authors.initials: CS
authors.lastname: Goldsmith
authors.forename: M Jaber
authors.initials: MJ
authors.lastname: Hossain
authors.forename: Yunho
authors.initials: Y
authors.lastname: Jang
authors.forename: Charles T
authors.initials: CT
authors.lastname: Davis
authors.forename: Ruben O
authors.initials: RO
authors.lastname: Donis
authors.forename: James
authors.initials: J
authors.lastname: Stevens
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D004285:Dogs; D031521:Fractionation, Field Flow / Q000379:methods*; D009980:Influenza A virus / Q000302:isolation & purification*; D008027:Light; D015203:Reproducibility of Results; D019562:Viral Load / Q000379:methods*
keywords: Field flow fractionation; Influenza virus; Multiangle light scattering; Vaccine
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