title: Enhanced detection of infectious airborne influenza virus.
abstract: Current screening methodologies for detecting infectious airborne influenza virus are limited and lack sensitivity. To increase the sensitivity for detecting infectious influenza virus in an aerosol sample, the viral replication assay was developed. With this assay, influenza virus is first amplified by replication in Madin-Darby canine kidney (MDCK) cells followed by detection with quantitative PCR (qPCR). Spanning a 20-h replication period, matrix gene expression levels from infectious virus were measured at several time points using qPCR and found to exponentially increase. Compared with the traditional culture-based viral plaque assay, the viral replication assay resulted in a 4.6 × 10(5) fold increase in influenza virus detection. Furthermore, viral replication assay results were obtained in half the time of the viral plaque assay. To demonstrate that the viral replication assay is capable of detecting airborne influenza virus, dilute preparations of strain A/WS/33 were loaded into a nebulizer, aerosolized within a calm-air settling chamber and subsequently collected using NIOSH Two-Stage Bioaerosol Samplers. At the most diluted concentration corresponding to a chicken embryo infectious dose 50% endpoint (CEID(50)) of 2.8E+02/ml, the viral replication assay was able to detect infectious influenza virus that was otherwise undetectable by viral plaque assay. The results obtained demonstrate that the viral replication assay is highly sensitive at detecting infectious influenza virus from aerosol samples.
authors.affiliation: Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 1095 Willowdale Road, M.S. 4020, Morgantown, WV 26505-2888, USA. FBlachere@cdc.gov
authors.forename: Francoise M
authors.initials: FM
authors.lastname: Blachere
authors.forename: Gang
authors.initials: G
authors.lastname: Cao
authors.forename: William G
authors.initials: WG
authors.lastname: Lindsley
authors.forename: John D
authors.initials: JD
authors.lastname: Noti
authors.forename: Donald H
authors.initials: DH
authors.lastname: Beezhold
mesh_terms: D000336:Aerosols; D000391:Air Microbiology; D000818:Animals; D002460:Cell Line; D004285:Dogs; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000302:isolation & purification / Q000502:physiology*; D009980:Influenza A virus / Q000302:isolation & purification / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000821:virology*; D057230:Limit of Detection; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D000336:Aerosols; D012367:RNA, Viral