title: Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression.
abstract: We describe a simple and expedited microneutralization assay for human metapneumovirus virus (HMPV) based on a recombinant HMPV expressing the enhanced green fluorescent protein (rHMPV-GFP). Test serum dilutions were incubated with fixed amounts of rHMPV-GFP and inoculated onto Vero cells, and the growth of non-neutralized rHMPV-GFP was visualized by fluorescent microscopy of living cells. A preliminary titer could be determined following 3 days of incubation. GFP expression was sufficient to be read by an automated scanner after 4-5 days of incubation, which also provided a permanent record. In comparison, the conventional serum neutralization assay requires a longer incubation time plus the additional steps of fixation and staining or immunostaining. rHMPV-GFP-based titers could be determined by the 50% infectivity endpoint method of Reed and Muench [Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoint. Am. J. Hyg. 27, 493-497], or by automated scanning and non-linear regression to determine the 50% endpoint of GFP fluorescence. The latter method was two- to three-fold more sensitive. This assay also permits automation and up-scaling, making it suitable for broad HMPV seroepidemiology studies and experiments that require large scale serology, such as vaccine studies.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Building 50, Room 6505, 50 South Drive, MSC 8007 Bethesda, MD 20892-8007, USA.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Biacchesi
authors.forename: Mario H
authors.initials: MH
authors.lastname: Skiadopoulos
authors.forename: Lijuan
authors.initials: L
authors.lastname: Yang
authors.forename: Brian R
authors.initials: BR
authors.lastname: Murphy
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.forename: Ursula J
authors.initials: UJ
authors.lastname: Buchholz
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D029121:Metapneumovirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000472:pathogenicity*; D009500:Neutralization Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014709:Vero Cells
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins