Rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression

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.