Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase

Abstract

BackgroundHuman metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. ObjectivesThe purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). Study designA recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50pfu/well of the recombinant virus at 4°C for 1h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. ResultsThe novel assay could be completed within 24h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. ConclusionsThe neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies.