Single-particle measurements of filamentous influenza virions reveal damage induced by freezing

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantity and characterise the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70°C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced both their concentration and median length. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by using the cryoprotectant DMSO. We recommend that functional studies of filaments characterise virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterisations of filaments that are required to understand their roles in natural influenza virus infections.