High-throughput super-resolution analysis of influenza virus pleomorphism reveals insights into viral spatial organization

Many viruses form highly pleomorphic particles; in influenza, these particles range from spheres of ~ 100 nm in diameter to filaments of several microns in length. Virion structure is of interest, not only in the context of virus assembly, but also because pleomorphic variations may correlate with infectivity and pathogenicity. We have used fluorescence super-resolution microscopy combined with a rapid automated analysis pipeline to image many thousands of individual influenza virions, gaining information on their size, morphology and the distribution of membrane-embedded and internal proteins. We observed broad phenotypic variability in filament size, and Fourier transform analysis of super resolution images demonstrated no generalized common spatial frequency patterning of HA or NA on the virion surface, suggesting a model of virus particle assembly where the release of progeny filaments from cells occurs in a stochastic way. Finally, we showed that in long filaments, viral RNP complexes are located preferentially within Archetti bodies, suggesting that these structures may play a role in virus transmission. Our approach therefore offers exciting new insights into influenza virus morphology and represents a powerful technique that is easily extendable to the study of pleomorphism in other pathogenic viruses. ### Competing Interest Statement The work was carried out using portable commercial microscopes from Oxford Nanoimaging, a company in which A.N.K. is a co-founder and shareholder.