Extended Data Fig. 4: Heterogeneity of RSV virions from different viral strains and origins.

RSV virion heterogeneity, with respect to the number of vRNPs and their DARPin-P state was assessed. To study individual virions, A549 cells expressing P^exo-fluoro and DARPin-P-fluoro were infected at low MOI (0.01-0.1) to ensure that the majority of cells were infected with a single virion. In all analyses, all vRNPs in the same infected cell was considered to originate from a single virion. Analysis was carried out at 4 hpi to ensure all vRNPs from individually infecting virions had split. a, b, RSV Long strain was passaged once in HEp-2, A549 and Vero cell lines as well as in air-liquid-interface (ALI) cultures of primary human nasal epithelium obtained from 2 healthy donors. Virions of these different cellular origins were assessed. Schematic summarizing experimental protocol (a) and quantification of vRNP numbers (b). c, Two RSV strains, RSV-X and RSV-A2, were evaluated. The number of vRNPs per virion were quantified. d, e, RSV virions present in nasal wash samples of RSV-infected individuals was assessed. The samples were obtained from participants of a controlled human infection (CHIM) study, where healthy adults were inoculated with the challenge strain RSV-A Memphis-37 (refs. ^57,58). Schematic (d) and quantification of vRNP numbers per virion (e) is shown. f, The DARPin-P state of virions produced in different cell lines and primary cells (see Extended Data Fig. 4a) is quantified. g, The DARPin-P state of virions from RSV-X and RSV-A2 is quantified. h, The DARPin-P state of virions from nasal wash samples (see Extended Data Fig. 4d) is quantified. i, The DARPin-P state of virions from two RSV clinical isolates is quantified. (b, c) Two-way ANOVA with Tukey’s multiple comparisons test compared if there was significant variation of the frequencies between different samples for each of the foci numbers. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.