Extended Data Fig. 6: Assessing vRNA integrity in RSV stocks.

The integrity of genomic viral RNA ((-)vRNA) was assessed by smFISH. Probe sets targeting different (-)vRNA regions allowed identification of fully intact vRNPs and those missing genomic regions. a, b, c, d, Three RSV WT stocks were analyzed for defective viral genomes (DVGs) using a probe set targeting the 5′ trailer sequence. Many copy-back DVGs (cbDVGs), the predominant DVG type in RSV, contain break/rejoin events near the 5′ end of the genome, deleting large regions of the 3′ (-)vRNA^28,29. Targeting the 5′ trailer region thus enables sensitive and specific detection of cbDVGs. Coupling the 5′ probe set with a 3′ region probe set distinguishes intact RSV genomes from DVGs. Virions and vRNPs were analyzed on glass. Virions were detergent treated to release vRNPs (see Methods). (a) Schematic of the smFISH-based DVG detection strategy. (b) Representative images showing probe labeling of virions and vRNPs in three RSV WT stocks. Colocalization of probe signals was quantified for virions (c) and vRNPs (d). e, f, g, h, i, j, k, l, m, The (-)vRNA integrity for passive vRNPs and PRCs in infected cells was assessed. Schematics (e, h, k), representative images (f, i, l) and quantification (g, j, m) for smFISH probe sets targeting upstream, middle and downstream regions of the (-)vRNA, respectively, are shown. Assessments were performed 4 h after viral inoculum addition in P^exo-fluoro/DARPin-P-fluoro cells. To prevent infection progression, the translation inhibitor emetine was added simultaneously with virus inoculation. (f, i, l) Scale bar, 10 µm. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.