Orsay Virus Infection Assay in C. elegans

Orsay viral preps were prepared as previously described (7 (link)). Synchronized L1 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 18 h at 20 °C (Fig 6A). For infection at the L4 stage (Fig 6B), synchronized animals were grown on rde-1 RNAi plates (1,000 animals per plate, 2 plates per strain) for 72 h (pals-17(*) mutants) or 48 h (all other strains) at 20 °C. rde-1 RNAi increases susceptibility to Orsay virus infection (78 (link), 79 (link)). L4 animals were top-plated with a mixture of Orsay virus, OP50-1 and M9, and incubated at 20 °C for 24 h. Following incubation, animals were collected and washed in M9, and subsequently fixed in 4% paraformaldehyde for 30 min. Fixed worms were washed and then stained at 46 °C overnight using FISH probes conjugated to the red Cal Fluor 610 fluorophore (Biosearch Technologies), targeting Orsay virus RNA1 and RNA2 (80 (link), 81 ). Analyses were performed visually using a Zeiss AxioImager M1 compound microscope; at least 300 animals were scored for the presence of the FISH fluorescent signal per strain per replicate. Due to relatively high variation in infection levels between replicates, the percentage of infected wild-type animals was normalized to one. Data analysis was performed using GraphPad Prism 9 software. Normalized data are shown in S1 Table.