Quantifying Orsay Virus Infection in C. elegans

Orsay virus filtrates were prepared as previously described [29 (link)]. A 1:5 dilution of Orsay virus filtrate was mixed with OP50-1, M9, and 1,000 synchronized L1 animals, and the mix was top-plated onto 6-cm NGM plates; at least two technical replicates (two plates) per genotype were set up per infection assay. Four independent infection assays were performed. Animals were infected at 20°C for 18 h before fixation in 4% paraformaldehyde. Fixed worms were incubated at 46°C overnight with two FISH probes, mixed equally, that were both conjugated to the red Cal Fluor 610 fluorophore and hybridize to either Orsay RNA1 or RNA2 genome segments (Biosearch Technologies). Samples were analyzed for Orsay virus infection using a AxioImager M1 compound microscope (Zeiss). 100 animals per genotype, per infection assay, were scored and the percentage of infected animals in the population was quantified.