Quantifying Orsay Virus Infection in C. elegans

Orsay virus isolate was prepared as previously described^{11 (link)}. For pals-5p::GFP expression analysis and FISH staining for infection level quantification, L1 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 12 h at 20 °C, whereas animals used for ZIP-1::GFP analysis were infected with a high dose of virus for 9 h at 20 °C. pals-5p::GFP reporter expression was analyzed in animals that were anesthetized with 10 mM levamisole. For FISH analysis, animals were collected and fixed in 4% paraformaldehyde for 15–45 min depending on the assay. Fixed worms were stained at 46 °C overnight using FISH probes conjugated to the red Cal Fluor 610 fluorophore, targeting Orsay virus RNA1. GFP imaging and FISH analysis were performed using Zeiss AxioImager M1 compound microscope. For qRT-PCR analyses, synchronized L4 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 24 h at 20 °C. RNA isolation and qRT-PCR analysis were performed as described below. ZIP-1::GFP expression was analyzed and imaged on a Zeiss LSM700 confocal microscope run by ZEN2010 software.