Orsay Virus Infection in Caenorhabditis elegans

Abstract

Orsay virus infection in the nematode Caenorhabditis elegans presents an opportunity to study host-virus interactions in an easily culturable, whole-animal host. Previously, a major limitation of C. elegans as a model for studying antiviral immunity was the lack of viruses known to naturally infect the worm. With the 2011 discovery of the Orsay virus, a naturally occurring viral pathogen, C. elegans has emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability of C. elegans enables mechanistic studies of antiviral immunity while the transparency of this animal allows for the observation of subcellular processes in vivo. Preparing infective virus filtrate and performing infections can be achieved with relative ease in a laboratory setting. Moreover, several tools are available to measure the outcome of infection. Here, we describe workflows for generating infective virus filtrate, achieving reproducible infection of C. elegans, and assessing the outcome of viral infection using molecular biology approaches and immunofluorescence. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of Orsay virus filtrate Support Protocol: Synchronize C. elegans development by bleaching Basic Protocol 2: Orsay virus infection Basic Protocol 3: Quantification of Orsay virus RNA1/RNA2 transcript levels by qRT-PCR Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

Keywords: C. elegans; Orsay virus; anti‐dsRNA immunofluorescence; virus filtrate preparation; virus infection.

Figure 1.

Overview of Basic Protocols 1–5. (A) Basic Protocol 1: Preparation of Orsay virus filtrate. (B) Basic Protocol 2: Orsay virus infection. (C) Basic Protocol 3: Quantification of Orsay virus RNA1 transcript levels by qRT-PCR. (D) Basic Protocol 4: Quantification of infection rate and fluorescence in situ hybridization (FISH) fluorescence intensity. (E) Basic Protocol 5: Immunofluorescent labeling of dsRNA in virus-infected intestinal tissue.

Figure 2.

Bead beater homogenization of infected worms. (A) After bead beating, the homogenate should be uniform with no large chunks remaining. Silicon carbide beads will settle at the bottom of the tube. (B) After separating out remaining worm tissue by centrifugation, the supernatant should then be filter-sterilized.

Figure 3.

Schematic of C. elegans intestinal dissection. Add worms to a glass slide and anesthetize with levamisole. Position the needle at an angle and cut at either or both of the indicated cut sites (orange dashed lines) by touching the worm with the sharp edge of the needle. A single touch should be sufficient to cause extrusion of the intestine (and gonad).

Figure 4.

Assessing outcomes of viral infection. (A) Percentage of animals expressing pals- 5p::GFP following inoculation with different dilutions of virus filtrate. A two-tailed t-test was used to calculate p-values; *p<0.05, **p<0.01. (B) qRT-PCR analysis of viral load in rde-1; jyIs8 and jyIs8 animals at 24 hpi. Animals were infected at the L4 stage. rde-1 mutants exhibit increased viral RNA1 transcript levels compared to rde-1 wild-type animals. Fold change in gene expression was determined relative to jyIs8. A one-tailed t-test was used to calculate p-values; *p<0.05. (C) Percentage of animals that show Orsay FISH staining at 18 hpi following L1 infection. The percentage of infected animals is significantly increased in rde-1 mutants compared to rde-1 wild- type animals. A two-tailed t-test was used to determine statistical significance; ***p<0.001. For all plots, each dot represents an experimental replicate; three to four independent experimental replicates were performed. Bars represent the mean across experimental replicates; error bars indicate standard deviation.

Figure 5.

Quantification of fluorescence by FISH staining. (A) Representative images of WT and pals-22(jy3) animals stained with CF610-conjugated Orsay FISH probes (red) targeting the RNA1 and RNA2 viral genomic segments. Animals were infected at the L1 stage, and images were taken at 18 hpi. Scale bar = 100 µm. (B) Quantification of background-corrected mean fluorescence in (A). Each dot represents an individual animal; 20 animals were analyzed for each strain. Horizontal lines in box-and-whisker plots represent median values, and the box reflects the 25^th to 75^th percentiles. A Mann-Whitney U test was used to determine statistical significance; ***p<0.001.

Figure 6.

Viral FISH and dsRNA staining in dissected intestines. (A) Representative images of intestines (white dotted outline) from uninfected or virus-infected animals. Viral RNA was visualized using Quasar 670-conjugated (far-red) FISH probes targeting the Orsay virus RNA1 and RNA2 genomic segments. Samples were incubated with the J2 antibody to visualize dsRNA (green). Scale bar = 25 µm. (B) Magnification of region outlined by white box in (A). White arrowheads indicate dsRNA forming ring-like structures in cells that exhibit Orsay FISH staining. Scale bar = 25 µm.