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Abstract Orsay virus infection in the nematodeCaenorhabditis eleganspresents an opportunity to study host‐virus interactions in an easily culturable, whole‐animal host. Previously, a major limitation ofC. elegansas 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. eleganshas emerged as a compelling model for research on antiviral defense. From the perspective of the host, the genetic tractability ofC. elegansenables 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 ofC. 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: SynchronizeC. elegansdevelopment 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
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This content will become publicly available on October 22, 2026
A robust absolute quantification protocol for potato virus Y based on reverse transcription-quantitative PCR
Standardized protocols for absolute quantification of potato virus Y (PVY) from potato tissue is critical for host-virus dynamic studies. Here, we developed a standardized protocol using a cloned viral sequence as standards to detect and quantify PVY. Starting with total RNA, concentrated via column-based kit, this protocol is able to detect approximately 50 viral copies/reaction from multiple PVY strains. Validation of this protocol confirmed linearity across 8 orders of magnitude with high repeatability, reproducibility and statistical robustness across three independent runs. This protocol offers reliable PVY quantification to manage potato crop health and enables comparative studies with other viral systems.
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- Award ID(s):
- 2119968
- PAR ID:
- 10644319
- Publisher / Repository:
- microPublication Biology
- Date Published:
- Journal Name:
- microPublication biology
- Volume:
- 2025
- ISSN:
- 2578-9430
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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