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Generation, Propagation, and Titering of Dicistrovirus From an Infectious Clone
Junzhou Shen1, Jibin Sadasivan1,2, Eric Jan1
1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
Bio-Protocol
|March 3, 2025
Summary
A new protocol efficiently generates and amplifies Cricket paralysis virus (CrPV) using infectious clones and S2 cells. This method enables accurate viral titration via focus-forming assays, crucial for studying insect-virus interactions and developing antiviral strategies.
Area of Science:
- Virology
- Molecular Biology
- Entomology
Background:
- Cricket paralysis virus (CrPV) is a significant arthropod pathogen within the Dicistroviridae family.
- Dicistrovirus infections threaten agriculture, impacting honey bees and shrimp.
- Drosophila melanogaster serves as a model for insect-virus-host interactions.
Purpose of the Study:
- To develop a streamlined protocol for generating and titering recombinant CrPV.
- To overcome limitations of traditional plaque assays for CrPV in Drosophila S2 cells.
- To facilitate research on CrPV pathogenesis, epidemiology, and antiviral development.
Main Methods:
- Generation of recombinant CrPV from an infectious clone.
- Sequential amplification of the virus in Drosophila Schneider 2 (S2) cells.
- Titration using an immunofluorescence-based focus-forming assay (FFA).
Main Results:
- Successful generation and propagation of recombinant CrPV in S2 cells.
- Demonstration of a rapid and reliable focus-forming assay (FFA) for CrPV titration.
- Quantification of focus-forming units (FFU) using high-throughput microscopy.
Conclusions:
- The presented protocol provides an efficient method for CrPV recombinant virus production and titration.
- This approach supports in-depth investigation of dicistrovirus biology.
- Enables accelerated development of effective antiviral strategies against CrPV.
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