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Development and optimization of plaque assays for rat coronaviruses
D J Gaertner1, D F Winograd, S R Compton
1Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06510.
Journal of Virological Methods
|June 1, 1993
Summary
Optimized agarose plaque assays improve rat coronavirus (RCV) quantification. Enhanced methods using DEAE-dextran and trypsin increase plaque visibility and yield for RCV research.
Area of Science:
- Virology
- Cell Biology
Background:
- Rat coronaviruses (RCVs) require efficient methods for isolation and quantification.
- Traditional plaque assays have limitations in RCV propagation and evaluation.
Purpose of the Study:
- To optimize plaque assay conditions for RCVs in L2 mouse fibroblasts.
- To enhance plaque visualization and facilitate virus stock production and quantification.
Main Methods:
- Evaluated Sephadex and agarose overlay plaque assays for RCVs.
- Optimized agarose plaque assay using diethylaminoethyl-dextran (DEAE-D) and trypsin.
- Tested DEAE-D and trypsin in pre-treatment, absorption, and overlay media.
Main Results:
- Agarose plaque assay with DEAE-D and trypsin significantly increased plaque numbers and visibility.
- Optimal conditions involved DEAE-D in pre-treatment and absorption media, and trypsin in absorption and overlay media.
- Demonstrated assay utility for isolating and quantifying RCVs, including sialodacryoadenitis virus (SDA) and Parker's rat coronavirus (PRCV).
Conclusions:
- Optimized agarose plaque assay provides a robust method for RCV quantification and isolation.
- Enhanced plaque assay facilitates the production of cloned virus stocks for RCV research.
- This method supports in vitro and in vivo studies of RCV biology.