FOR REVIEW ONLY UNIT 15C.1 Mammalian Reoviruses: Propagation, Quantification, and Storage Alicia Berard 1 and Kevin M. Coombs 1 1 Department of Medical Microbiology and Infectious Diseases, University of Manitoba and Manitoba Centre for Proteomics and Systems Biology Winnipeg, Manitoba, Canada ABSTRACT Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infec- tions. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This unit describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described. Curr. Protoc. Microbiol. 14:15C.1.1-15C.1.18. C  2009 by John Wiley & Sons, Inc. Keywords: RNA virus  double-stranded RNA  non-enveloped virus  mouse cell culture  cesium chloride ultracentrifugation  plaque assay INTRODUCTION Mammalian reoviruses are pathogens that present as gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical dis- ease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. Studies with reoviruses can provide insight into these more dangerous members. This unit describes techniques on culturing mouse fibroblast L929 cell lines, in which most of the mammalian reovirus studies take place (Support Protocol 1), as well as propagating, quantifying, and storing reovirus. Basic Protocol 1 describes the propagation of reovirus in mouse L929 cell lines. The titration, or quantification, of the virus is described in Basic Protocol 2. Methods for virus storage are explained in Basic Protocol 3. CAUTION: Mammalian reoviruses are Biosafety Level 2 (BSL-2) pathogens. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microor- ganisms. See UNIT 1A.1 and other pertinent resources (APPENDIX 1) for more information. BASIC PROTOCOL 1 PROPAGATION OF MAMMALIAN REOVIRUSES IN CELL CULTURE FROM VIRUS STOCKS Mouse L292 cells (ATCC #CCL-1) are ??preferred?? to use for in vitro cell culture and manipulation of mammalian reoviruses. The propagation of reovirus stocks is performed by a series of passages. Typical virus passaging is described below. Materials Neutral red–stained plate containing virus plaques (see Basic Protocol 2) Gel saline (see recipe) 25-cm 2 tissue culture flask with pre-attached L929 cell monolayer (see Support Protocol 1) Current Protocols in Microbiology 15C.1.1-15C.1.18, August 2009 Published online August 2009 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/9780471729259.mc15c01s14 Copyright C  2009 John Wiley & Sons, Inc. Animal RNA Viruses 15C.1.1 Supplement 14