Avian Reoviruses: Propagation, Quantification, and Storage

Anh Tran1, Alicia Berard1, Kevin M. Coombs1

1 University of Manitoba and Manitoba Centre for Proteomics and Systems Biology, Winnipeg, Manitoba, Canada
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.2
DOI:  10.1002/9780471729259.mc15c02s14
Online Posting Date:  August, 2009
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Abstract

Avian reoviruses (ARVs) are pathogens that cause significant morbidity among commercial poultry. ARVs are prototypic representatives of non‐enveloped viruses that can cause cell‐cell fusion. They belong to the Reoviridae family, which contains many highly pathogenic viruses. ARVs are ubiquitous in commercial poultry and are frequently isolated from the gastrointestinal and respiratory tracts of chickens with acute infections. The virus causes a range of disease states in chicken, including viral arthritis/tenosynovitis, gastroenteritis, hepatitis, myocarditis, “pale bird syndrome,” runting‐stunting syndrome, and respiratory illness. This unit describes avian reovirus propagation, quantification, and storage. Curr. Protoc. Microbiol. 14:15C.2.1‐15C.2.16. © 2009 by John Wiley & Sons, Inc.

Keywords: RNA virus; double‐stranded RNA; non‐enveloped virus; quail cell culture; plaque assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Avian Reoviruses in Cell Culture from Virus Stocks
  • Alternate Protocol 1: Propagation of Avian Reoviruses from Single Plaque Isolation
  • Basic Protocol 2: Quantification of Avian Reoviruses by Plaque Assay with Neutral Red Staining
  • Alternate Protocol 2: Quantification of Avian Reoviruses by Plaque Assay with Crystal Violet Staining
  • Basic Protocol 3: Storage of Avian Reoviruses
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Propagation of Avian Reoviruses in Cell Culture from Virus Stocks

  Materials
  • Avian reovirus stock (obtained from ATCC or clinical samples)
  • Gel saline (see recipe)
  • Quail fibrosarcoma (QM5) cells, set up 1 to 2 days previously in a 75‐cm2 flask (see appendix 4G), and 95% to 98% confluent
  • Complete 1× M199 medium, prewarmed to 37°C (see recipe)
  • Low‐speed benchtop centrifuge
  • 15‐ml conical tubes, sterile
  • 10‐ml serological pipets, sterile
  • 2‐ml cryovials, sterile
NOTE: A confluent monolayer is considered to be ≥95% confluent without being over confluent, unless otherwise stated (see appendix 4G and Fig. for further details on cellular confluency).

Alternate Protocol 1: Propagation of Avian Reoviruses from Single Plaque Isolation

  Materials
  • Plate of neutral red–stained avian reovirus plaques (see protocol 3)
  • Gel saline (see recipe), sterile
  • QM5 cells, set up 1 to 2 days previously in 25‐cm2 flasks (see appendix 4G), and 95‐98% confluent.
  • Complete 1× M199 medium, prewarmed to 37°C (see recipe)
  • Penicillin‐streptomycin (see recipe)
  • Amphotericin B (see recipe)
  • Small rubber bulb for Pasteur pipet
  • Disposable, cotton‐plugged, glass Pasteur pipets, sterile
  • 1‐dram (dm) or 2‐dm glass vials, sterile
  • 5‐ml serologic pipets, sterile
  • 15‐ml conical tube, sterile
  • 2‐ml cryovials
NOTE: A confluent monolayer is considered to be ≥95% confluent without being over confluent unless otherwise stated (see appendix 4G and Fig. for further details on cellular confluency).NOTE: A separate flask will be required for each picked plaque. The following protocol describes manipulation of each picked plaque; repeat for each picked plaque.

Basic Protocol 2: Quantification of Avian Reoviruses by Plaque Assay with Neutral Red Staining

  Materials
  • Complete 2× M199 medium (see recipe)
  • 2% (w/v) agar (Difco Bacto)
  • Penicillin‐streptomycin (see recipe), optional
  • Amphotericin B (optional; see recipe)
  • Avian reovirus stock (obtained from ATCC or clinical samples)
  • Gel saline (see recipe)
  • Quail fibrosarcoma (QM5) cells, set up 1 to 2 days previously in 6‐well plates (see appendix 4G), and 95% to 98% confluent
  • 2% (w/v) neutral red staining solution
  • 2× PBS ( appendix 2A)
  • Microwave
  • 37°, 62°, 42°, and 50°C water baths
  • Biosafety cabinet, certified for aseptic procedures
  • Dilution tubes
  • Micropipettors
  • 100‐ to 1000‐µl pipet tips
  • 20‐ to 200‐µl pipet tips
  • 10‐ml pipets
  • Vortex
NOTE: All steps can be done on a benchtop unless otherwise indicated.

Alternate Protocol 2: Quantification of Avian Reoviruses by Plaque Assay with Crystal Violet Staining

  • 2% formaldehyde (see recipe)
  • 0.05% crystal violet staining solution (see recipe)
  • Milli‐Q or double‐distilled purified water
  • Small metal or plastic scoop
  • Paper towels
NOTE: All steps can be done on a benchtop unless otherwise indicated. Follow appropriate disposure guidelines for biohazard wastes.
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Figures

Videos

Literature Cited

Literature Cited
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