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Avian Reoviruses: Propagation, Quantification, and Storage

Anh Tran¹, Alicia Berard¹, Kevin M. Coombs¹

¹University 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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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 ³95% confluent without being over confluent, unless otherwise stated (see appendix 4G and Fig. 15C.2.1 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 Basic Protocol 2)
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 ³95% confluent without being over confluent unless otherwise stated (see appendix 4G and Fig. 15C.2.1 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

Additional Materials (also see Basic Protocol 2)

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

Figure 15C.2.1

QM5 cell monolayers at different degrees of confluence; (A) ~30%; (B) ~97%; (C) just-confluent (100%); (D) overly-confluent (>100%). Note that cell packing in overly confluent cells leads to formation of “rivers.”

View Image
Figure 15C.2.2

Schematic diagram of picking ARV plaques on a neutral red–stained agar plate. The inset shows the view from over top of the infected plate. The white stars represent viral plaques. The recommended distance between each picked plaque should be ³1 cm.

View Image
Figure 15C.2.3

Schematic diagram of 10-fold serial dilution from a concentrated virus stock.

View Image
Figure 15C.2.4

Neutral red–stained plaque assay of ARV strains (A) 138 and (B) 176. The black arrows indicate ARV plaques. Dilutions go from lowest (left) to highest (right). These plaque assays were carried out in 6-well culture plates with only three wells shown here for each clone.

View Image
Figure 15C.2.5

Illustration on the removal of the agar overlay from a well, which is a necessary step in crystal violet staining.

View Image
Figure 15C.2.6

Crystal violet–stained plaque assay of ARV strains (A) 138 and (B) 176. The black arrows indicate ARV plaques. Dilutions go from lowest (left) to highest (right). These plaque assays were carried out in 6-well culture plates with only three wells shown here for each clone.

View Image

Literature Cited

Literature Cited
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