Culturing, Storage, and Quantification of Rotaviruses

Michelle Arnold1, John T. Patton1, Sarah M. McDonald1

1 Laboratory of Infectious Diseases, NIAID/NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.3
DOI:  10.1002/9780471729259.mc15c03s15
Online Posting Date:  November, 2009
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Abstract

Group A rotaviruses (RVs) infect the young of numerous animal species and cause acute gastroenteritis. Cultivation of animal and human RVs in cells requires proteolytic activation of the viral attachment protein using trypsin. Continuous cell lines, such as rhesus monkey kidney cells, as well as primary monkey kidney cells, are routinely used for the growth and characterization of RVs. Isolation and cultivation of human RVs from clinical fecal specimens is difficult and adaptation to growth in vitro requires multiple rounds of passage in primary cells. Following growth, RV stocks can be purified by centrifugation, if required, and quantified using plaque assay or fluorescence focus assay. This unit describes easily applicable procedures for the culturing, storage, and quantification of RVs. Curr. Protoc. Microbiol. 15:15C.3.1‐15C.3.24. © 2009 by John Wiley & Sons, Inc.

Keywords: rotavirus; RV; infection; cell culture; purification; plaque assay; fecal specimen; MA104 cells; AGMK cells; trypsin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Rotavirus in MA104 Cell Culture from Virus Stocks
  • Alternate Protocol 1: Propagation of Rotavirus in Primary AGMK Roller Cell Culture from Virus Stocks
  • Alternate Protocol 2: Propagation of Rotavirus in Cell Culture from Clinical Fecal Specimens
  • Basic Protocol 2: Semi‐Purification of Rotaviruses Using Ultracentrifugation
  • Alternate Protocol 3: Purification of Rotavirus Using Cesium Chloride Gradients
  • Basic Protocol 3: Quantification of Rotavirus by Plaque Assay
  • Alternate Protocol 4: Quantification of Rotavirus by Fluorescence Focus Assay
  • Basic Protocol 4: Purification of Rotavirus from Plaques
  • Basic Protocol 5: Storage of Rotaviruses
  • Support Protocol 1: Growth and Maintenance of MA104 Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation of Rotavirus in MA104 Cell Culture from Virus Stocks

  Materials
  • MA104 cells confluent in a 150‐cm2 flask (∼1.5 × 107 cells; see the protocol 10)
  • 1× serum‐free Medium 199 (see recipe)
  • RV stock vial (105 to 108 PFU/ml; see protocol 9)
  • 2 mg/ml trypsin in PBS (see recipe)
  • 1× serum‐free Medium 199 (see recipe) with 0.5 µg/ml trypsin (see recipe)
  • 1× complete Medium 199 (see recipe)
  • 15‐ and 50‐ml conical tubes
  • Vortex (optional)
  • 37°C water bath
  • Platform rocker
  • Inverted light microscope
  • −80°C freezer
  • Table‐top centrifuge
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Alternate Protocol 1: Propagation of Rotavirus in Primary AGMK Roller Cell Culture from Virus Stocks

  • AGMK cells in a round‐bottom glass tube (Diagnostic HYBRIDS, cat. no. 46‐0600A)
  • Roller drum for 37°C incubator
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Alternate Protocol 2: Propagation of Rotavirus in Cell Culture from Clinical Fecal Specimens

  • Clinical fecal sample
  • AGMK cells in a round‐bottom glass tube (Diagnostic HYBRIDS, cat. no. 46‐0600A)
  • 1.5‐ml microcentrifuge tubes
  • Roller drum for 37°C incubator
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work and all handling of clinical specimens should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Basic Protocol 2: Semi‐Purification of Rotaviruses Using Ultracentrifugation

  Materials
  • 240 ml of clarified RV stock (from twelve 150‐cm2 flasks of infected MA104 cells; see protocol 1)
  • 35% (w/v) sucrose in TNC buffer (see recipe)
  • TNC buffer (see recipe)
  • Ultra‐Clear 25 × 89–mm ultracentrifuge tubes (Beckman)
  • 2‐ml and 50‐ml pipets
  • Beckman ultracentrifuge with an SW32Ti rotor (or equivalent)
  • 15‐ml conical tube
  • Sonicator
NOTE: If preparing a sterile RV stock, perform the purification protocol in a class II biological safety cabinet and all equipment and solutions should be sterile. If preparing a nonsterile RV stock, perform the purification protocol on the laboratory bench.

Alternate Protocol 3: Purification of Rotavirus Using Cesium Chloride Gradients

  Materials
  • 240 ml of RV‐infected cell lysate (from twelve 150‐cm2 flasks of infected MA104 cells; see protocol 1)
  • TNC buffer (see recipe)
  • Trichlorotrifluoroethane (Freon)
  • Cesium chloride (CsCl), Ultra‐Pure, Optical Grade
  • Ultra‐Clear 25 × 89–mm ultracentrifuge tubes (Beckman)
  • Beckman ultracentrifuge with SW32Ti and SW55Ti rotors (or equivalent)
  • Sorvall Omni‐mixer or vortex
  • 50‐ml conical tubes
  • Benchtop centrifuge
  • Sonicator
  • Refractometer
  • Ultra‐Clear 13 × 51–mm ultracentrifuge tubes (Beckman)
  • Inverted light source
  • 1.5‐ml microcentrifuge tubes
NOTE: If preparing a sterile RV stock, perform the purification protocol in a class II biological safety cabinet and all equipment and solutions should be sterile. If preparing a non‐sterile RV stock, perform the purification protocol on the laboratory bench.CAUTION: This protocol requires the use of Freon, which is a colorless liquid that has known toxic properties and is an ozone‐depleting substance. Freon should be handled in a chemical fume hood and according to the Materials Safety and Data Sheet (MSDS) provided by the manufacturer. In addition, Freon should be disposed of appropriately.

Basic Protocol 3: Quantification of Rotavirus by Plaque Assay

  Materials
  • MA104 cells confluent in a 150‐cm2 flask (∼1.5 × 107 cells)
  • 1× complete Medium 199 (see recipe)
  • RV stock (unknown titer; see Basic Protocols protocol 11 and protocol 42)
  • 2 mg/ml trypsin in PBS (see recipe)
  • 1× serum‐free Medium 199 (see recipe)
  • 2× serum‐free EMEM (see recipe)
  • 1.2% (w/v) agarose (see recipe)
  • 0.33% (w/v) neutral red (contains 3.3 g neutral red/liter PBS, sterile filtered, cell culture grade; store at 4°C for up to 1 year; Sigma‐Aldrich, cat. no. N2889)
  • 6‐well tissue culture plates
  • 37° and 55°C water baths
  • 15‐ and 50‐ml conical tubes
  • Vortex
  • 10‐, 200‐, and 1000‐µl pipets and tips
  • 5‐, 10‐, and 25‐ml pipets
  • Rocking platform
  • Light box
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator, unless otherwise specified.

Alternate Protocol 4: Quantification of Rotavirus by Fluorescence Focus Assay

  • 1× Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 100% methanol
  • Anti‐RV antibody (e.g., Santa Cruz Biotechnology, cat. no. SC‐101363)
  • 3% bovine serum albumin (BSA) in PBS
  • FITC‐conjugated secondary antibody (can be purchased from Invitrogen and Sigma‐Aldrich)
  • 96‐well tissue culture plates
  • Multichannel pipettor
  • 1.5‐ml microcentrifuge tubes
  • Inverted fluorescence microscope with fluorescein isothiocyanate (FITC) filter

Basic Protocol 4: Purification of Rotavirus from Plaques

  Materials
  • RV stock to be plaque purified
  • 1× serum‐free Medium 199 (see recipe)
  • Marker
  • 1.5 ml microcentrifuge tubes
  • Pasteur pipets with rubber bulbs
  • Additional reagents and equipment for preparing a plaque assay ( protocol 6)
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator.

Basic Protocol 5: Storage of Rotaviruses

  Materials
  • MA104 cells (ATTC #CRL‐2378.1)
  • 1× Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.05% Trypsin/0.1% EDTA solution (or alternative disassociating reagent)
  • 1× complete Medium 199 (see recipe)
  • Trypan blue solution (optional)
  • 5‐, 10‐, and 25‐ml pipets
  • Inverted light microscope
  • 150‐cm2 tissue culture flasks
NOTE: All equipment and solutions coming in contact with cells must be sterile and sterile technique should be used. Cell culture work should be conducted in a class II biological safety cabinet.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator, unless otherwise specified.
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Figures

  •   FigureFigure 15.C0.1 Cartoon of two types of RV particles in a CsCl gradient. Following centrifugation of RV in a CsCl gradient, two distinct, cloudy, whitish‐colored bands will be visible. Shining a light on the gradient will facilitate visualization of the bands. The upper band consists of infectious, triple‐layered particles (TLPs; density ∼1.36 g/cm3) and the lower band consists of non‐infectious, double‐layered particles (DLPs; density ∼1.38 g/cm3). Harvest the upper band as a source of purified RV.
  •   FigureFigure 15.C0.2 Example of RV plaque assay. These images depict the results of a plaque assay using the animal RV strain SA11‐4F, which grows well in cell culture. At 48 hr post‐infection, the cells were stained with neutral red. The image on the top was taken 4 hr after staining. The image on the bottom is a plate that was incubated an additional 16 hr after staining. The sizes of the plaques for this virus grew substantially during the overnight incubation, and are considered large at 3 days post‐infection. Many strains of rotavirus will make plaques that take additional days to form and are smaller in size. To calculate the titer for this plaque assay, the plate on the bottom was used, and the titer was calculated as: 15 plaques × 1/(10−7) × 1/(1 ml) = 1.5 × 108 PFU/ml.

Videos

Literature Cited

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Key References
   Gray, J. and Desselberger, U. 2000. Rotaviruses: Methods and Protocols. Humana Press, Totowa, New Jersey.
  This book describes several useful methods and protocols for studying rotavirus biology.
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