Bluetongue Virus (BTV): Propagation, Quantification, and Storage

Joseph K.K. Li1

1 Department of Biology, Utah State University, Logan, Utah
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15C.4
DOI:  10.1002/9780471729259.mc15c04s24
Online Posting Date:  February, 2012
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Abstract

As an obligate intracellular parasite, the genome of the Bluetongue virus (BTV) contains ten double‐stranded RNA segments which are encapsidated by viral proteins, forming “transport vesicles” that can transmit the viral progeny cell‐to‐cell efficiently and that can also be transmitted animal‐to‐animal by a biting midge. BTV is a cytoplasmic virus, and its five major steps of viral infection: attachment, entry, uncoating, assembly, and release, occur only in the cytosol within the infected host cell. Viral replication, suppression of cellular processes, and subsequent pathological damage disrupt many cellular pathways, leading to cellular apoptosis. All of these steps are under very rapid, tight, and efficient control. BTV infects both domestic and wild ruminants, especially sheep, but not humans. BTV is also the prototype in the Orbivirus genus of the Reoviridae family, and has been studied very extensively for the last 25 years. The experimental protocols presented here describe most of the methods that have been used routinely and reproducibly in our lab for our studies of the BTV biosystems. Curr. Protoc. Microbiol. 24:15C.1.1‐15C.1.18. © 2012 by John Wiley & Sons, Inc.

Keywords: Bluetongue viruses; growth and propagation; molecular cloning and expression

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Thawing Cryopreserved Cells for Growth of Bluetongue Viruses
  • Basic Protocol 2: Cryopreservation of Tissue Culture Cells
  • Basic Protocol 3: Cell Subculture for BTV Infection
  • Basic Protocol 4: Determination of the Infectivity of BTV by Plaque Assay
  • Basic Protocol 5: Plaque‐Purification of Infectious BTV Grown with Either Vero or L Cells
  • Basic Protocol 6: Amplification of Plaque‐Purified BTV Stock
  • Basic Protocol 7: Isolation and Purification of BTV
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Thawing Cryopreserved Cells for Growth of Bluetongue Viruses

  Materials
  • Minimum essential medium/Earle's balanced salt solution (MEM/EBSS; Hyclone, or Thermo Fisher Scientific, cat. no. SH30024.02); once a bottle is open for use, discard after 7 to 8 weeks.
  • Fetal bovine serum (FBS; Hyclone, or Thermo Fisher Scientific, cat. no. SH30088.03)
  • 10 mM MEM nonessential amino acids supplement (NEAA; Invitrogen, cat. no. 11140050)
  • 100 mM MEM sodium pyruvate supplement (MSP; Invitrogen, cat. no. 11360070)
  • Tissue culture cell lines from the American Type Culture Collection (ATCC), frozen in cryovials:
    • BHK‐21 cells (Syrian golden hamster kidney; ATCC #CCL‐10)
    • L cells (mouse subcutaneous connective tissue; ATCC #CRL‐2648)
    • Vero cells (African green monkey; ATCC #CCL‐81)
    • HeLa cells (human cervix adenocarcinoma; ATCC #CCL‐2)
    • A498 human kidney cells (human kidney carcinoma; ATCC #HTB‐44)
    • HEP‐G2 cell (human liver hepatocellular carcinoma; ATCC #HB‐8065)
    • A549 cells (human lung carcinoma; ATCC #CCL‐185)
    • HEK 293 cells (transformed human embryonic kidney; ATCC #CRL‐2405)
  • 70% (v/v) isopropanol
  • Tissue cultureware (Corning, 25‐, 75‐, and 150‐cm2 flasks; 6‐ and 12‐well plates; and 850‐cm2 roller bottles)
  • Tissue‐culture 37°C water‐jacketed incubator, with humidified air and 5% to 6% CO 2 (Model Isotemp, Thermo Fisher Scientific or Forma Scientific)
  • 42°C water bath
  • Kimwipes EX‐L (Kimberly‐Clark)
NOTE: Clean and rinse all glassware with double‐distilled water (ddH 2O; see Critical Parameters and Troubleshooting) before autoclaving them at 100°C under 15 lb./in.2 pressure for 35 min.

Basic Protocol 2: Cryopreservation of Tissue Culture Cells

  Materials
  • Cells growing in 75‐cm2 tissue culture flasks (see protocol 1 and protocol 3)
  • Fetal bovine serum (FBS; Hyclone, or Thermo Fisher Scientific, cat. no. SH30088.03)
  • Dimethylsulfoxide (DMSO; J.T. Baker)
  • Isopropyl alcohol (C 3H 8O; Mallinckrodt)
  • Liquid N 2 (optional)
  • 15‐ and 50‐ml tissue culture centrifugation (CFG) tubes with caps (Corning, cat. no. 430304)
  • Beckman Allegra 6R centrifuge
  • Nonpyrogenic sterile polypropylene cryogenic vials (Corning, cat. no. 430488)
  • Nalgene Cryo 1°C freezing container (Nalgene, cat. no. 5100‐0001)
  • Additional reagents and equipment for trypsinizing cells ( protocol 3), preparation of growth medium ( protocol 1, step 1), and trypan blue exclusion test for cell viability ( protocol 3)

Basic Protocol 3: Cell Subculture for BTV Infection

  Materials
  • Cells growing in 75‐cm2 tissue culture flasks (see protocol 1 and protocol 3)
  • MEM/EBSS medium without FBS, or 1× HEPES or 1× PBS ( appendix 2A), 37°C (optional)
  • 0.25% trypsin/0.02% EDTA solution (e.g., Invitrogen)
  • Trypsin neutralizing solution: growth medium (see protocol 1, step 1) containing 5% to 10% FBS)
  • 0.5% trypan blue or 0.5% neutral red in 1× PBS (see appendix 2A for PBS)
  • Inverted tissue culture microscope
  • Tissue‐culture 37°C water‐jacketed incubator, with humidified air and 5% to 6% CO 2 (Model Isotemp, Thermo Fisher Scientific or Forma Scientific)
  • 15‐ml tissue culture centrifugation (CFG) tubes with caps (Corning, cat. no. 430304)
  • Beckman Allegra 6R centrifuge
  • Hemacytometer (or flow cytometry instrumentation)
  • Additional reagents and equipment for preparation of growth medium ( protocol 1, step 1)

Basic Protocol 4: Determination of the Infectivity of BTV by Plaque Assay

  Materials
  • L cells (mouse subcutaneous connective tissue; ATCC #CRL‐2648) or Vero cells (African green monkey; ATCC #CCL‐81)
  • United States BTV 17, 13, 11, 10, and 2 viruses (Arthropod‐Borne Animal Disease Research Laboratory)
  • MEM/EBSS medium without FBS
  • 2× MEM/EBSS (see recipe)
  • 2% Ultrapure low‐melting‐point (LMP) agarose (see recipe)
  • Refined Noble agar (Invitrogen)
  • Methylcellulose (Sigma, cat. no M‐0512)
  • 25 µg/ml DEAE‐dextran
  • Crystal violet and neutral red (Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 6‐well flat‐bottom plates
  • Hemacytometer
  • Tissue‐culture 37°C water‐jacketed incubator, with humidified air and 5% to 6% CO 2 (Model Isotemp, Thermo Fisher Scientific or Forma Scientific)
  • Sonicator
  • 40°C water bath
  • Additional reagents and equipment for preparation of growth medium ( protocol 1, step 1)

Basic Protocol 5: Plaque‐Purification of Infectious BTV Grown with Either Vero or L Cells

  Materials
  • L cells (mouse subcutaneous connective tissue; ATCC #CRL‐2648) or Vero cells (African green monkey; ATCC #CCL‐81)
  • United States BTV 17, 13, 11, 10, and 2 viruses (Arthropod‐Borne Animal Disease Research Laboratory)
  • MEM/EBSS medium without FBS
  • 2% Ultrapure low‐melting‐point (LMP) agarose (see recipe)
  • 0.01% (w/v) neutral red (or trypan blue or crystal violet), filter sterilized
  • 2×MEM/EBSS (see recipe)
  • 12‐ or 6‐well flat‐bottom plates (Corning)
  • Tissue‐culture 37°C water‐jacketed incubator, with humidified air and 5% to 6% CO 2 (Model Isotemp, Thermo Fisher Scientific or Forma Scientific)
  • 40°C water bath
  • Additional reagents and equipment for preparation of growth medium ( protocol 1, step 1)

Basic Protocol 6: Amplification of Plaque‐Purified BTV Stock

  Materials
  • BHK‐21 cells (Syrian golden hamster kidney; ATCC #CCL‐10) growing in 25‐ or 75‐cm2 tissue culture plates (Corning, cat. no. 430168 or 430720, respectively)
  • MEM/EBSS medium containing 5% FBS and 10% FBS
  • Tissue‐culture 37°C water‐jacketed incubator, with humidified air and 5% to 6% CO 2 (Model Isotemp, Thermo Fisher Scientific or Forma Scientific)
  • Cell scraper
  • Beckman Allegra 6R centrifuge

Basic Protocol 7: Isolation and Purification of BTV

  Materials
  • BHK‐21 cells (Syrian golden hamster kidney; ATCC #CCL‐10)
  • 1.0 M NaCl
  • 30% (w/v) PEG‐6000 (Sigma, cat. no. P‐2139), sterile
  • TEN buffer: 0.01 M Tris⋅Cl ( appendix 2A), 0.01 M NaCl, 1 mM EDTA, pH 7.5 ( appendix 2A)
  • Sucrose
  • 850‐cm2 roller bottles (Becton Dickinson; Falcon cat. no. 3027)
  • 450‐cm2 PETG cell culture flasks (In Vitro, Ventura; cat. no. 4450‐05)
  • Disposable cell scrapers with handle length of 39 cm for roller bottles (Fisher Scientific, cat. no. 08‐773‐3)
  • 250‐ml CFG bottles (Corning, cat. no. 25350‐250)
  • Beckman Allegra 6R centrifuge
  • VirSonic Model 50 sonicator (Virtis) or Sonic Vibra‐Cell (http://www.sonics.com/)
  • Sterile filters with 0.22‐µm pores (BD Falcon, cat. no. 7105; or use 0.22‐µm‐pore‐size sterile PES filters from Whatman; see Critical Parameters and Troubleshooting)
  • Cryogenic vials with nonpyrogenic polypropylene sterile round bottom (Corning,
  • cat. no. 430487)
  • Tissue culture conical flask (Corning, cat. no. 431175)
  • Magnetic stirrer
  • Ultracentrifuge tubes and ultracentrifuge
  • Sonic Vibra‐Cell (Sonics & Material Inc.)
  • Additional reagents and equipment for preparation of medium ( protocol 1, step 1) and
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Literature Cited

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