Methods for Growing and Titrating African Swine Fever Virus: Field and Laboratory Samples

Angel L. Carrascosa1, M. Jose Bustos1, Patricia de Leon1

1 Centro de Biologia Molecular “Severo Ochoa” (CSIC‐UAM), Universidad Autonoma de Madrid, Madrid, Spain
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 26.14
DOI:  10.1002/0471143030.cb2614s53
Online Posting Date:  December, 2011
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Abstract

Growing African swine fever virus (ASFV) isolates obtained mainly from the field, but also engineered in the laboratory, is a critical step for diagnosis, titration, or virus infection studies. This unit describes a set of methods and protocols to produce and titrate any ASFV strain in cell cultures. The procedures include (1) basic techniques to prepare virus-sensitive target cells; (2) strategies for growth, concentration, and purification of virus stocks; and (3) the semi-quantitative (end dilution) and quantitative (plaque) assays for the determination of viral titers, and the use of different ASFV-sensitive cells as targets for virus production and titration. Curr. Protoc. Cell Biol. 53:26.14.1-26.14.25. © 2011 by John Wiley & Sons, Inc.

Keywords: African swine fever virus (ASFV); susceptibility; production; purification; titration; hemadsorption; cytopathic effect; plaque assay; target cells; Vero; COS-1; IPAM; WSL; swine macrophages

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Swine Peripheral Blood Monocytes
  • Support Protocol 1: Preparation of Swine Serum
  • Alternate Protocol 1: Preparation of Swine Alveolar Macrophages
  • Alternate Protocol 2: Preparation of Established Cell Lines
  • Basic Protocol 2: Growth and Purification of Susceptible Cells (Vero and Cos-1) in Roller Bottles
  • Basic Protocol 3: Virus Purification (Percoll Method)
  • Basic Protocol 4: Infectivity Assay: Hemadsorption on Swine Monocytes/Macrophages
  • Alternate Protocol 3: Cytopathic Effect on Swine Macrophages
  • Alternate Protocol 4: Plaque Assay on Swine Macrophages
  • Basic Protocol 5: Plaque Assay on Established Cell Lines (Vero and Cos-1 Cells)
  • Basic Protocol 6: Infection of Sensitive Cell Lines by Different ASFV Isolates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Swine Peripheral Blood Monocytes

 Materials
  • Swine (large white, 20 to 30 kg)
  • 70% ethanol
  • 100-ml glass bottles containing anticoagulant and antibiotics (40 IU/ml heparin/100 IU/ml penicillin/0.1 mg/ml streptomycin)
  • Phosphate-buffered saline (PBS; see recipe)
  • Heparin
  • Swine serum (see Support Protocol 1)
  • Erythrocyte lysis buffer (see recipe)
  • Dulbecco's modified Eagle medium (DMEM; appendix 2A)
  • Restraining ropes
  • Atraumatic needles (22- or 23-G; Novartis) connected to a Multifix Mini peristaltic pump with a sterile tygon tube (or to a vacutainer)
  • 37°C incubator
  • Refrigerated centrifuge

Support Protocol 1: Preparation of Swine Serum

 Materials
  • Restrained pig
  • 250-ml glass bottles
  • 37°C incubator
  • Gauze
  • 50-ml conical centrifuge tubes (Falcon)
  • Batch filtration device (Sartorius model SM16263/67)
  • Sterile membranes (Millipore) with pore mean sizes of 1.2, 0.45, and 0.22 µm

Alternate Protocol 1: Preparation of Swine Alveolar Macrophages

 Additional Materials (also see Basic Protocol 1)
  • Swine (large white, 20 to 30 kg)
  • Ketamine (Ketolar 50; Sigma Chemicals)
  • Midazolam (Dormicum; Sigma Chemicals)
  • Atropine (Atropine B; Sigma Chemicals)
  • Fetal calf serum, heat inactivated, cold
  • DMSO (dimethyl sulfoxide; Hybri-Max, Sigma, cat. no. D2650)
  • Mechanical cutters (scalpels, scissors, sternal saw)
  • Sternal retactor
  • Hemostatic clamps and forceps
  • 1-liter sterile bottles
  • 2-ml cryotubes (Nunc, cat. no. 375418)
  • Cryo cooler (Nalgene Cryo 1°C freezing container, cat. no. 5100-0001)
  • 37°C water bath

Alternate Protocol 2: Preparation of Established Cell Lines

 Additional Materials (also see Alternate Protocol 1)
  • Trypsin-EDTA solution (see recipe)
  • Established cell lines from monkey (Vero and COS-1) or from porcine (IPAM and WSL) species and their corresponding cell medium (see recipes for IPAM cell medium, Vero and COS-1 cell medium, and WSL cell medium)
  • 37°C, 5% CO2 humidified incubator
  • Tissue culture plates (10-, 5-, or 2-cm diameter dishes or multi-well plates)

Basic Protocol 2: Growth and Purification of Susceptible Cells (Vero and Cos-1) in Roller Bottles

 Materials
  • Vero or COS-1 cells (10-mm confluent plates)
  • Vero/COS-1 cell medium (see recipe), 37°C
  • ASFV
  • Saline or PBS (see recipe)
  • Sterile 1000-ml glass bottles (e.g., borosilicate 3.3; VWR, cat. no. 215-1595)
  • CO2 injecting device
  • Sterile cotton-plugged pipets
  • Roller apparatus (e.g., Modular Cell Production Model III, Wheaton Instruments, for up to 90 bottles) integrated into an incubator (Hotpack model 1650, with forced-air circulation)
  • Centrifuge tubes
  • Additional reagents and equipment for trypsinizing cells (see Alternate Protocol 2)

Basic Protocol 3: Virus Purification (Percoll Method)

 Materials
  • Percoll (GE Healthcare, cat. no. 17-0891-01)
  • 10× PBS (see recipe)
  • Extracellular ASFV concentrated in PBS (see Basic Protocol 2)
  • Tris-sucrose solution (10 mM Tris×Cl (pH 7.5)/0.25 M sucrose)
  • Sephacryl S-1000 superfine (GE Healthcare, cat. no. 17-0476-01)
  • 10% trichloroacetic acid (TCA), cold
  • Liquid N2
  • 26-ml polycarbonate ultracentrifuge bottles (with liquid-tight cap assembly, 25 × 89–mm)
  • Refrigerated ultracentrifuge
  • Fixed-angle ultracentrifuge T865 rotor (Sorvall) and Sorvall AH-650 rotor
  • Pasteur pipets
  • Vacuum
  • Chromatographic column (e.g., 1-cm diameter, 20-cm high, 15-ml total volume; Pharmacia Fine Chemicals)
  • Fraction collector (laboratory-made or commercial; e.g., LKB 2112 Redirack)
  • Spectrophotometer (e.g., Shimadzu, model no. UV-1201)

Basic Protocol 4: Infectivity Assay: Hemadsorption on Swine Monocytes/Macrophages

 Materials
  • Swine erythrocyte suspension (see Basic Protocol 1) or frozen macrophages (see Alternate Protocol 1)
  • DMEM (appendix 2A)
  • Autologous or homologous (HAD-compatible) swine serum (see Support Protocol 1)
  • Virus samples (see Basic Protocol 3)
  • Heparinized swine peripheral blood (see Basic Protocol 1)
  • PBS (see recipe)
  • Microtest I plates (60-well, Falcon 3034)
  • Electronic adjustable pipet
  • Hamilton microsyringe coupled to an automatic repeating dispenser
  • 37°C, CO2 incubator
  • 96-well plates (e.g., Falcon, Costar, or Nunc)
  • Contrast-phase inverted microscope and hemacytometer

Alternate Protocol 3: Cytopathic Effect on Swine Macrophages

 Additional Materials (also see Basic Protocol 4)
  • 2% crystal violet in 5% formaldehyde

Alternate Protocol 4: Plaque Assay on Swine Macrophages

 Additional Materials (also see Basic Protocol 4)
  • Swine serum (see Support Protocol 1)
  • Solid agar-medium solution (see recipe)
  • 2% crystal violet (prepared in 5% formaldehyde)
  • 24-well plates
  • Filter paper

Basic Protocol 6: Infection of Sensitive Cell Lines by Different ASFV Isolates

 Materials
  • Cell lines (COS-1, IPAM, WSL, and swine alveolar macrophages) and corresponding cell culture media
  • ASFV isolates (Ba71V, EP153R, E70, NHV, and Lisbon 60)
  • 24-well plates
  • 37°C incubator
  • Ultrasonic bath (e.g., Bransonic 12)
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Figures

  •  FigureFigure 26.14.1 Porcine lungs as extracted from the thoracic cavity (A) and distended by filling with PBS (B).
    View Image
  •  FigureFigure 26.14.2 Purification of ASFV. Gel filtration on Sephacryl S-1000. Extracellular ASFV purified by Percoll sedimentation and subjected to chromatography on Sephacryl S1000 column. Determination of Percoll by precipitation with 10% TCA. Virus protein detected in the excluded fractions and Percoll particles in the included volume.
    View Image
  •  FigureFigure 26.14.3 Micrographs of extracellular ASFV particles purified by Percoll sedimentation. (A) Negative staining, low magnification. (B) Negative staining, high magnification. (C) Ultra-thin section.
    View Image
  •  FigureFigure 26.14.4 Hemadsorption in ASFV-infected cells. (A) Peripheral blood monocytes incubated for 2 days after removing the non-adherent cells. (B) A similar culture after ASFV infection and addition of swine erythrocytes to develop HAD rosettes.
    View Image
  •  FigureFigure 26.14.5 Calculating HADU50: A practical case.
    View Image
  •  FigureFigure 26.14.6 Titration of an ASFV sample by cytopathic effect assay. Cultures of swine alveolar macrophages in 96-well plate, inoculated with serial ten-fold dilutions of the virus sample and stained with crystal violet 5 days after infection. With the data scored in this case, and considering a volume of 0.02 ml of inoculum per well, the titer of the virus sample is: 1/10-5.375 × 0.02 = 1.18 × 106 TCID50/ml.
    View Image
  •  FigureFigure 26.14.7 Plaque assay of ASFV samples on swine macrophages. Swine alveolar cells grown in 24-well plates (390,000 cells/cm2) were infected with ten-fold dilutions of virus samples, overlaid with agar-medium and incubated 5 days at 37°C, before stained with crystal violet.
    View Image
  •  FigureFigure 26.14.8 Plaques developed in COS cell monolayers by different ASFV isolates. COS-1 cells grown in 24-well plates (120,000 cells/well) were infected with ten-fold dilutions of virus samples, overlaid with agar-medium and incubated 5 days at 37°C, before stained with crystal violet.
    View Image
  •  FigureFigure 26.14.9 Production of infective virus in four virus-susceptible cells infected with different ASFV isolates. Cell cultures infected at an MOI of 3 pfu/cell were collected at 3 to 4 days after infection and the total virus titrated in COS-1 cell monolayers.
    View Image

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Literature Cited

Literature Cited
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    Carrascosa, A.L., Santaren, J.F., and Vinuela, E. 1982. Production and titration of African swine fever virus in porcine alveolar macrophages. J. Virol. Methods 3:303-310.
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