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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 protocol 2)
  • 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

  • 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

  • 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 reciperecipes for IPAM cell medium, Vero and COS‐1 cell medium, and WSL cell medium)
  • 37°C, 5% CO 2 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)
  • CO 2 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 protocol 4)

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 protocol 5)
  • 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 N 2
  • 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 protocol 1) or frozen macrophages (see protocol 3)
  • DMEM ( appendix 2A)
  • Autologous or homologous (HAD‐compatible) swine serum (see protocol 2)
  • Virus samples (see protocol 6)
  • Heparinized swine peripheral blood (see 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, CO 2 incubator
  • 96‐well plates (e.g., Falcon, Costar, or Nunc)
  • Contrast‐phase inverted microscope and hemacytometer

Alternate Protocol 3: Cytopathic Effect on Swine Macrophages

  • 2% crystal violet in 5% formaldehyde

Alternate Protocol 4: Plaque Assay on Swine Macrophages

  • Swine serum (see protocol 2)
  • Solid agar‐medium solution (see recipe)
  • 2% crystal violet (prepared in 5% formaldehyde)
  • 24‐well plates
  • Filter paper

Basic Protocol 5: Plaque Assay on Established Cell Lines (Vero and Cos‐1 Cells)

  Materials
  • Cell lines (COS‐1, IPAM, WSL, and swine alveolar macrophages) and corresponding cell culture media
  • ASFV isolates (Ba71V, ΔEP153R, E70, NHV, and Lisbon 60)
  • 24‐well plates
  • 37°C incubator
  • Ultrasonic bath (e.g., Bransonic 12)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aguero, M., Fernandez, J., Romero, L., Sanchez Mascaraque, C., Arias, M., and Sanchez‐Vizcaino, J.M. 2003. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. J. Clin. Microbiol. 41:4431‐4434.
   Barderas, M.G., Wigdorovitz, A., Merelo, F., Beitia, F., Alonso, C., Borca, M.V., and Escribano, J.M. 2000. Serodiagnosis of African swine fever using the recombinant protein p30 expressed in insect larvae. J. Virol. Methods. 89:129‐136.
   Bustos, M.J., Nogal, M.L., Revilla, Y., and Carrascosa, A.L. 2002. Plaque assay for African swine fever virus on swine macrophages. Arch. Virol. 147:1453‐1459.
   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.
   Carrascosa, A.L., del Val, M., Santaren, J.F., and Vinuela, E. 1985. Purification and properties of African swine fever virus. J. Virol. 54:337‐344.
   Casal, I., Enjuanes, L., and Vinuela, E. 1984. Porcine leukocyte cellular subsets sensitive to African swine fever virus in vitro. J. Virol. 52:37‐46.
   Dixon, L.K., Abrams, C.C., Bowick, G., Goatley, L.C., Kay‐Jackson, P.C., Chapman, D., Liverani, E., Nix, R., Silk, R., and Zhang, F. 2004. African swine fever virus proteins involved in evading host defence systems. Vet. Immunol. Immunopathol. 100:117‐134.
   Enjuanes, L., Carrascosa, A.L., Moreno, M.A., and Vinuela, E. 1976. Titration of African swine fever (ASF) virus. J. Gen. Virol. 32:471‐477.
   Galindo, I., Almazán, F., Bustos, M.J., Viñuela, E., and Carrascosa, A.L. 2000. African Swine Fever Virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells. Virology 266:340‐351.
   Garcia, R., Almazan, F., Rodriguez, J.M., Alonso, M., Vinuela, E., and Rodriguez, J.F. 1995. Vectors for the genetic manipulation of African swine fever virus. J. Biotechnol. 40:121‐131.
   Granja, A.G., Nogal, M.L., Hurtado, C., Del Aguila, C., Carrascosa, A.L., Salas, M.L., Fresno, M., and Revilla, Y. 2006. The viral protein A238L inhibits TNF‐{alpha} expression through a CBP/p300 transcriptional coactivators pathway. J. Immunol. 176:451‐462.
   Hurtado, C., Bustos, M.J., and Carrascosa, A.L. 2010. The use of COS‐1 cells for studies of field and laboratory African swine fever virus samples. J. Virol. Methods 164:131‐134.
   Oura, C.A., Powell, P.P., and Parkhouse, R.M. 1998. Detection of African swine fever virus in infected pig tissues by immunocytochemistry and in sity hybridisation. J. Virol. Methods 72:205‐217.
   Parker, J. and Plowright, W. 1968. Plaque formation by African swine fever virus. Nature 219:524‐525.
   Pastor, M.J., Laviada, M.D., Sanchez‐Vizcaino, J.M., and Escribano, J.M. 1989. Detection of African swine fever virus antibodies by immunoblotting assay. Can. J. Vet. Res. 53:105‐107.
   Pastor, M.J., Arias, M., Alcaraz, C., De Diego, M., and Escribano, J.M. 1992. A sensitive dot immunobinding assay for serodiagnosis of African swine fever virus with application in field conditions. J. Vet. Diagn. Invest. 4:254‐257.
   Vinuela, E. 1985. African swine fever virus. Curr. Top. Microbiol. Immunol. 116:151‐170.
   Wilkinson, P.J. 1989. African swine fever virus. In Virus Infections of Porcines (M.B. Pensaert, ed.) pp. 17‐37. Elsevier Sciences Publishers, Amsterdam.
   Zsak, L., Borca, M.V., Risatti, G.R., Zsak, A., French, R.A., Lu, Z., Kutish, G.F., Neilan, J.G., Callahan, J.D., Nelson, W.M., and Rock, D.L. 2005. Preclinical diagnosis of African swine fever in contact‐exposed swine by a real‐time PCR assay. J. Clin. Microbiol. 43:112‐119.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library