Sindbis Virus: Propagation, Quantification, and Storage

Raquel Hernandez1, Christine Sinodis1, Dennis T. Brown1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15B.1
DOI:  10.1002/9780471729259.mc15b01s16
Online Posting Date:  February, 2010
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Abstract

The prototype of the Alphaviruses, Sindbis virus, has a broad host range. In nature, Sindbis virus shuttles from an insect vector to a vertebrate host and back to the insect vector in a complex transmission cycle. Sindbis virus must, therefore, be able to replicate in two biochemically and genetically divergent hosts, invertebrates and vertebrates. In the laboratory, Sindbis grows to high titers in a large variety of cultured cells of both vertebrate and invertebrate origin. Sindbis virus is easily titered for infectivity on several mammalian cell lines as well as certain mosquito cells. Full‐length cDNA clones of several strains of Sindbis virus are available from which infectious RNA can be synthesized, making possible the genetic manipulation of the virus. Transfection of mammalian and insect cells by electroporation has facilitated expression of RNA mutants in the cell lines of choice. Curr. Protoc. Microbiol. 16:15B.1.1‐15B.1.41. © 2010 by John Wiley & Sons, Inc.

Keywords: Sindbis virus; SVHR; plaque assay; focus assay; MTT titration; infection; transfection; insect cell culture; mammalian cell culture; Alphavirus

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Infection of BHK or CEF Cells
  • Basic Protocol 2: Sindbis Virus Purification
  • Alternate Protocol 1: BHK Cell Transfection
  • Basic Protocol 3: Assaying Virus by Plaque Formation on BHK Cells
  • Alternate Protocol 2: Assaying Virus by Focus Formation on BHK or Mosquito Cells
  • Alternate Protocol 3: Fluorescent Focus Assay on BHK or Mosquito Cells
  • Support Protocol 1: Propagation of BHK Cells
  • Support Protocol 2: Preparation of Primary Chick Embryo Fibroblast (CEF) Culture
  • Basic Protocol 4: Vero Cell MTT Assay of Alphavirus Production
  • Support Protocol 3: Propagation of Vero Cells
  • Basic Protocol 5: Mosquito Cell Monolayer Infection
  • Alternate Protocol 4: Mosquito Cell Infection in Suspension
  • Alternate Protocol 5: Mosquito Cell Transfection
  • Support Protocol 4: Propagation of Mosquito Cells
  • Support Protocol 5: BHK and Mosquito Cell Storage
  • Support Protocol 6: Transcription Reaction for the Production of Infectious Sindbis Virus RNA
  • Basic Protocol 6: Assay of Virus by Plaque Formation on C7‐10 Mosquito Cells
  • Basic Protocol 7: Purification of Virus from Plaques
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Infection of BHK or CEF Cells

  Materials
  • Baby hamster kidney (BHK) cells (ATCC #CCL10) or CEF cells, growing in culture ( protocol 7 or protocol 8)
  • 1× complete EMEM medium (see recipe)
  • Sindbis virus (SVHR) stock (see note below), titered (see protocol 4)
  • 1× PBS‐D (see recipe for 10×) containing 3% FBS (heat‐inactivated at 56°C; see appendix 2A)
  • Glycerol, sterile
  • Liquid nitrogen
  • Platform rocker
  • 25‐ or 75‐cm2 culture flasks (the authors have found that vented flasks are less susceptible to contamination with mold than the standard flasks)
  • 15‐ or 50‐ml conical polypropylene centrifuge tubes, sterile
NOTE: It is standard practice to grow a stock of virus from which additional virus stocks will be grown prior to any additional work with the virus. This practice avoids the production of defective interfering particles which will accumulate upon successive serial passage of high concentrations of virus. Generally, an MOI of 0.01 is required for production of this stock virus, referred to as the “stock stock,” i.e., the primary source from which stock virus is grown. This “stock stock” does not need to be gradient purified. In the event that it is suspected that the virus source is contaminated by other virus strains or by mutants, plaque purification (see protocol 18) may be required. Generally, infection at an MOI of 0.01, which is the method used to replenish the “stock stock,” is sufficient to suppress the production of defective interfering particles. Stock virus may be produced from any cell line of choice; stock virus grown in BHK cells yields a high titer and has the advantage that the progress of infection can be followed by evaluating the cytopathic effect.NOTE: Sindbis virus is very sticky and will bind to glass and plastic surfaces. The authors have found that Corning polypropylene tubes (microcentrifuge tubes, self‐standing, and conical) bind less virus than other brands of plastic.

Basic Protocol 2: Sindbis Virus Purification

  Materials
  • Virus supernatant (see protocol 1, step 8)
  • 15% and 35% (w/v) potassium tartrate in PBS‐D (see recipe for PBS‐D), 4°C
  • 1× PBS‐D (see recipe for 10×), 4°C
  • Beckman UltraClear centrifuge tubes (25 × 89–mm)
  • Beckman ultracentrifuge with SW28 rotor and buckets (or equivalent), prechilled to 4°C
  • Ring stands
  • Small tube clamps
  • Hand‐held low‐intensity (e.g., 20‐W) lamp
  • 30‐ to 100‐ml gradient former (e.g., Bio‐Rad model 385)

Alternate Protocol 1: BHK Cell Transfection

  Materials
  • Baby hamster kidney (BHK) cells (ATCC #CCL‐10), growing in culture (see protocol 7)
  • 1× complete EMEM medium (see recipe) without gentamicin
  • 1× PBS‐D (see recipe for 10×; use RNase‐free PBS‐D as indicated in protocol)
  • 0.25% trypsin (see recipe)
  • Versene solution (see recipe)
  • RNA transcript(s) from cDNA(s) of interest (see protocol 16)
  • 75‐ and 25‐cm2 tissue culture flasks
  • Siliconized, low‐retention RNase‐free microcentrifuge tubes (Fisher, cat. no. 02‐681‐331)
  • Aerosol‐barrier pipet tips
  • Electroporator (Bio Rad Gene Pulser)
  • Electroporation cuvettes, 2‐cm gap width
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, ) and harvesting of virus (see protocol 1)

Basic Protocol 3: Assaying Virus by Plaque Formation on BHK Cells

  Materials
  • Baby hamster kidney (BHK) cells (ATCC #CCL‐10), growing in culture (see protocol 7)
  • 1× and 2× complete EMEM medium (see recipe)
  • SVHR diluent (see recipe) or TM diluent for BHK cell assay (see recipe)
  • Virus stock to be titered (e.g., protocol 1, protocol 2, or protocol 3)
  • 2% agarose (see recipe)
  • 2× PBS‐D (see recipe for 10×)
  • 2% neutral red stock solution (see recipe)
  • 20 mM HEPES, pH 7.2 to 7.4 (see recipe; for less stable virus)
  • 25‐cm2 tissue culture flasks (the authors have found that vented flasks are less susceptible to contamination with mold than the standard flasks)
  • Dilution tubes: 13 × 100–mm borosilicate glass test tubes
  • Dilution tube rack
  • Platform rocker
  • Light box

Alternate Protocol 2: Assaying Virus by Focus Formation on BHK or Mosquito Cells

  Materials
  • Virus stock to be titered (e.g., protocol 1, protocol 2, or protocol 3)
  • 1× PBS‐D (see recipe for 10×) containing 3% FBS (heat‐inactivated at 56°C; see appendix 2A)
  • Baby hamster kidney (BHK) cells (see protocol 7) or mosquito cells (see protocol 11), growing in culture in 24‐well plates (see protocol introduction)
  • 2% carboxymethylcellulose (CMC; see recipe): dilute 1:1 in appropriate 2× medium for either BHK ( protocol 7) or mosquito ( protocol 14) cells
  • 1× phosphate buffered saline (PBS; appendix 2A), pH 7.4, containing 0.05% (v/v) Tween 20
  • 80% (v/v) methanol
  • Antibody dilution buffer: 5% (w/v) powdered (Carnation) skim milk in 1× PBS, with and without 0.05% (v/v) Tween 20
  • Primary antibody: anti–Sindbis virus antibody (usually rabbit; Cocalico Biologicals; http://www.cocalicobiologicals.com/)
  • Goat anti‐rabbit horseradish peroxidase (HRP)–labeled antibody (Sigma, cat. no. A‐8924)
  • TrueBlue peroxidase substrate system (TrueBlue Peroxidase Substrate—Kirkegaard & Perry Laboratories, cat. no. 50‐78‐02)

Alternate Protocol 3: Fluorescent Focus Assay on BHK or Mosquito Cells

  • 100% methanol
  • PBS‐BSA: 1× PBS ( appendix 2A) containing 0.2% bovine serum albumin (BSA)
  • Fluorescently labeled secondary antibody: goat anti rabbit Alexa Fluor 488 labeled antibody (Invitrogen, cat. no. A‐11017) for rabbit primary anti‐Sindbis antibody
  • Inverted fluorescent microscope

Support Protocol 1: Propagation of BHK Cells

  Materials
  • Baby hamster kidney (BHK) cells (ATCC #CCL‐10), growing in culture
  • 1× PBS‐D (see recipe)
  • 0.25% trypsin (see recipe)
  • Versene solution (see recipe)
  • 1× complete EMEM medium
  • 25‐ or 75‐cm2 tissue culture flasks
  • Conical tubes

Support Protocol 2: Preparation of Primary Chick Embryo Fibroblast (CEF) Culture

  Materials
  • Fertile chicken eggs, 10 days old (Spafas; http://www.criver.com/)
  • 70% ethanol
  • 1× phosphate‐buffered saline (PBS) with calcium and magnesium (see recipe)
  • 0.25% trypsin
  • Versene solution (see recipe)
  • 1× complete EMEM (see recipe)
  • Strong light source (75 to 100 W)
  • Small sterile beaker
  • Sharp scissors and forceps, sterile
  • 50‐ml conical tubes
  • 37°C water bath
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for counting cells (Strober, ), subculturing BHK cells (see protocol 7), and freezing cells (see protocol 15)

Basic Protocol 4: Vero Cell MTT Assay of Alphavirus Production

  Materials
  • Vero cells ( protocol 10)
  • 1× EMEM complete Vero media (see recipe)
  • Virus stock ( protocol 1, protocol 2, or protocol 3)
  • MTT labeling reagent (see recipe)
  • MTT solubilizing reagent (see recipe)
  • 96‐well Corning Cellbind culture plates (Costar 3300)
  • Plate reader with 500‐ to 700‐nm excitation and emission capability
  • Computer with Excel spreadsheet software
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic techniques should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 3: Propagation of Vero Cells

  Materials
  • Mosquito cells (e.g., U4.4 cells; laboratory isolate; contact authors at ), growing in tissue culture in 75‐cm2 flasks (also see protocol 14)
  • 1× PBS‐D (see recipe)
  • Serum‐free M&M or EMEM medium (e.g., Invitrogen; also see reciperecipes)
  • Sindbis virus (SVHR) stock, titered (see protocol 4 or protocol 176)
  • Fresh 1× complete Mitsuhashi and Maramorosch (M&M) medium (see recipe) or 1× complete EMEM medium (see recipe)
  • Glycerol
  • Neutral‐pH buffer (PBS‐D or culture medium buffered to pH 7.2 to 7.4 using 10 to 20 mM HEPES)
  • 28°C humidified 5% CO 2 incubator (CO 2 required only for use with EMEM)
  • 75‐cm2 tissue culture flask
  • Additional reagents and equipment for counting cells (Strober, ) and infecting cell cultures with Sindbis virus and harvesting and storing the virus (see protocol 1)

Basic Protocol 5: Mosquito Cell Monolayer Infection

  • Mosquito cells (e.g., U4.4 cells; laboratory isolate; contact authors at ), growing in tissue culture in 75‐cm2 flasks at ∼6 × 107 cells/flask (also see protocol 14)
  • Fresh 1× complete Mitsuhashi and Maramorosch (M&M) medium (see recipe) or 1× complete EMEM medium (see recipe)
  • Sindbis virus (SVHR) stock, titered (see protocol 4)
  • Liquid nitrogen
  • 75‐ and 25‐cm2 tissue culture flasks
  • Humidified 28°C, 5% CO 2 incubator (CO 2 required only for use with EMEM)
  • Additional reagents and equipment for counting cells (Strober, )

Alternate Protocol 4: Mosquito Cell Infection in Suspension

  Materials
  • Mosquito cells (e.g., U4.4 cells; laboratory isolate; contact authors at ), growing in tissue culture in 75‐cm2 flasks (also see protocol 14)
  • 1× PBS‐D (see recipe for 10×)
  • HBS buffer (see recipe), RNase‐free, ice‐cold
  • RNA transcript(s) from cDNA(s) of interest (see protocol 16)
  • 1× complete Mitsuhashi and Maramorosch (M&M) medium (see recipe)
  • 1 M MOPS buffer (see recipe; for use with mosquito cell cultures and mutant viruses requiring additional buffering)
  • 50‐ml conical polypropylene centrifuge tubes
  • Aerosol‐barrier pipet tips, RNase free
  • RNase‐free microcentrifuge tubes
  • Electroporation cuvettes 2‐cm gap width
  • Electroporator (e.g., BioRad Gene Pulser)
  • Additional reagents and equipment for counting cells (Strober, ) and harvesting virus (see protocol 1)

Alternate Protocol 5: Mosquito Cell Transfection

  Materials
  • Mosquito cells—U4.4 and C7‐10 (contact authors at ) and C6/36 cells (ATCC #CRL‐1660)—growing in semisuspension
  • Medium: 1× complete Mitsuhashi and Maramorosch (M&M) medium (see recipe) or 1× complete EMEM medium (see recipe)
  • Tissue culture flasks
  • Humidified 28°C, 5% CO 2 incubator (CO 2 required only for use with EMEM)

Support Protocol 4: Propagation of Mosquito Cells

  Materials
  • BHK cells (ATCC #CCL‐10)
  • 0.25% trypsin (see recipe)
  • Versene solution (see recipe)
  • 1× complete EMEM medium
  • Freezing medium (see recipe) made with EMEM for BHK cells or with either M&M or EMEM for mosquito cells, depending on optimal growth medium
  • Mosquito cells: U4.4 and C7‐10 (contact authors at ) and C6/36 cells (ATCC #CRL‐1660)
  • Cryotubes
  • Nalgene Cryo 1°C freezing container
  • Liquid nitrogen freezer

Support Protocol 5: BHK and Mosquito Cell Storage

  Materials
  • RNase‐free H 2O (Gilman, ; also see appendix 2A)
  • Linearized cDNA of interest: do not treat with RNase
  • 10× transcription buffer (see recipe)
  • dNTPs: ATP, UTP, GTP, and CTP ( appendix 2A)
  • m7GpppG (New England Biolabs)
  • RNase inhibitor (Ambion)
  • SP6 RNA polymerase (New England Biolabs)
  • Ethidium bromide staining solution ( appendix 2A)
  • RNase‐free DNase
  • RNase‐free tubes and pipets
  • 40°C water bath

Support Protocol 6: Transcription Reaction for the Production of Infectious Sindbis Virus RNA

  Materials
  • C7‐10 mosquito cells (laboratory isolate; contact authors at ) growing in 75‐cm2 tissue culture flasks
  • 1× and 2× serum‐free EMEM medium (see recipe)
  • TM diluent for C7‐10 plaque assay (see recipe)
  • SVHR sample to be titered (any cell source)
  • 2× complete EMEM (see recipe)
  • 2% agarose (see recipe)
  • 1× PBS‐D (see recipe) containing 20 mM MOPS (prepare using 1 M MOPS buffer; see recipe)
  • 2% neutral red stock solution (see recipe)
  • 25‐cm2 tissue culture flasks
  • Humidified 28°C 5% CO 2 incubator (CO 2 required only for use with EMEM)
  • Dilution tubes: 13 × 100–mm borosilicate glass test tubes
  • Dilution tube rack
  • Platform rocker
  • Additional materials for counting cells (Strober, )

Basic Protocol 6: Assay of Virus by Plaque Formation on C7‐10 Mosquito Cells

  Materials
  • Virus stock to be purified (see above protocols)
  • Virus diluent: 1× PBS‐D (for wild‐type SVHR; see recipe) or TM diluent for BHK or C7‐10 plaque assay as appropriate (for mutants; see reciperecipes)
  • Soldering iron
  • Sterile Pasteur pipets and tubes
  • Additional reagents and equipment for plaque assay (see protocol 4 for BHK cells or protocol 17 for mosquito cells)
NOTE: All solutions and equipment that comes into contact with living cells must be sterile and aseptic technique must be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
   Brown, D.T., Smith, J.F., Gliedman, J.B., Riedel, B., Filtzer, D., and Renz, D. 1976. Morphogenesis of Sindbis virus in cultured mosquito cells. In Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture (E. Kurstak and K. Maramorosch, eds.) pp. 35‐48. Academic Press, New York.
   Gilman, M. 2002. Preparation of cytoplasmic RNA from tissue culture cells. Curr. Protoc. Mol. Biol. 58:4.1.1‐4.1.5.
   Heldt, C.L., Hernandez, R., Mudiganti, U., Gurgel, P.V., Brown, D.T., and Carbonell, R.G. 2006. A colorimetric assay for viral agents that produce cytopathic effects. J. Virol. Methods 135:56‐65.
   Hernandez, R., Lee, H., Nelson, C., and Brown, D.T. 2000. A single deletion in the membrane‐proximal region of the Sindbis virus glycoprotein E2 endodomain blocks virus assembly. J. Virol. 74:4220‐4228.
   Hernandez, R., Luo, T., and Brown, D.T. 2001. Exposure to low pH is not required for penetration of mosquito cells by Sindbis virus. J. Virol. 75:2010‐2013.
   Hernandez, R., Sinodis, C., Horton, M., Ferreira, D., Yang, C., and Brown, D.T. 2003. Deletions in the transmembrane domain of a sindbis virus glycoprotein alter virus infectivity, stability, and host range. J. Virol. 77:12710‐12719.
   Igarashi, A. 1978. Isolation of a Singh Aedes albopictus cell clone sensitive to Dengue and Chickungunya viruses. J. Gen. Virol. 40:531‐544.
   Lilijestrom, P. and Garoff, H. 1991. Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor. J. Virol. 65:147‐154.
   Liu, L.N., Lee, H., Hernandez, R., and Brown, D.T. 1996. Mutations in the endo domain of Sindbis virus glycoprotein E2 block phosphorylation, reorientation of the endo domain, and nucleocapsid binding. Virology 222:236‐246.
   Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Extraction, Purification and Analysis of mRNA from Eukaryotic Cells. In Molecular Cloning a Laboratory Manual, Vol. 1 (T. Maniatis, E.F. Fritsch, and J. Sambrook, eds.). p. 190. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
   Miller, M.L., and Brown, D.T. 1992. Morphogenesis of Sindbis virus in three subclones of Aedes albopictus (mosquito) cells. J. Virol 66:4180‐4190.
   Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55‐63.
   Phinney, B.S. and Brown, D.T. 2000. Sindbis virus glycoprotein E1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections. J. Virol. 74:9313‐9316.
   Phinney, B.S., Blackburn, K., and Brown, D.T. 2000. The surface conformation of Sindbis virus glycoproteins E1 and E2 at neutral and low pH, as determined by mass spectrometry‐based mapping. J. Virol. 74:5667‐5678.
   Rice, C.M., Levis, R., Strauss, J.H., and Huang, H.V. 1987. Production of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature‐sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61:3809‐3819.
   Sarver, N. and Stollar, V. 1977. Sindbis virus‐induced cytopathic effect in clones of Aedes albopictus (Singh) cells. Virology 80:390‐400.
   Scheefers‐Borchel, U., Scheefers, H., Edwards, J., and Brown, D.T. 1981. Sindbis virus maturation in cultured mosquito cells is sensitive to actinomycin D. Virology 110:292‐301.
   Sefton, B.M. and Keegstra, K. 1974. Glycoproteins of Sindbis virus: Preliminary characterization of the oligosaccharides. J. Virol. 14:522‐530.
   Singh, K.R.P. 1967. Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci. India 36:506‐508.
   Strauss, J.H. and Strauss, E.G. 2002. Family Togaviridae. In Viruses and Human Disease (J.H. Strauss and E.G. Strauss, eds.), pp. 76‐84. Academic Press, San Diego.
   Strober, W. 1997. Monitoring cell growth. Curr. Protoc. Immunol. 21:A.3A.1‐A.3A.2.
   Struhl, K. 1998. Synthesizing proteins in vitro by transcription and translation of cloned genes. Curr. Protoc. Mol. Biol. 48:10.17.1‐10.17.5.
   Voytas, D. 1991. Agarose gel electrophoresis. Curr. Protoc. Immunol. 2:10.4.1‐10.4.8.
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