Herpes Simplex Virus: Propagation, Quantification, and Storage

John A. Blaho1, Elise R. Morton1, Jamie C. Yedowitz1

1 Mount Sinai School of Medicine, New York, New York
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 14E.1
DOI:  10.1002/9780471729259.mc14e01s00
Online Posting Date:  October, 2005
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Abstract

Herpes simplex virus (HSV) is the prototype of a family of large, enveloped, double‐stranded DNA viruses, the Herpesviridae, which cause significant morbidity and mortality in humans. Productive replication of HSV in cells in culture results in definitive changes in cellular physiology and metabolism, ultimately leading to lysis. These definitive aspects of viral‐host interactions enable diagnosis of HSV infections. In this unit, a series of methods are described for the propagation, quantification, and storage of HSV. Infectious center assays are used to measure the titers of HSV stocks. In addition, immunological methods are described for documenting the accumulation of viral polypeptides in infected whole cell extracts, as well as in situ using indirect immunofluorescence. These techniques should be beneficial to basic research virologists utilizing standard laboratory HSV strains, as well as clinical microbiologists interested in characterizing HSV isolated from patients.

Keywords: HSV; cells; medium; plaques; immunoblots; indirect immunofluorescence

     
 
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Table of Contents

  • Basic Protocol 1: Preparing Virus Stocks
  • Basic Protocol 2: Determining Virus Titers
  • Basic Protocol 3: Picking Virus Plaques for Plaque Purification
  • Support Protocol 1: Splitting Cells
  • Support Protocol 2: Freezing Cells
  • Support Protocol 3: Thawing Cells
  • Basic Protocol 4: Preparing Whole Infected Cell Extracts for Immunoblotting
  • Basic Protocol 5: Preparing and Running DATD‐Acrylamide Gels with HSV Whole Infected Cell Extracts for Immunoblotting
  • Basic Protocol 6: Detection of HSV Proteins by Immunoblotting
  • Basic Protocol 7: Using Indirect Immunofluorescence to Localize Viral Proteins within Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparing Virus Stocks

  Materials
  • Cell line of choice (e.g., Vero; see Table 14.1.1) grown to confluence in 75‐cm2 tissue culture flasks (see protocol 4 for culture techniques)
  • 199V medium (see recipe)
  • HSV stock (ATCC), titered (see protocol 2)
  • DMEM/5% NBCS medium (see recipe)
  • Sterile milk (see recipe)
  • Freezer box, cardboard (optional)
  • 15‐ml conical tubes, sterile
  • Probe sonicator (e.g., Branson Sonifier) with microtip
  • Screw‐capped cryovials
  • Additional reagents and equipment for titering virus (see protocol 2)
    Table 4.0.1   MaterialsExamples of Commonly Used HSV‐Permissive Cell Lines

    Cell line ATCC Growth conditions Uses
    Vero(African Green Monkey Kidney Cells) #81‐CCL DMEM containing serum, passage every 3–4 days, 37°C, 5% CO 2 Growing virus stocks and titering Indirect immunofluorescence
    HEp‐2(Human Epithelial Carcinoma Cells) #23‐CCL DMEM containing serum, passaged every 3–4 days, 37°C, 5% CO 2 Preparation of infected cell extracts Indirect immunofluorescence

Basic Protocol 2: Determining Virus Titers

  Materials
  • Cell line of choice (e.g., Vero)
  • DMEM/5% FBS medium (see recipe)
  • 199V medium (see recipe)
  • Virus stock (see protocol 1)
  • 20 mg/ml pooled human immunoglobulin (Sigma) in distilled H 2O
  • Phosphate‐buffered saline with potassium (KPBS; see recipe)
  • Methanol
  • KaryoMax Giemsa Stain stock solution (Invitrogen)
  • 25‐cm2 tissue culture flasks
  • Plugged, sterile pipet tips
  • Fine‐point marking pen
  • Inverted microscope
  • Additional reagents and equipment for splitting cells (see protocol 4)

Basic Protocol 3: Picking Virus Plaques for Plaque Purification

  Materials
  • Virus stock (see protocol 1)
  • DMEM/5% NBCS medium (see recipe)
  • 199V medium (see recipe)
  • 20 mg/ml pooled human immunoglobulin (Sigma) in distilled H 2O
  • Sterile milk (see recipe)
  • 1% (w/v) agarose in KPBS (see recipe for KPBS; store at 4°C)
  • Phosphate‐buffered saline containing potassium (KPBS; see recipe), sterile
  • 25‐cm2 tissue culture flasks
  • Plugged, sterile pipet tips
  • Fine‐point marking pen
  • 15‐ml snap‐cap tubes
  • Bent Pasteur pipet (see recipe)
  • Hand‐held battery‐operated pipetting device (e.g., Pipet‐Aid; Drummond Scientific)
  • Probe sonicator (e.g., Branson Sonifier) with microtip
  • Additional reagents and equipment for splitting cells (see protocol 4)

Support Protocol 1: Splitting Cells

  Materials
  • DMEM/5% FBS medium (see recipe)
  • Cells: Vero or HEp‐2 (Table 14.1.1)
  • Phosphate‐buffered saline with potassium (KPBS; see recipe), sterile
  • 70% ethanol
  • Trypsin‐EDTA (Invitrogen; also see recipe)
  • Culture vessels: 75‐ or 25‐cm2 tissue culture flasks

Support Protocol 2: Freezing Cells

  Materials
  • Phosphate‐buffered saline with potassium (KPBS; see recipe), sterile
  • DMEM/5% NBCS medium (see recipe)
  • Dimethylsulfoxide (DMSO), sterile
  • 70% ethanol
  • Confluent flasks of Vero or HEp‐2 cells (see protocol 4)
  • Trypsin‐EDTA (Invitrogen; also see recipe)
  • Liquid nitrogen
  • Tabletop clinical centrifuge
  • 1.5‐ml cryovials
  • Liquid nitrogen tank or freezer

Support Protocol 3: Thawing Cells

  Materials
  • DMEM/5% FBS medium with antibiotics (see recipe)
  • Cryovial containing frozen cells (see protocol 5, or purchase from ATCC), maintained at −70°C or below
  • 70% ethanol
  • 75‐cm2 flask (or alternative culture vessel)
  • Inverted tissue culture microscope

Basic Protocol 4: Preparing Whole Infected Cell Extracts for Immunoblotting

  Materials
  • DMEM/5% FBS medium (see recipe)
  • 199V medium (see recipe)
  • Phosphate‐buffered saline with potassium (KPBS; see recipe), 4°C
  • Buffer A with protease inhibitors (see recipe), 4°C
  • Bio‐Rad Protein Assay solution
  • Platform rocker
  • Cell scraper: preferably 12 in. (∼30 cm) long
  • 6‐ml tubes
  • Tabletop clinical centrifuge
  • Probe sonicator (e.g., Branson Sonifier) with microtip
  • Spectrophotometer
  • Additional reagents and equipment for splitting cells (see protocol 4), infecting cells (see protocol 1)

Basic Protocol 5: Preparing and Running DATD‐Acrylamide Gels with HSV Whole Infected Cell Extracts for Immunoblotting

  Materials
  • Mild laboratory detergent
  • 70% ethanol
  • Petroleum jelly
  • 1.4 µg/ml ammonium persulfate
  • Protein gel solutions A, B, and C (see recipe)
  • 20% (w/v) SDS ( appendix 2A)
  • TEMED
  • 30% acrylamide/bisacrylamide (see recipe)
  • 1× protein running buffer (see recipe for 10×)
  • Whole infected cell extract (see protocol 7)
  • Buffer A (see recipe)
  • 4× disruption buffer (see recipe)
  • Gel‐forming apparatus:
    • Glass plates for 20 × 20–cm gel
    • 2‐mm spacers
    • Combs
    • Large binder clips
  • Syringes
  • Vertical electrophoresis apparatus, power supply, and cables
  • Bent needles for removing bubbles from electrophoresis chamber
  • Boiling water bath

Basic Protocol 6: Detection of HSV Proteins by Immunoblotting

  Materials
  • Gel containing separated HSV proteins (see protocol 8)
  • Transfer buffer with and without SDS (see recipes)
  • 0.1% Ponceau S (optional; see recipe)
  • Phosphate‐buffered saline (KPBS; see recipe)
  • 5% milk/KPBS (see recipe)
  • Tris‐buffered saline (TBS; see recipe)
  • Primary antibodies: HSV‐specific antibodies are available from the Rumbaugh‐Goodwin Institute for Cancer Research (http://www.rgicr.org/)
  • Secondary antibody, alkaline phosphatase–conjugated (Sigma or Fisher); fluorescently tagged antibodies may be obtained from Molecular Probes
  • 1% BSA/KPBS (see recipe)
  • Tris‐buffered saline with Tween 20 (TBST; see recipe)
  • AP buffer (see recipe)
  • 15 mg/ml nitroblue tetrazolium chloride (NBT) in 70% dimethylformamide (DMF)
  • 30 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP) in 100% DMF
  • Whatman no. 1 filter paper
  • 0.45‐µm nitrocellulose transfer membrane
  • Plastic dish large enough to accommodate gel
  • Flat glass plate slightly larger than gel
  • Electroblotting apparatus: tank transfer system including transfer cassette and power supply (e.g., Bio‐Rad)

Basic Protocol 7: Using Indirect Immunofluorescence to Localize Viral Proteins within Cells

  Materials
  • Ethanol
  • Cells of choice, growing in tissue culture
  • DMEM/5% FBS medium (see recipe)
  • Virus stock (see protocol 1)
  • 199V medium (see recipe)
  • DMEM/5% NBCS medium (see recipe)
  • Phosphate‐buffered saline (KPBS; see recipe)
  • 2.5% paraformaldehyde (see recipe)
  • Acetone, −20°C
  • 10 µg/ml human immunoglobulin in 1% BSA/KPBS (see recipe for 1% BSA/KPBS)
  • Primary antibody
  • 1% BSA/KPBS (see recipe)
  • Fluorescently conjugated secondary antibody
  • ProLong Antifade Kit (Molecular Probes)
  • Clear nail polish
  • 25‐mm2 coverslips
  • 6‐well or 33‐mm2 tissue culture dishes
  • Dark plastic box
  • Glass microscope slides
  • Additional reagents and equipment for splitting cells (see protocol 4)
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Figures

Videos

Literature Cited

   Pomeranz, L.E. and Blaho, J.A. 1999. Modified VP22 localizes to the cell nucleus during synchronized herpes simplex virus type 1 infection. J. Virol. 73:6769‐6781.
   Pomeranz, L.E. and Blaho, J.A. 2000. Assembly of infectious Herpes simplex virus type 1 virions in the absence of full‐length VP22. J. Virol. 74:10041‐10054.
   Roizman, B. and Knipe, D.M. 2001. Herpes simplex viruses and their replication. In Virology, 4th ed. (D.M. Knipe and P.M. Howley, eds.) pp. 2399‐2459. Lippincott‐Raven, Philadelphia.
Key References
   Brown, S.M. and MacLean, A.R. 1998. Methods in Molecular Medicine, Vol. 10: Herpes Simplex Virus Protocols. Humana Press, Totowa, N.J.
  This review volume provides additional technical information for the analysis of HSV in cell culture and animal systems
Internet Resources
   http://www.stdgen.lanl.gov/stdgen
  Web site maintained by the Los Alamos National Laboratory Bioscience Division that includes compilation and analysis of molecular sequence information pertaining to sexually transmitted bacteria and viruses. Dynamic graphics and extended analyses are available for all organisms. Annotation is accomplished by a combination of automation and hand review of each record.
   http://darwin.bio.uci.edu/∼faculty/wagner
  Home page of Dr. Edward Wagner, Professor of Microbiology and Molecular Genetics at the University of California–Irvine. It provides an introduction to HSV. Here one can explore herpes virus research with Dr. Wagner on the following topics: herpes simplex virus (HSV); the HSV genome; HSV replication; use of DNA microarrays to analyze gene expression in HSV‐infected cells; microarray analysis of cellular transcript abundance as a function of HSV infection; temporal patterns of HSV‐2 transcripts; analysis of herpes simplex virus promoters; structural properties of herpes simplex virus promoters; and latent infections by HSV.
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