Lymphocytic Choriomeningitis Virus (LCMV): Propagation, Quantitation, and Storage

Raymond M. Welsh1, Mina O. Seedhom1

1 University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15A.1
DOI:  10.1002/9780471729259.mc15a01s8
Online Posting Date:  February, 2008
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Abstract

Lymphocytic choriomeningitis virus (LCMV) is an enveloped, ambisense RNA virus and the prototypic virus of the arenavirus group. It can cause viral meningitis and other ailments in humans, but its natural host is the mouse. The LCMV/mouse model has been useful for examining mechanisms of viral persistence and the basic concepts of virus‐induced immunity and immunopathology. This unit discusses strain differences and biosafety containment issues for LCMV. Recommendations are made for techniques for propagating LCMV to high titers to quantify it by plaque assay and PCR techniques and to preserve its infectivity by appropriate storage. Curr. Protoc. Microbiol. 8:15A.1.1‐15A.1.11. © 2008 by John Wiley & Sons, Inc.

Keywords: BHK cell; defective‐interfering (DI) virus; lymphocytic choriomeningitis virus; PCR; plaque; plaque‐forming unit (pfu); Vero cell

     
 
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Table of Contents

  • Introduction
  • Safety Considerations
  • Basic Protocol 1: Propagation of LCMV
  • Basic Protocol 2: Quantitation of LCMV Infectious Units by Plaque Assay
  • Basic Protocol 3: Quantitation of LCMV mRNA by Quantitative Polymerase Chain Reaction (qRT‐PCR)
  • Basic Protocol 4: Storage of LCMV
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Propagation of LCMV

  Materials
  • Baby hamster kidney (BHK) cells, lines 21 or 21/13s
  • BHK propagation medium
  • Lymphocytic choriomeningitis virus (LCMV; MOI of 0.03 to 0.1 pfu/cell for harvesting after 48 hr or MOI of 0.003 to 0.01 pfu/cell for harvesting after 72 hr)
  • 75‐cm2 (T‐75) or 150‐cm2 (T‐150) tissue culture flasks, or tissue culture roller bottles
  • Plastic centrifuge tubes of appropriate size
  • Refrigerated centrifuge, 4°C

Basic Protocol 2: Quantitation of LCMV Infectious Units by Plaque Assay

  Materials
  • Vero cells (African green monkey kidney cells; ATCC)
  • Eagle's MEM with 10% (v/v) heat‐inactivated (56°C for 30 min) fetal bovine serum (FBS)
  • Lymphocytic choriomeningitis virus (LCMV) sample to be tested
  • Vero cell propagation medium (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2× LCMV plaque assay medium (see recipe)
  • 1% (w/v) Seakem agarose‐ME (Lonza), recently boiled (in a microwave)
  • 1% (w/v) neutral red (aqueous solution); store up to several months at 4°C
  • 6‐well petri plates
  • 96‐well microtiter plates
  • Platform rocker

Basic Protocol 3: Quantitation of LCMV mRNA by Quantitative Polymerase Chain Reaction (qRT‐PCR)

  Materials
  • Test sample (cells infected with LCMV)
  • SuperScript First‐Strand Synthesis for RT‐PCR (Invitrogen)
  • Double‐distilled (dd) H 2O
  • 500 mM Tris buffer
  • 5 µg/µl bovine serum albumin (BSA)
  • 30 mM MgCl 2
  • 2.5 mM (each) dNTPs
  • SYBR Green I nucleic acid stain, 10,000× concentration (Molecular Probes)
  • 10 µM LCMV GP forward primer: 5′‐TGC CTG ACC AAA TGG ATG ATT‐3′
  • 10 µM LCMV GP reverse primer: 5′‐CTG CTG TGT TCC CGA AAC ACT‐3′
  • 10 µM beta‐actin forward primer: 5′‐CGA GGC CCA GAG CAA GAG AG‐3′
  • 10 µM beta‐actin reverse primer: 5′‐CGG TTG GCC TTA GGG TTC AG‐3′
  • 1 µM fluorescein (Bio‐Rad)
  • 10 µM LCMV Taq Man MGB oligo: 6FAM‐TTG CTG CAG AGC TT MGBNFQ (Applied Biosystems)
  • 5 U/µl Taq DNA polymerase (Promega)
  • 2‐ml Phase Lock Gel tubes, heavy (Eppendorf)
  • iCycler iQ PCR plates, 96 well (Bio‐Rad)
  • iCycler iQ Optical Tape (Bio‐Rad)
  • iCycler iQ real‐time PCR detection system (Bio‐Rad)
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Figures

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Literature Cited

Literature Cited
   Cao, W., Henry, M.D., Borrow, P., Yamada, H., Elder, J.H., Ravkov, E.V., Nicho, S.T., Compans, R.W., Campbell, K.P., and Oldstone, M.B. 1998. Identification of alpha‐dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science 282:1999‐2000.
   Ciurea, A., Klenerman, P., Hunziker, L., Horvath, E., Senn, B.M., Ochsenbein, A.F., Hengartner, H., and Zinkernagel, R.M. 2000. Viral persistence through selection of neutralizing antibody‐escape variants. Proc. Natl. Acad. Sci. U.S.A. 97:2749‐2754.
   Lee, K.J. and de la Torre, J.C. 2002. Reverse genetics of arenaviruses. Curr. Top. Microbiol. Immunol. 262:175‐193.
   Lewicki, H.A., Tishon, A., Borrow, P., Evans, C.F., Gairin, J.E., Hahn, K.M., Jewell, D.A., Wilson, I.A., and Oldstone, M.B. 1995. CTL escape viral variants. 1. Generation and molecular characterization. Virology 210:29‐40.
   Miller, A.T., Wilcox, H.M., Lai, Z., and Berg, L.J. 2004. Signaling through Itk promotes T helper 2 differentiation via negative regulation of T‐bet. Immunity. 21:67‐80.
   Moskophidis, D., Lechner, F., Pircher, H., and Zinkernagel, R.M. 1993. Virus persistence in acutely infected immunocompetent mice by exhaustion of antiviral cytotoxic effector cells. Nature 362:758‐761.
   Roberts, T.J., Lin, Y., Spence, P.M., Van Kaer, L., and Brutkiewicz, R.R. 2004. CD1d1‐dependent control of the magnitude of an acute antiviral immune response. J. Immunol. 172:3454‐3461.
   Welsh, R.M. 2000. Lymphocytic choriomeningitis virus as a model for the study of cellular immunology. In Effects of Microbes on the Immune System (M. W. Cunningham and R. J. Fujinami, eds.), pp. 289‐312. Lippincott, Williams & Wilkins, Philadelphia.
   Welsh, R.M. and Oldstone, M.B. 1978. Inhibition of immunological injury of cultured cells infected with lymphocytic choriomeningitis virus: Role of defective interfering virus in regulating viral antigenic expression. J. Exp. Med. 145:1449‐1468.
   Zajac, A.J., Blattman, J.N., Murali‐Krishna, K., Sourdive, J.D., Suresh, M., Altman, J.D., and Ahmed, R. 1998. Viral immune evasion due to persistence of activated cells without effector function. J. Exp. Med. 188:2205‐2213.
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