Growth and Quantification of MERS‐CoV Infection

Christopher M. Coleman1, Matthew B. Frieman1

1 University of Maryland School of Medicine, Microbiology and Immunology, Baltimore, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15E.2
DOI:  10.1002/9780471729259.mc15e02s37
Online Posting Date:  May, 2015
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Abstract

Middle East respiratory syndrome coronavirus (MERS‐CoV) is an emerging highly pathogenic respiratory virus. Although MERS‐CoV only emerged in 2012, we and others have developed assays to grow and quantify infectious MERS‐CoV and RNA products of replication in vitro. MERS‐CoV is able to infect a range of cell types, but replicates to high titers in Vero E6 cells. Protocols for the propagation and quantification of MERS‐CoV are presented. © 2015 by John Wiley & Sons, Inc.

Keywords: coronavirus; virology; MERS; infection; quantification; plaque assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of MERS‐CoV
  • Basic Protocol 2: Quantification of MERS‐CoV by TCID50
  • Basic Protocol 3: Quantification of MERS‐CoV by Plaque Assay
  • Basic Protocol 4: Quantification of MERS‐CoV RNA Products of Replication
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Growth of MERS‐CoV

  Materials
  • Vero E6 cells (ATCC #1568)Keep aliquots of Vero E6 cells frozen in liquid nitrogen.
  • Vero E6 cell growth medium (see recipe)
  • MERS‐CoV (GenBank accession #KC776174.1, MERS‐CoV‐Hu/Jordan‐N3/2012)
  • MERS‐CoV can be obtained as a frozen stock from Kanta Subbarao (NIH, Bethesda, MD).
  • 175‐cm2 (T‐175) tissue culture flasks
  • 1.5‐ml screw‐cap tubes
  • Additional reagents and equipment for basic cell culture techniques including trypsinization (Phelan, )

Basic Protocol 2: Quantification of MERS‐CoV by TCID50

  Materials
  • Vero E6 cells (ATCC #1568)Keep aliquots of Vero E6 cells frozen in liquid nitrogen.
  • Vero E6 cell growth medium (see recipe)
  • MERS‐CoV (see protocol 1)
  • 4% paraformaldehyde in H 2O
  • 0.05% (w/v) crystal violet in 20% methanol
  • Tissue culture‐treated flat‐bottomed 96‐well plates
  • Optional: Untreated round‐bottomed 96‐well plates for sample dilution
  • Optional: Light box to view the plates
  • Additional reagents and equipment for basic cell culture techniques (Phelan, )

Basic Protocol 3: Quantification of MERS‐CoV by Plaque Assay

  Materials
  • Vero E6 cells (ATCC #1568)Keep aliquots of Vero E6 cells frozen in liquid nitrogen.
  • Vero E6 cell growth medium (see recipe)
  • MERS‐CoV (see protocol 1)
  • Phosphate‐buffered saline (PBS; )
  • 2× supplemented MEM (see recipe)
  • 1.6% agarose, suitable for tissue culture (see recipe)
  • Optional: 0.5% neutral red in PBS with 0.85% NaCl
  • Tissue culture‐treated flat‐bottomed 6‐well plates
  • Titer tubes or other tubes suitable for sample dilution
  • Additional reagents and equipment for basic cell culture techniques (Phelan, )

Basic Protocol 4: Quantification of MERS‐CoV RNA Products of Replication

  Materials
  • Cell culture extract or homogenized tissue in Trizol (or similar) from which RNA will be obtained
  • RNA extraction reagents or kit
  • Optional: Reagents for cDNA synthesis (e.g., RevertAid reverse transcriptase; Thermo Scientific)
  • MERS‐CoV primer/probe sets described in Table 15.2.1
  • TaqMan master mix
  • Optical 96‐well plates for PCR, and sealing tape
  • qPCR machine capable of reading at least 3 TaqMan probe dyesThe method in this protocol utilizes ABY, FAM, and VIC probe dyes, however, other dye combinations may be used based on the investigator's preference.
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Figures

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Literature Cited

Literature Cited
   Chan, J.F. , Chan, K.H. , Choi, G.K. , To, K.K. , Tse, H. , Cai, J.P. , Yeung, M.L. , Cheng, V.C. , Chen, H. , Che, X.Y. , Lau, S.K. , Woo, P.C. , and Yuen, K.Y. , 2013. Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: Implications for disease pathogenesis and clinical manifestation. J. Infect. Dis. 207:1743‐1752.
   Coleman, C.M. , Matthews, K.L. , Goicochea, L. , and Frieman, M.B. , 2013. Wild type and innate immune deficient mice are not susceptible to the Middle East Respiratory Syndrome Coronavirus. J. Gen. Virol. 95(Pt 2):408‐412.
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