Influenza: Propagation, Quantification, and Storage

Amanda L. Balish1, Jacqueline M. Katz1, Alexander I. Klimov1

1 Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15G.1
DOI:  10.1002/9780471729259.mc15g01s29
Online Posting Date:  May, 2013
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Abstract

Influenza viruses are negative‐sense, single‐stranded, enveloped RNA viruses belonging to the family Orthomyxoviridae. Three types exist, influenza A, B, and C. All infect humans, but only A and B are major human pathogens. Influenza type A viruses are divided into subtypes based on genetic and antigenic differences in the two surface spike proteins, hemagglutinin (HA) and neuraminidase (NA). The appropriate cell lines to be used for isolation of influenza A or B viruses depend on the clinical information and the host of origin. MDCK cells are the preferred cell line for isolation of human influenza viruses from clinical specimens. Curr. Protoc. Microbiol. 29:15G.1.1‐15G.1.24. © 2013 by John Wiley & Sons, Inc.

Keywords: human influenza; Madin Darby canine kidney; MDCK; embryonated chicken eggs; EID50; TCID50; plaque‐forming units

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Propagation of Influenza Viruses in Cell Culture from Virus Stocks
  • Alternate Protocol 1: Propagation of Influenza Viruses in Other Cell Lines
  • Alternate Protocol 2: Propagation of Influenza Virus in Cell Culture from Primary Clinical Specimens
  • Alternate Protocol 3: Propagation of Influenza Virus from Clinical Specimens in Shell Vials
  • Basic Protocol 2: Propagation of Influenza Viruses in Embryonated Chicken Eggs from Virus Stocks
  • Alternate Protocol 4: Propagation of Influenza Virus in Embryonated Chicken Eggs from Primary Clinical Specimens
  • Basic Protocol 3: Quantification of Influenza Viruses by Hemagglutination Assay
  • Basic Protocol 4: Quantification of Influenza Viruses by 50% Tissue Culture Infectious Dose Assay
  • Basic Protocol 5: Quantification of Influenza Viruses by 50% Egg Infectious Dose Assay
  • Basic Protocol 6: Quantification of Influenza Viruses by Plaque Assay
  • Basic Protocol 7: Storage of Influenza Viruses
  • Alternate Protocol 5: Lyophilization of Influenza Viruses
  • Support Protocol 1: Growth and Maintenance of MDCK Cell Line
  • Support Protocol 2: Candling 10‐Day Embryonated Chicken Eggs
  • Support Protocol 3: Preparation of Primary Influenza Virus Specimens
  • Support Protocol 4: Standardization of Red Blood Cells for Hemagglutination‐Based Assays
  • Support Protocol 5: Calculation of Infectious Dose 50 Titers by the Reed‐Muench Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Propagation of Influenza Viruses in Cell Culture from Virus Stocks

  Materials
  • Influenza virus stock
  • Madin‐Darby canine kidney (MDCK) cells confluent in a 25‐cm2 flask (see protocol 13)
  • Phosphate‐buffered saline (PBS) containing potassium ( appendix 2A)
  • cDMEM/7.5% BSA (see recipe)
  • Influenza virus growth medium (see recipe)
  • 5‐ and 10‐ml pipets, sterile
  • 33° to 37°C incubator
  • 15‐ml tubes, sterile (Falcon or Corning)
  • 2‐ml cryovials, sterile (Nunc)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used. All of the following steps must be performed in a class‐II biosafety cabinet.

Alternate Protocol 1: Propagation of Influenza Viruses in Other Cell Lines

  • Primary clinical specimen (see protocol 15)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used. All of the following steps must be performed in a class‐II biosafety cabinet.

Alternate Protocol 2: Propagation of Influenza Virus in Cell Culture from Primary Clinical Specimens

  • MDCK shell vials (Diagnostic Hybrids)
  • Primary clinical specimen (see protocol 15)
  • Microscope
  • 15‐ml tubes
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used. All of the following steps must be performed in a class‐II biosafety cabinet.

Alternate Protocol 3: Propagation of Influenza Virus from Clinical Specimens in Shell Vials

  Materials
  • Influenza virus stock
  • Egg diluent: antibiotic‐supplemented KPBS or tryptose phosphate broth (see recipes)
  • 9‐ to 11‐day‐old embryonated chicken eggs (see protocol 14)
  • 70% ethanol
  • Glue (Elmers), nonsterile
  • 21‐G, 1 1/2‐in. and 18‐G, 1/2‐in. needles
  • 1‐ml syringe
  • 33° to 37°C incubator
  • Forceps, sterile
  • 10‐ml pipets
  • 50‐ml tubes, sterile (Falcon or Corning)
  • 2‐ml cryovials, sterile (Nunc)
  • Additional reagents and equipment for performing a hemagglutination test (see protocol 7)
NOTE: All equipment and solutions coming into contact with eggs must be sterile and proper sterile technique should be used accordingly.

Basic Protocol 2: Propagation of Influenza Viruses in Embryonated Chicken Eggs from Virus Stocks

  • Primary clinical specimen (see protocol 15)
  • Egg candler (KUHL)

Alternate Protocol 4: Propagation of Influenza Virus in Embryonated Chicken Eggs from Primary Clinical Specimens

  Materials
  • Phosphate‐buffered saline (PBS) containing potassium ( appendix 2A)
  • Influenza virus stock
  • Standardized turkey red blood cells (see protocol 16)
  • 96‐well V‐shaped microtiter plate (Nunc)
NOTE: Keep the virus stock on ice or at 4° to 8°C during the HA test to maintain virus infectivity.

Basic Protocol 3: Quantification of Influenza Viruses by Hemagglutination Assay

  Materials
  • MDCK cells in 96‐well tissue culture plate (see protocol 13)
  • Influenza virus growth medium (see recipe)
  • Influenza virus stock
  • 96‐well tissue culture plate (Costar)
  • Inverted microscope
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a 35° to 37°C, 5% CO 2 humidified incubator.

Basic Protocol 4: Quantification of Influenza Viruses by 50% Tissue Culture Infectious Dose Assay

  Materials
  • Egg diluent (see recipe)
  • Influenza virus stock
  • 9‐ to 11‐day‐old embryonated chicken eggs (see protocol 14)
  • Standardized turkey red blood cells (see protocol 16)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Forceps, sterile
  • 96‐well V‐shaped microtiter plate (Nunc)
  • Additional reagents and equipment for inoculating eggs (see protocol 5)
NOTE: All equipment and solutions coming into contact with embryonated eggs must be sterile and proper sterile technique should be used accordingly.

Basic Protocol 5: Quantification of Influenza Viruses by 50% Egg Infectious Dose Assay

  Materials
  • 2× plaque assay medium (see recipe)
  • 1.6% (w/v) agarose solution
  • Influenza virus stock
  • Madin‐Darby canine kidney (MDCK) cells confluent in 6‐well tissue culture plates (see protocol 13)
  • Plaque assay wash medium (see recipe)
  • 2 mg/ml TPCK‐trypsin working stock (see recipe)
  • 70% ethanol
  • 0.3% crystal violet solution
  • 37° and 56°C water baths
  • Forceps, sterile
  • Inverted microscope
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used accordingly.

Basic Protocol 6: Quantification of Influenza Viruses by Plaque Assay

  Materials
  • Madin‐Darby canine kidney (MDCK) cells (ATCC# CCL‐34) grown confluent in a 75‐cm2 flask
  • Trypsin/EDTA (Invitrogen)
  • Heat‐inactivated fetal bovine serum (FBS; appendix 2A)
  • MDCK growth medium (see recipe)
  • 75‐ and/or 25‐cm2 tissue culture flasks or 6‐ or 96‐well tissue culture plates (Corning)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 4A)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used.NOTE: All cell culture incubations are performed in a 35°C to 37°C in 5% CO 2, in humidified conditions.

Basic Protocol 7: Storage of Influenza Viruses

  Materials
  • Specimen in collection vials (e.g., from nasal, throat, or combined nasal/throat swabs; or nasopharyngeal, nasal, or throat aspirates or washings)
  • 10 mg/ml gentamicin (Invitrogen)
  • Vortex mixer
  • Centrifuge
  • 2‐ to 4‐mm glass beads (VWR)
  • Polypropylene microcentrifuge tubes, sterile
  • Cryovials (Nunc)
NOTE: Clarified supernatants can be used to directly inoculate cell culture flasks or eggs.

Alternate Protocol 5: Lyophilization of Influenza Viruses

  Materials
  • Whole blood mixed 1:1 in Alsever's solution ( appendix 2A)
  • Phosphate‐buffered saline (PBS) containing potassium ( appendix 2A)
  • Cotton gauze, sterile
  • 50‐ and 15‐ml conical centrifuge tubes
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 4A)
NOTE: All equipment and solutions coming into contact with cells must be sterile and proper sterile technique should be used.
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Figures

Videos

Literature Cited

Literature Cited
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