Influenza Viruses

Disease Modeling and Laboratory Methods

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• Publisher: Wiley-Blackwell
• Year: 2013
• Print ISBN: 9781118829493
• eBook ISBN: 9781118829479
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Microbiology
• Index: Biological Sciences

Influenza virus infections are a serious health problem. Each year, about 500 million people are infected by the virus, resulting in about 500, 000 deaths worldwide. The occurrence of new influenza subtypes has caused severe pandemics, including the 2009 swine flu (vH1N1). In addition, highly pathogenic viruses, like subtypes H7N7 and H5N1, also called the fowl plague and bird flu, respectively, represent enormous economic threats to livestock farming. This book begins with descriptions of the molecular make-up of influenza viruses, their replication cycles and functions of viral proteins. A history of major influenza pandemics is provided as is a detailed article discussing how viral growth and decay is mathematically modeled to evaluate the biological parameters governing interaction between host and virus. Several laboratory protocols describe how influenza virus is handled and used in animal models to study host-pathogen interactions and test potential new therapies.

This e-book — a curated collection from eLS, WIREs, and Current Protocols — offers a fantastic introduction to the field of influenza research for students or interdisciplinary collaborators.

John Wiley & Sons, Ltd.

Current Protocols in Microbiology 15G.1.1-15G.1.24, May 2013

Published online May 2013 in Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/9780471729259.mc15g01s29

Copyright © 2013 John Wiley & Sons, Inc.

UNIT 15G.1

Influenza: Propagation, Quantification, and Storage

Amanda L. Balish,¹ Jacqueline M. Katz,¹ and Alexander I. Klimov¹

¹Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia

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

INTRODUCTION

This unit covers several techniques for propagating, quantifying, and storing human influenza A viruses from existing stocks (see Basic Protocols 1 and 2) or from primary clinical specimens (see Alternate Protocols 2 and 4). Virus isolation is a highly sensitive and useful technique for the identification of viral infections. An important advantage of virus isolation is the amplification of the virus from the original specimen, making it available for further antigenic and genetic characterization and for drug susceptibility testing, if required. Successful influenza virus diagnosis depends largely upon the quality of the specimen and the conditions under which it is stored and transported. Specimens collected for viral isolation should ideally be collected within three days of the onset of clinical symptoms. Influenza viruses are quantified by several virus titration methods. The hemagglutination assay HA (see Basic Protocol 3) quantifies the viral particles regardless of infectivity. The HA titer is the reciprocal of the dilution of virus in the last well with complete hemagglutination. An HA unit (HAU) is defined as the amount of virus needed to agglutinate an equal volume of red blood cells. To determine the amount of infectious particles in a sample, the 50% tissue culture infectious dose assay (see Basic Protocol 4), 50% egg infectious dose assay (see Basic Protocol 5), or plaque assay (see Basic Protocol 6) methods are used. After isolating and quantifying human influenza, the product must be properly stored to maintain virus viability.

CAUTION: The protocols presented in this unit are for use with contemporary human influenza virus subtypes, which must be handled under Biosafety Level 2 (BSL-2) conditions. For biosafety levels recommended for noncontemporary or novel human or nonhuman influenza viruses, refer to the influenza agent summary statement in the Biosafety in Microbiological and Biomedical Laboratories manual published by the Centers for Disease Control and Prevention and National Institutes of Health (http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf). All work with infectious influenza virus should be conducted in a class II biological safety cabinet. See UNIT 1A.1 and other resources (APPENDIX 1B) for additional information.

IMPORTANT NOTE: Use of trade names and commercial sources is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.

PROPAGATION OF INFLUENZA VIRUSES IN CELL CULTURE FROM VIRUS STOCKS

Madin-Darby canine kidney (MDCK) cells (ATCC# CCL-34) are the preferred host for the isolation and characterization of influenza A and B viruses, but not influenza C viruses due to the incompatibility of sialic acid moieties on the cell surface with the viral receptor specificity. Influenza viruses are also able to replicate in a few primary, diploid, and continuous cell cultures. The propagation of influenza viruses in primary monkey kidney (PMK) cells (Viro-Med Laboratories or BioWhittaker) and R-mix fresh cells (Diagnostic Hybrids) is presented in Alternate Protocol 1. For preparation of a virus stock from a primary clinical specimen, refer to Alternate Protocol 2. The protocol described below is for the propagation of human influenza A and B virus stocks in MDCK cell culture.

Materials

Influenza virus stock

Madin-Darby canine kidney (MDCK) cells confluent in a 25-cm² flask (see Support Protocol 1))

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.

1. Thaw vial of influenza virus stock in cool water.

To reduce loss of infectivity, maintain virus on ice or at 4° to 8°C once thawed.

2. Remove the MDCK growth medium using a sterile 10-ml pipet and wash the MDCK monolayer two times with 5 ml of room temperature PBS and once with cDMEM/7.5% BSA, removing washes with sterile 10-ml pipets.

This cell line requires the cells to be confluent, i.e., completely covering the surface of the flask, before the virus is inoculated. If the monolayer is overgrown (i.e., cells overlapping), it is less sensitive to virus infection.

Fetal bovine serum (FBS) inhibits viral entry and must be removed for efficient infection of cells.

Do not add medium directly onto the monolayer as this may disrupt the monolayer.

3. Dilute virus samples in influenza cDMEM/7.5% BSA.

Use 1:5 to 1:1000 dilutions of virus to achieve optimal growth.

4. Inoculate the flask with 200 µl of virus and rotate to cover monolayer with inoculum. Inoculum should carefully be added to the monolayer so as to not disrupt the cells.

5. Incubate the inoculated flasks for a minimum of 30 min or up to 1 hr at 35° to 37°C to allow the inoculum to adsorb.

Incubation for >1 hr may cause the MDCK cell monolayer to dry out and result in reduced cell viability.

6. Add 6 ml influenza virus growth medium to the inoculated flasks.

Do not add medium directly onto the monolayer as this may disrupt the monolayer.

7. Incubate flasks at 33°C (optimal for influenza B) to 37°C (optimal for influenza A), observing the MDCK monolayer for cytopathic effect (CPE) daily. Harvest cell culture supernatant when at least 75% of the cell monolayer is exhibiting CPE.

Typical CPE by influenza viruses include rounding up of infected cells and detachment from culture flask.

100% cytopathic effect is optimal for recent seasonal influenza A viruses.

8. To harvest, decant the medium in the flask into a 15-ml conical tube. Centrifuge the supernatant 15 min at 300 × g, 4°C, to pellet cellular debris. Transfer clarified supernatant into a fresh 15-ml tube.

A 0.5% stabilizer such as glycerol, gelatin or bovine albumin fraction V (7.5%) is recommended prior to freezing isolated virus.

9. Dispose of tissue culture flask(s) in an appropriate biological waste container.

10. Dispense the supernatant into sterile 2-ml cryovials and store up to 1 year at −70° to −80°C or in liquid nitrogen at −135° to −150°C for optimal viability for long-term storage (see Basic Protocol 7).

PROPAGATION OF INFLUENZA VIRUSES IN OTHER CELL LINES

The Madin-Darby canine kidney (MDCK) cell line is the preferred cell line for the isolation and propagation of influenza viruses (see Basic Protocol 1). If unable to maintain or purchase the MDCK cell line, other cell lines may be used to isolate and propagate influenza viruses. R-mix fresh cells (Diagnostic Hybrids) and primary monkey kidney (PMK) cells (Viro-Med Laboratories or BioWhittaker) are widely used by diagnostic laboratories for the isolation of many human respiratory viruses. R-mix fresh cells are a combination of the human adenocarcinoma (A549) and mink lung (Mv1Lu) cell lines, and is an alternate for MDCK cells in the growth and characterization of i...

Table of contents

• Cover
• Introduction
• Disease Modeling
• Protocols
• Further Reading