influenza: propagation, quantification, and storage

UNIT 15G.1Influenza: Propagation, Quantification,and Storage

This unit covers several techniques for propagating, quantifying, and storing human in-fluenza A viruses from existing stocks (see Basic Protocols 1 and 2) or from primaryclinical specimens (see Alternate Protocols 2 and 4). Virus isolation is a highly sensitiveand useful technique for the identification of viral infections. An important advantageof virus isolation is the amplification of the virus from the original specimen, making itavailable for further antigenic and genetic characterization. Influenza viruses are quanti-fied either by a “unit” of hemagglutination, which is not a measure of an absolute amountof virus but is an operational unit dependent on the method used for the hemaggluti-nation assay titration (see Basic Protocol 3), or by determining infectious units usingthe 50% tissue culture infectious dose assay (see Basic Protocol 4), 50% egg infectiousdose assay (see Basic Protocol 5), or plaque assay (see Basic Protocol 6). After isolatingand quantifying human influenza, the product must be properly stored to maintain virusviability.

CAUTION: The protocols presented in this unit are for use with contemporary hu-man influenza virus subtypes, which must be handled under Biosafety Level 2 (BSL-2)conditions. For biosafety levels recommended for noncontemporary or nonhuman in-fluenza viruses, refer to the influenza agent summary statement in the Biosafety inMicrobiological and Biomedical Laboratories manual published by the Centers for Dis-ease Control and Prevention and National Institutes of Health (see interim guidelines athttp://www.cdc.gov/flu/pdf/h2n2bsl3.pdf). All work with infectious influenza virus shouldbe conducted in a class II biological safety cabinet. See UNIT 1A.1 and other pertinent re-sources (APPENDIX 1B) for additional information.

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

BASICPROTOCOL 1

PROPAGATION OF INFLUENZA VIRUSES IN CELL CULTURE FROMVIRUS STOCKS

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

Materials

Influenza virus stockMadin Darby canine kidney (MDCK) cells confluent in a 75-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)

Contributed by Kristy J. Szretter, Amanda L. Balish, and Jacqueline M. KatzCurrent Protocols in Microbiology (2006) 15G.1.1-15G.1.22Copyright C© 2006 by John Wiley & Sons, Inc.

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Supplement 3

Influenza:Propagation,

Quantification,and Storage

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Supplement 3 Current Protocols in Microbiology

10- and 25-ml pipets, sterile33◦ to 37◦C incubator50-ml tubes, sterile (Falcon or Corning)2-ml cryovials, sterile (Nunc)

NOTE: All equipment and solutions coming into contact with cells must be sterile andproper sterile technique should be used.

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

To reduce loss of infectivity, maintain virus at 4◦C once thawed.

2. Remove the MDCK growth medium using a sterile 25-ml pipet and wash theMDCK monolayer two times with 10 ml of room temperature PBS and once withcDMEM/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 theflask, 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 infectionof cells.

3. Dilute virus samples in influenza virus growth medium.

Use a 1:10 to 1:1000 dilution of virus to achieve optimal growth.

4. Inoculate flask with 1 ml virus and rotate to cover monolayer with inoculum. Takecare not to add medium directly onto the monolayer as this may disrupt the cells.

5. Incubate inoculated flasks for a minimum of 30 min or up to 1 hr at 37◦C.

Incubation for >1 hr may cause the MDCK cell monolayer to dry out.

6. Add 20 ml influenza virus growth medium to the inoculated flasks.

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

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

8. Centrifuge supernatant 15 min at 300 × g, 4◦C, to pellet cellular debris. Transferclarified supernatant to a fresh 50-ml tube.

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

10. Dispense supernatant into 2-ml aliquots in sterile 2-ml cryovials and store up to1 year at −70◦ to −80◦C or in liquid nitrogen at −135◦ to −150◦C for optimalviability after long-term storage (see Basic Protocol 7).

ALTERNATEPROTOCOL 1

PROPAGATION OF INFLUENZA VIRUSES IN OTHER CELL LINES

The Madin Darby canine kidney (MDCK) cell line is the preferred cell line for theisolation and propagation of influenza viruses (see Basic Protocol 1). If unable to maintainor purchase the MDCK cell line, other cell lines may be used to isolate and propagateinfluenza viruses. R-mix fresh cells (Diagnostic Hybrids) and primary monkey kidney(PMK) cells (Viro-Med Laboratories or BioWhittaker) are widely used by diagnosticlaboratories for the isolation of many human respiratory viruses. R-mix fresh cells are acombination of the human adenocarcinoma (A549) and mink lung (Mv1Lu) cell lines,and are an alternate for MDCK cells in the growth and characterization of influenzaviruses. PMK cells do not require the addition of tosyl phenylalanyl chloromethyl ketone

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Current Protocols in Microbiology Supplement 3

(TPCK)-trypsin for virus growth, but do require the addition of 0.5% FBS to mediuminstead of BSA. Limitations of PMK cells include lower viral titers, contamination byadventitious simian agents such as foamy viruses, and batch-to-batch variability.

ALTERNATEPROTOCOL 2

PROPAGATION OF INFLUENZA VIRUS IN CELL CULTURE FROMPRIMARY CLINICAL SPECIMENS

Specimens for virus isolation should be kept at 4◦C immediately after collection andinoculated into susceptible cell cultures as soon as possible. If the specimen cannotbe processed within 48 to 72 hr, the specimen should be frozen up to 1 year at orbelow −70◦C. Primary influenza specimens require the addition of antibiotics prior toinoculating into cell culture (see Support Protocol 3). Due to the unknown amount ofinfluenza virus in the clinical specimen, it may require multiple passages in cell culturefor the virus to grow. Typically, two passages may follow the initial inoculation before aspecimen is considered to be negative by virus isolation.

Additional Materials (also see Basic Protocol 1)

Primary clinical specimen (see Support Protocol 3)


1. Thaw vial of primary clinical specimen in cool water.

Keep virus at 4◦C once thawed to reduce loss of infectivity.

2. Remove the MDCK growth medium using a sterile 25-ml pipet and wash the MDCKmonolayer two times with 10 ml of room temperature PBS and one time withcDMEM/7.5% BSA, removing washes with sterile 10-ml pipets.


3. Dilute virus samples in influenza virus growth medium.

Use a 1:10 to 1:1000 dilution of virus to achieve optimal growth.

4. Inoculate flask with 200 µl virus and rotate to cover monolayer with inoculum. Takecare not to add medium directly onto the monolayer as this may disrupt the cells.

5. Incubate inoculated flasks for 30 min to 1 hr at 37◦C.

After 1 hr the monolayer will begin to dry out.

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◦ to 37◦C, observing the MDCK monolayer for cytopathic effects(CPE) daily.

If type of influenza is unknown, incubate at 37◦C.

8. Harvest cell culture supernatant into 50-ml tubes, when at least 75% of the cellmonolayer is exhibiting CPE.

Typical CPE by influenza viruses include rounding up of infected cells and detachmentfrom culture flask. If no CPE is present, perform a hemagglutination assay (see BasicProtocol 3). If HA titer is <8 HAU, then 600 µl of undiluted supernatant should bepassaged into a new MDCK flask. Primary clinical specimens should be passaged up tothree times. If no HA titer is detected after three passages, the virus should be consideredunrecoverable (i.e., influenza virus was not recovered from the specimen by this method).



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

10. Centrifuge supernatant 15 min at 300 × g, 4◦C, to pellet cellular debris. Transferclarified supernatant to a fresh 50-ml tube.

11. Dispense supernatant into 2-ml aliquots in sterile 2-ml cryovials and store <1 year at−70◦ to −80◦C or at −135◦ to −150◦C for long-term storage. (see Basic Protocol 7).

ALTERNATEPROTOCOL 3

PROPAGATION OF INFLUENZA VIRUS FROM CLINICAL SPECIMENS INSHELL VIALS

MDCK shell vials (Diagnostic Hybrids) are an alternative for the isolation of humaninfluenza viruses from primary clinical material. Using the shell vial assay significantlyreduces the length of time for the detection of virus in primary specimens. With thistechnique, cell monolayers are grown on coverslips contained in flat-bottomed shell vials.This type of culture vessel allows for centrifugation of the cultures after inoculation toenhance viral infection of the cells, increase sensitivity to virus isolation and allow fora shortened turnaround time for specimen identification. Detection can be accomplishedwithin 48 to 72 hr after inoculation, prior to the development of CPE, by the identificationof viral antigens synthesized in the early stages of replication. Any negative result doesnot rule out viral etiology.

Commercially available MDCK shell vials should have a 70% to 90% confluent mono-layer and be free of contamination. Slightly subconfluent monolayers of MDCK cellsare preferred for virus isolation. Three shell vials should be inoculated per specimen andeach run of shell vials should contain an uninoculated shell vial to serve as a negativecontrol.


MDCK shell vials (Diagnostic Hybrids)Primary clinical specimen (see Support Protocol 3)15-ml tubes

1. Carefully remove MDCK growth medium with sterile pipet tips so as not to disturbmonolayer, viewing it under a microscope.

The monolayer may be viewed under a microscope by tilting the vial at a slight angle todetermine if the cells are completely covering the bottom of the vial.

2. Inoculate each shell vial monolayer with ∼0.2 ml primary clinical specimen.

This volume is sufficient to cover the monolayer in a shell vial. Do not pipet inoculumdirectly onto the monolayer as this may disrupt the cells.

3. Centrifuge shell vials for 30 to 60 min at 700 × g, at room temperature (24◦ to 27◦C).

Centrifugation of shell vials allows for more efficient absorption of virus into the cellmonolayer.

4. Add 1 ml influenza virus growth medium and incubate 72 hr at 33◦ to 35◦C.

These are the optimal temperatures for isolation of influenza viruses in shell vials.

5. Observe the shell vials during the 72-hr incubation and harvest supernatant into15-ml tubes at the first signs of cytopathic effect (CPE). Dispose of tissue culturematerials in an appropriate biological waste container.


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6. Centrifuge supernatant 15 min at 300 × g, 4◦C, to pellet cellular debris. Transferclarified supernatant to a fresh 15-ml tube. Dispense supernatant into 2-ml aliquotsin 2-ml sterile cryovials and store <1 year at −70◦ to −80◦C or at −135◦ to −150◦Cfor long-term storage (see Basic Protocol 7).

The coverslips in the shell vials can be used for immunofluorescence staining.

BASICPROTOCOL 2

PROPAGATION OF INFLUENZA VIRUSES IN EMBRYONATED CHICKENEGGS FROM VIRUS STOCKS

In the past, influenza viruses were often isolated in embryonated eggs; however, today, themajority of laboratories use cell culture due to availability and ease of isolation of somecontemporary human influenza strains in mammalian cells versus embryonated eggs.Nevertheless, many laboratory-adapted viruses and all vaccine strains are egg-grown andmay be propagated readily in 10- to 11-day-old embryonated chicken eggs.

Materials

Influenza virus stockEgg diluent: antibiotic-supplemented KPBS or tryptose phosphate broth (see

recipes)10- to 11-day-old embryonated chicken eggs (see Support Protocol 2)70% ethanolGlue (Elmers), nonsterile

22-G, 1 1/2-in. and 18-G, 1/2-in. needles1-ml syringe33◦ to 35◦C incubatorForceps, sterile10-ml pipets50-ml plastic concical tubes2-ml cryovials, sterile (Nunc)

Additional reagents and equipment for performing a hemagglutination test (seeBasic Protocol 3)

NOTE: All equipment and solutions coming into contact with eggs must be sterile andproper sterile technique should be used accordingly.

Inoculate eggs1. Dilute influenza virus stock in egg diluent.

The concentration of the seed stock will determine how dilute the inoculum should be.Typically, a 1:100 to 1:1000 dilution of virus will yield optimal virus growth (∼104 to 106

50% egg infectious dose (EID50) or 0.01 to 0.1 HAU of virus).

Either KPBS or tryptose phosphate broth–based diluent can be used in eggs.

2. Arrange three eggs with the airsac up. Spray tops of eggs with 70% ethanol.

Three to five eggs are recommended to produce sufficient volume of allantoic fluid forfuture use. Also, eggs may die during the incubation time due to bacterial contamination,virulence of inoculated virus, etc., therefore, the more eggs inoculated, the better thechance to obtain a viable stock of virus. One egg produces 3 to 10 ml allantoic fluid and0.1 to 1 ml amniotic fluid.

Allow the alcohol to evaporate before proceeding to next step.

3. Punch a small hole in the shell over the air sac using an 18-G, 1/2-in. needle anddispose of needle in an appropriate sharps container.

4. Aspirate 0.6 ml diluted virus sample into a 1-ml syringe with a 22-G, 11/2-in. needle.



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Figure 15G.1.1 Inoculation of a chicken egg with influenza virus.

5. Insert the needle at a 45◦ angle into the allantoic cavity and inoculate 0.2 ml influenzavirus dilution; repeat with the other two eggs (Fig. 15G.1.1).

6. Discard syringe and needle into a sharps safety container.

7. Seal the hole punched in the eggshell with a drop of glue.

Care should be taken not to contaminate glue in bottle with virus.

8. Incubate human influenza A virus for 48 hr at 33◦ to 35◦C and influenza B virusesfor 72 hr at 33◦ to 35◦C.

Harvest virus from infected eggs9. Chill eggs overnight (8 to 24 hr) at 4◦C to halt embryo viability and minimize the

flow of blood into the allantoic fluid during harvest.

Eggs may be quick-chilled for 30 min in a −20◦C freezer. However, this may cause theallantoic fluid to contain some blood.

10. With sterile forceps, break the shell over the air sac and push aside the allantoicmembrane with the forceps, taking care not to break the yolk.

11. Using a 10-ml pipet, aspirate the allantoic fluid and place in a labeled 50-ml plasticconical tube. Dispose of eggs in an appropriate biological waste container.

12. Centrifuge tubes 5 min at 500 × g, 4◦C, to pellet any blood cells and tissue fragments.Transfer clarified fluid into fresh 50-ml tube. Keep on ice (4◦C).

13. Perform a hemagglutination test (see Basic Protocol 3).

14. Dispense allantoic fluid into 2-ml aliquots in 2-ml sterile cryovials and store<1 year at −70◦ to −80◦C or at −135◦ to −150◦C for long-term storage (see BasicProtocol 7).

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ALTERNATEPROTOCOL 4

PROPAGATION OF INFLUENZA VIRUS IN EMBRYONATED CHICKENEGGS FROM PRIMARY CLINICAL SPECIMENS

With original clinical material, the amniotic sac is inoculated at the same time as theallantoic cavity. Virus inoculated into the amniotic sac that surrounds the embryo canreplicate directly in the embryonic tissue and the resulting virus is released into theamniotic fluid. However, the amniotic sac will yield only a small volume of fluid andthus subsequent passage into the allantoic cavity may be required to produce sufficientvolume for testing.


Primary clinical specimen (see Support Protocol 3)Egg candler (KUHL)

1. Dilute primary clinical specimen in egg diluent.

The concentration of the seed stock will determine how dilute the inoculum should be.Typically, an original specimen will be diluted 1:10 to achieve optimal virus growth.

2. Inoculate three eggs per specimen; label eggs to clearly identify original sample.

3. Punch a small hole in the shell over the air sac using an 18-G, 1/2-in. needle anddispose of needle in appropriate sharps container.

4. Aspirate 0.6 ml clinical specimen into a 1-m syringe with a 22-G, 11/2-in. needle.

5. Using an egg candler apparatus, hold the egg up to light source and locate the embryo.

6. Insert the needle into the amniotic sac and inoculate 0.1 ml primary clinical specimeninto the sac. Withdraw the needle about 1/2-in. and inoculate 0.2 ml primary clinicalspecimen into the allantoic cavity (Fig. 15G.1.1).

7. Repeat with remaining clinical specimen and eggs.

8. Discard syringe and needle into a sharps safety container.

9. Seal the holes punched in the egg shells with a drop of glue.

Care should be taken not to contaminate glue in bottle with virus.

10. Incubate the eggs for 48 to 72 hr at 33◦ to 37◦C.

The incubation temperature is dependent on the virus being grown (i.e., influenza A at37◦C and influenza B at 33◦C).

11. Harvest the allantoic fluid as described in Basic Protocol 2, steps 9 through 11. Keeptubes on ice (4◦C).

12. Invert the egg so that the embryo and amniotic sac hang down and are clearly visibleand separate from the egg yolk. Using a 1-ml syringe and 22-G, 1 1/2-in. needle,pierce the air sac and remove as much amniotic fluid as possible from around theembryo. Dispose of eggs in an appropriate biological waste container.

Keep allantoic and amniotic samples separate as the amniotic fluid may contain morevirus than the allantoic fluid. The volume of allantoic fluid recovered may vary from 5 to10 ml, while the volume of the amniotic fluid recovered may vary from 0.1 ml to 1 ml.

The amniotic cavity provides a richer source of susceptible cells for virus replication andis used to help viruses from primary specimens grow more efficiently.

13. Centrifuge tubes 5 min at 500 × g, 4◦C, to pellet any blood cells and tissue fragments.Transfer clarified fluid into fresh tube. Keep tubes on ice (4◦C).



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14. Perform a hemagglutination test on both the allantoic and amniotic fluids. (see BasicProtocol 3).

If hemagglutination test is negative or titer is <8 HAU, passage the specimen two moretimes before reporting inability to recover virus from the specimen. If HA test results intiters >8 HAU, specimens can be dispensed into aliquots and stored.

15. Dispense allantoic and amniotic fluid into 2-ml aliquots in 2-ml sterile cryovials andstore <1 year at −70◦ to −80◦C or at −135◦ to −150◦C for long-term storage. (seeBasic Protocol 7).

BASICPROTOCOL 3

QUANTIFICATION OF INFLUENZA VIRUSES BY HEMAGGLUTINATIONASSAY

Hemagglutination, or the ability to bind red blood cells, is a property of all influenzaviruses that can be utilized as a rapid assay for determining the presence of virus insamples. Because the hemagglutination (HA) assay is dependent on the amount ofhemagglutinin on the surface of influenza viruses and not the ability of the virus toreplicate, this assay quantifies viral particles regardless of their infectivity. The highestdilution of virus that causes complete hemagglutination is considered the HA titration endpoint. The HA titer is the reciprocal of the dilution of virus in the last well with completehemagglutination. A “unit” of hemagglutination is not a measure of an absolute amountof virus, but is an operational unit dependent on the method used for HA titration. AnHA unit (HAU) is defined as the amount of virus needed to agglutinate an equal volumeof a standardized red blood cell suspension. This protocol uses turkey red blood cellsas an example as these cells can be agglutinated by recent human influenza viruses aswell as laboratory-adapted strains of human origin. Refer to Support Protocol 4 for adiscussion of other types of red blood cells that can be used for hemagglutination assays.Hemagglutination units can be utilized when using inactivated influenza in experiments.For determining infectious units, use the 50% tissue culture infectious dose assay (seeBasic Protocol 4), 50% egg infectious dose assay (see Basic Protocol 5), or the plaqueassay (see Basic Protocol 6).

Materials

Phosphate-buffered saline (PBS) containing potassium (APPENDIX 2A)Influenza virus stock (e.g., allantoic or amniotic fluid)Standardized turkey red blood cells (see Support Protocol 4)96-well V-bottom microtiter plate (Nunc)

NOTE: Keep virus stock on ice (4◦C) during the HA test to maintain virus infectivity.

1. Pipet 50 µl PBS into wells 2 through 12 across a 96-well V-bottom plate(Fig. 15G.1.2).

2. Pipet 100 µl influenza virus stock into the first column of the 96-well plate.

3. Perform a two-fold dilution series across the 96-well plate by transferring 50 µlbetween wells, disposing of the final 50 µl from the last well.

4. Add 50 µl standardized turkey red blood cells to all wells. Tap the plate gently tomix.

Approximately 5 ml standardized turkey red blood cells will be needed per 96-well plateused in this assay.

5. Incubate 96-well plate for 30 min at room temperature (24◦ to 27◦C).

Avian red blood cells in V-well microtiter plates require a 30-min incubation at roomtemperature, whereas mammalian red blood cells require a U-well microtiter plate witha 60-min incubation at room temperature.

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Figure 15G.1.2 Plate layout for hemagglutination titration assay.

Figure 15G.1.3 Reading a hemagglutination titration assay plate. Positive samples will look pinkas the red blood cells are held in solution by the virus. Negative samples will look clear with a reddot, since the red blood cells settle to the bottom of the V-bottom plate.

6. Observe endpoint of agglutination and record titer per 50 µl of sample (Fig. 15G.1.3).

Red blood cells will settle to the bottom of the V-bottom well in negative samples, whilered blood cells will agglutinate in positive samples. The endpoint should be read as thelast well showing complete agglutination.

7. Store virus stocks with desired HAU titers (see Alternate Protocol 4, step 14). Disposeof materials in an appropriate biological waste container.

BASICPROTOCOL 4

QUANTIFICATION OF INFLUENZA VIRUSES BY 50% TISSUE CULTUREINFECTIOUS DOSE ASSAY

The 50% tissue culture infectious dose (TCID50) assay is a method to measure the amountof infectious virus in a sample by determining the highest dilution of the sample thatcan infect 50% of cells in culture. In this procedure, the virus sample is diluted across a96-well tissue culture plate containing MDCK cells. The titration should be performedin quadruplicate.

Materials

MDCK cells in 96-well tissue culture plate (see Support Protocol 1)Influenza virus growth medium (see recipe)Influenza virus stock

96-well tissue culture plate (Costar)Inverted microscope



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NOTE: All equipment and solutions coming into contact with cells must be sterile andproper sterile technique should be used accordingly.

NOTE: All culture incubations are performed in a 37◦C, 5% CO2 humidified incubator.

1. Remove MDCK growth medium from plate and wash cells by adding 350 µl influenzavirus growth medium to all wells. Aspirate the medium and repeat washing step.Care should be taken not to disrupt the cell monolayer.

2. Add 100 µl influenza virus growth medium to all wells, except the first column, ofa 96-well tissue culture plate.

3. Thaw influenza virus stock and dilute 1:100 in influenza virus growth medium.

4. Add 146 µl diluted virus to first column of 96-well tissue culture plate and perform a1/2 log10 dilution series by transferring 46 µl between wells, disposing of final 46 µlafter the eleventh column. Do not add virus to the final column as this is the cellcontrol for the assay.

5. Incubate plates 2 hr at 37◦C.

6. Remove inoculum and gently wash once with 250 µl influenza virus growth medium.

7. Add 200 µl influenza virus growth medium to all wells and incubate up to 72 hr at37◦C, observing for endpoints in cytopathic effect (CPE).


8. Examine wells for presence or absence of CPE using an inverted microscope.

To confirm results, 50 µl cell culture supernatant can be harvested into a 96-well V-bottom microtiter plate and tested for presence of virus by performing a spot HA assay(see Basic Protocol 5).

9. Record endpoint of CPE and determine TCID50 titer per 100 µl using the Reed-Muench method (see Support Protocol 5).

BASICPROTOCOL 5

QUANTIFICATION OF INFLUENZA VIRUSES BY 50% EGG INFECTIOUSDOSE ASSAY

The 50% egg infectious dose (EID50) assay is a method to measure the amount ofinfectious virus in a sample by determining the highest dilution of the sample thatcan infect 50% of eggs. In this procedure, the virus sample is serially diluted prior toinoculation of eggs and a hemagglutination test is performed on undiluted allantoic fluidto determine the presence of virus in the eggs.

Materials

Egg diluent (see recipe)Influenza virus stock10- to 11-day-old embryonated chicken eggs (see Support Protocol 2)Standardized turkey red blood cells (see Support Protocol 4)

1.5-ml microcentrifuge tubes, sterileForceps, sterile96-well V-bottom plate (Nunc)

Additional reagents and equipment for inoculating eggs (see Basic Protocol 2)

NOTE: All equipment and solutions coming into contact with embryonated eggs mustbe sterile and proper sterile technique should be used accordingly.

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1. Label ten 1.5-ml microcentrifuge tubes 1 through 10 and fill each with 450 µl eggdiluent.

2. Pipet 50 µl influenza virus stock into 450 µl egg diluent in the first tube and performa ten-fold dilution down to 10−10 by transferring 50 µl between tubes.

Changing tips between tubes will decrease the chance of sample carryover and will resultin a more accurate titration of the virus.

3. Inoculate three eggs with 100 µl per dilution as described in Basic Protocol 2, steps 1to 8. Incubate eggs 48 hr at 35◦C (optimal for influenza A) or 72 hr at 33◦C (optimalfor influenza B).

If the type of virus is unknown, incubate eggs at 35◦C.

4. Chill eggs overnight at 4◦C or for 30 min at −20◦C before harvesting.

5. With sterile forceps, break the shell over the air sac and push aside the allantoicmembrane with the forceps.

6. Pipet 50 µl allantoic fluid from each egg to a corresponding well in a 96-wellV-bottom plate.

V-bottom or U-bottom plates are used to allow for better settling of the red blood cells.

7. Add 50 µl standardized turkey red blood cells to all wells and incubate 30 min atroom temperature (24◦ to 27◦C).

Approximately 5 ml standardized turkey red blood cells will be needed per 96-well plateused in this assay.

8. Observe endpoint of agglutination and record.

Red blood cells will settle to the bottom of the V-bottom well in negative samples, whilered blood cells will agglutinate in positive samples.

9. Determine the EID50 titer per 100 µl by the Reed-Muench method (see SupportProtocol 5).

BASICPROTOCOL 6

QUANTIFICATION OF INFLUENZA VIRUSES BY PLAQUE ASSAY

Because influenza viruses cause cytopathic effect (CPE) and death of the infected cells,they may form plaques or circular zones of lysed cells on a monolayer. The plaqueassay is a method to measure the amount of infectious virus in a sample by determiningthe number of plaque forming units on a MDCK cell monolayer. At a high dilution ofvirus stock, each plaque represents the zone of cells infected by a single virus particle.Therefore, the titer of a virus stock can be calculated in plaque forming units per milliliter.The titration should be carried out in duplicate.

Materials

2× plaque assay medium (see recipe)1.6% (w/v) agarose solutionInfluenza virus stockMadin-Darby canine kidney (MDCK) cells confluent in 6-well tissue culture plates

(see Support Protocol 1)Plaque assay wash medium (see recipe)2 mg/ml TPCK-trypsin working stock (see recipe)70% ethanol0.3% crystal violet solution

37◦ and 56◦C water bathsForceps, sterileInverted microscope



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NOTE: All equipment and solutions coming into contact with cells must be sterile andproper sterile technique should be used accordingly.

1. Before beginning the plaque assay, warm 2× plaque assay medium in a 37◦C waterbath and place 1.6% agarose solution in a 56◦C water bath.

The 2× plaque assay medium should be warm enough so as not to solidify the 1.6%agarose solution when mixed together; also it should not be too hot, which will killthe MDCK cell monolayer when the media solution is added. The 1.6% agarose so-lution should be kept at 56◦C to keep the solution from solidifying before use. Whenboth solutions are mixed together the temperature of the resulting solution should besuitable for immediate addition to the MDCK cell monolayer while maintaining cellviability.

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

Keep virus at 4◦C once thawed to reduce loss of infectivity.

3. Remove the MDCK growth medium from the 6-well tissue culture plates andwash the MDCK monolayer three times with room temperature plaque assay washmedium.

For wash steps, either a sterile 10-ml pipet or 1000-µl pipettor may be used to removeand add the medium. Do not pipet medium directly onto the monolayer as this may disruptthe cells.


4. Perform a ten-fold dilution series starting at 10−1 and diluting virus samples down to10−10 in plaque assay wash medium. Change disposable pipet or pipet tips betweentubes to decrease the chance of sample carryover.

5. Inoculate 6-well tissue culture wells in duplicate with 100 µl diluted virus sampleand rotate tissue culture plate to cover monolayer with inoculum. Take care not toadd medium directly onto the monolayer as this may disrupt the cells.

6. Incubate inoculated plates for a minimum of 30 min or up to 1 hr at 37◦C.

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

7. Wash wells two times with room temperature plaque assay wash medium.

For wash steps, either a sterile 10-ml pipet or 1000-µl pipettor may be used to removeand add the medium. Do not pipet medium directly onto the monolayer as this may disruptthe cells.

Inoculum does not need to be removed before washing MDCK cell monolayer.

8. Add 1 µl of 2 mg/ml TPCK-trypsin working stock to 2× plaque assay mediumbefore proceeding to the next step.

TPCK-trypsin should not be added to the 2× plaque assay medium prior to this step toensure optimal enzymatic activity.

9. Mix 1:1 2× plaque assay medium with 1.6% agarose solution and immediatelyadd 2 ml of agarose medium solution to each inoculated well. Let solidify at roomtemperature (24◦ to 27◦C).

For one 6-well tissue culture plate, mix together 7 ml of 2× plaque assay medium with7 ml of 1.6% agarose solution.

10. Incubate 6-well tissue culture plates at 37◦C. Use an inverted microscope to observethe MDCK monolayer for plaque formation daily.

Plaques will appear as small clear areas in the monolayer.

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11. After 72 hr, carefully remove the agar plug from each well using sterile forceps anddisposing of the agar in a biological waste container. Take care to avoid scratchingthe MDCK cell monolayer or letting the agar plugs rotate during removal, as thiswill make it difficult to accurately count plaques.

12. Pipet 2 ml of 70% ethanol into each well and incubate 20 min at room temperature(24◦ to 27◦C) to fix the MDCK cell monolayer.

Do not pipet 70% ethanol directly onto the monolayer as this may disrupt the cells.

13. Remove ethanol and add 1 ml crystal violet solution to each well. Incubate 10 minat room temperature (24◦ to 27◦C) to stain MDCK cell monolayer.

14. Remove crystal violet solution and wash wells with water to rinse away excess stainsolution.

15. Let plates dry overnight at room temperature (24◦ to 27◦C) before counting plaques.

16. Count plaques in each well and determine the plaque forming units (pfu) per milliliterusing the following formula: pfu/ml = (no. of plaques × dilution factor × 10).

Plaques suitable for counting will appear as discrete clear circular zones on the back-ground of the purple stained MDCK cell monolayer. At low dilution of virus, the monolayermay be completely destroyed, resulting in no or minimal purple staining in the wells. Thepfu/ml calculation should be based on the dilution of virus sample that gives >10 plaquesper well but is still countable (i.e., <150 plaques).

BASICPROTOCOL 7

STORAGE OF INFLUENZA VIRUSES

Clinical specimens for viral isolation should be placed at 4◦C and transported to thelaboratory promptly. If clinical specimens are to be transported to the laboratory within2 days, the specimens may be kept at 4◦C; otherwise they should be frozen at or below−70◦C until transported to the laboratory. Influenza virus stocks should be stored at−70◦ to −80◦C and thawed just before use. For long-term storage, virus stocks shouldbe kept at −135◦C or in liquid nitrogen (−150◦C). Once thawed, virus stocks should bekept on ice/4◦C and used that day. To prevent loss of infectivity, repeated freezing andthawing must be avoided.

ALTERNATEPROTOCOL 5

LYOPHILIZATION OF INFLUENZA VIRUSES

Influenza viruses can be lyophilized for stable long-term storage at 4◦C. Stabilizers needto be added to viruses grown in tissue culture, whereas viruses grown in eggs do notrequire an additional stabilizer. Make a 1:1 solution of tissue culture–grown virus stockwith a 5% milk solution (5 g powdered dairy milk in 100 ml PBS, filter sterilize using a0.45-µm filtration unit). Once virus stocks are properly stabilized, follow the manu-facturer’s instructions for lyophilization procedures specific to the equipment available.The lyophilization process may cause a loss of infectivity of the virus stock. Therefore,lyophilization should only be used if lower viral titers will not adversely affect assays orno other long-term storage option is available.

SUPPORTPROTOCOL 1

GROWTH AND MAINTENANCE OF MDCK CELL LINE

Madin-Darby canine kidney (MDCK) cells (ATCC# CCL-34) are the preferred cell linefor the isolation and characterization of influenza A and B viruses, but not influenza Cviruses due to the incompatibility of sialic acid moieties on the cell surface with the viralreceptor specificity. This protocol describes how to maintain the MDCK cell line for usein the cell-based protocols presented in this unit.



15G.1.14


Materials

Madin-Darby canine kidney (MDCK) cells (ATCC# CCL-34) grown confluent in a75-cm2 flask (see Support Protocol 1)

Trypsin/EDTA (GIBCO)Heat-inactivated fetal bovine serum (FBS; APPENDIX 2A)MDCK growth medium (see recipe)

75- 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 culture incubations are performed in a humidified 37◦C, 5% CO2 incubator.

1. Decant growth medium from a confluent 75-cm2 flask of MDCK cells, and add 5 mltrypsin/EDTA prewarmed to 37◦C.

2. Gently rock the flask to distribute the trypsin/EDTA over the entire MDCK cellmonolayer. Remove the trypsin/EDTA with a pipet.

3. Repeat one time with an additional 5 ml of trypsin/EDTA.

4. Add 1 ml prewarmed trypsin/EDTA, ensuring that entire cell monolayer is bathedin trypsin/EDTA and incubate flask at 37◦C until all cells detach from the plasticsurface (∼5 to 10 min).

Gentle shaking or tapping of the flask may help cells detach.

5. Add 1 ml FBS to inactive trypsin/EDTA.

6. Add 8 ml MDCK growth medium to flask, pipetting gently to break up clumps ofcells.

7. Remove cell suspension and centrifuge 10 min at 200 × g, 4◦C.

8. Resuspend cell pellet in 2 ml MDCK growth medium and count cells with a hema-cytometer (APPENDIX 4A). Adjust the concentration accordingly.

For 75-cm2 flasks, 5 ml of a 1 × 105 cells/ml suspension added to 20 ml of MDCKgrowth medium will result in a confluent monolayer in 2 to 3 days. For 96-well tissueculture plates, 100 µl of 1.5 × 105 cells/ml suspension per well will result in a confluentmonolayer after an overnight (18- to 22-hr) incubation.

9. Incubate flasks in a 37◦C incubator.

MDCK cell lines should not be passaged indefinitely. Relatively low passage number(e.g., 20 to 30 passages) after establishing the line from frozen stocks will ensure thatcells retain their susceptibility to respiratory viruses. As such, a working stock of low-passage cells should be kept in liquid nitrogen as a source for renewing the cell line. Foroptimum results, the cells should be in log growth phase.

SUPPORTPROTOCOL 2

CANDLING 10-DAY EMBRYONATED CHICKEN EGGS

Candling chicken eggs is a standard procedure performed to ensure that only healthyembryonated eggs are used for the egg-based protocols presented in this unit. Use ofdead, broken, or nonembryonated eggs will result in little to no virus growth and as suchshould be detected and removed from the supply of eggs to be used in experiments. Eggsshould be used at an age of 9 to 11 days, because the 10-day-old chick embryo is oldenough to support efficient virus growth and optimal volumes of fluid containing virusmay be recovered. In addition, embryonated eggs <12 days of age have not yet developedadequate innate immunity competency, which can inhibit virus growth.

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15G.1.15


Figure 15G.1.4 Viable and nonviable chicken eggs as apparent from candling. This black andwhite facsimile of the figure is intended only as a placeholder; for full-color version of figure go tohttp://www.currentprotocols.com

In a darkened room, hold the airsac end of the egg to the candler light source andgently rotate it while observing the morphology of the chick embryo. A healthy embryohas a well-defined airsac, free-flowing membranes, uniform vein development andreacts to the light of the candler. It is very important to screen out broken, unhealthy,very porous, or dead embryos, as these eggs will not support efficient influenza virusgrowth. Figure 15G.1.4 depicts viable and non-viable chicken eggs as viewed by aKUHL candler. Alternatively, refer to the instruction manual of the egg candler for moreinstructions.

NOTE: Egg incubations are performed in a 34◦C, 50% humidity incubator with rotationfor eggs <9 days old. Cell culture incubators can be used for eggs >9 days old, but theCO2 must be turned off or the embryos will suffocate.

SUPPORTPROTOCOL 3

PREPARATION OF PRIMARY INFLUENZA VIRUS SPECIMENS

The success of virus diagnosis largely depends on the quality of the specimen andthe conditions for transport and storage of the specimen before it is processed in thelaboratory. Specimens for isolation of respiratory viruses in cell cultures and for thedirect detection of viral antigens or nucleic acids should generally be taken during thefirst 3 days after onset of clinical symptoms.

Materials

Specimen in collection vials (e.g., from nasal, throat, or combined nasal/throatswabs; or nasopharyngeal, nasal, or throat aspirates or washings)

10 mg/ml gentamicin (GIBCO)

2- to 4-mm glass beads (VWR)Polypropylene microcentrifuge tubes, sterileCryovials (Nunc)

NOTE: Clarified supernatants can be used to directly inoculate cell culture flasks or eggs.



15G.1.16


From nasal, throat, or combined nasal/throat swabs:

1a. Vortex collection vial and express the fluid in the swab.

2a. Remove swab from the collection vial and add 0.2 ml of 10 mg/ml gentamicin.

3a. Incubate 15 min at room temperature (24◦ to 27◦C).

4a. Centrifuge collection vial 5 min at 400 × g, 4◦C.

5a. Remove the clarified supernatant, dispense into 0.5- to 2-ml aliquots, and store<1 year at −70◦ to −80◦C or at −135◦ to −150◦C for long-term storage (see BasicProtocol 7).

From nasopharyngeal, nasal, or throat aspirates or washings:

1b. Break clumps of mucus by adding 2- to 4-mm glass beads to the specimen andvortexing.

2b. Add 0.1 ml of 10 mg/ml gentamicin/ml of specimen.

3b. Transfer the specimen to a clean tube and centrifuge 10 min at 400 × g, 4◦C.

4b. Remove the clarified supernatant, dispense into 0.5- to 2-ml aliquots, and store<1 year at −70◦ to −80◦C or at −135◦ to −150◦C for long-term storage (see BasicProtocol 7).

SUPPORTPROTOCOL 4

STANDARDIZATION OF RED BLOOD CELLS FORHEMAGGLUTINATION-BASED ASSAYS

Human influenza A, B, and C viruses agglutinate red blood cells (RBC) of several avianand animal species as they bind sialic acid on the surface of red blood cells. Naturalvariation of circulating human influenza viruses and repeated passaging of specimensin the laboratory can result in the alteration in the ability of the virus to agglutinatecertain species’ RBC. As such, samples may need to be tested in hemagglutination-based assays using RBCs from different species of animals before determining thatno virus is present. Table 15G1.1 outlines the species-specific concentration of RBC,type of microtiter plate, incubation time, and appearance of control cells when usedto detect influenza A and B viruses in these assays. Accurate determination of RBCconcentration is necessary as low concentration will overestimate and high concentrationwill underestimate the concentration of virus. Also, standardization of RBC concentrationis critical for obtaining reproducible results between assays. This support protocol iscommonly used for standardization of RBC regardless of species origin.

Materials

Whole blood mixed 1:1 in Alsever’s solution (APPENDIX 2A)Phosphate-buffered saline (PBS) containing potassium (APPENDIX 2A)Cotton gauze, sterile50-ml conical centrifuge tube

Table 15G.1.1 Properties of RBC from Different Species

Chicken Turkey Guinea pig Human type O

Concentration 0.5% 0.5% 0.75% 0.75%

Microtiter plate well shape V V U U

Incubation time at 25◦C 30 min 30 min 1 hr 1 hr

Appearance of control cells Button Button Halo Halo

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15G.1.17


Additional reagents and equipment for counting cells using a hemacytometer(APPENDIX 4A)


1. Filter ∼5 ml whole blood through a piece of sterile cotton gauze into a 50-ml conicalcentrifuge tube. Centrifuge tube 10 min at 200 × g, 4◦C.

2. Aspirate the plasma and buffy layer. Add 50 ml PBS and mix gently.

3. Centrifuge 5 min at 200 × g, 4◦C, and aspirate supernatant. Repeat PBS washes twoadditional times.

4. Resuspend RBC to final volume of 12 ml with PBS. Centrifuge 10 min at 200 × g,4◦C.

5. Estimate volume of packed RBC and dilute to approximate concentration with PBS.

6. Determine actual concentration of RBC with a hemacytometer (APPENDIX 4A) andadjust the concentration accordingly.

The final concentration of chicken and turkey RBC is 0.5%. Guinea pig and human “O”RBC is 0.75%. A higher concentration of guinea pig and human type O RBC is desirablefor complete settling of RBC and hence optimal discrimination between agglutinationand non-agglutination.

SUPPORTPROTOCOL 5

CALCULATION OF INFECTIOUS DOSE 50 TITERS BY THEREED-MUENCH METHOD

The Reed-Muench method (Reed and Muench, 1938) can be utilized to determine thedilution of virus sample required to yield a 50% positive result. The advantage to usingthis method rather than a 100% endpoint is that the 50% endpoint is less affected byvariations within the titration experiment. It is important to note that the unit of infectivitymeasured by this endpoint method may require more than one infectious particle. Withthe Reed-Muench method, it will be necessary to use a large number of small groupsof test samples at different dilutions (see Basic Protocols 4 and 5 for set up of titrationexperiments). At the end of the titration experiment, determine the number of positiveand negative samples at each dilution. Using these numbers, a percentage of infectedsamples can be calculated for each dilution. The dilution that would infect 50% of testsamples is estimated using the following formulas:

proportional distance formula = [(% positive value >50%) − 50%]/[(% positive value >50%) − (% positive value <50%)]

Knowing the proportional distance between dilutions, the 50%-endpoint can be calculatedusing the exact dilutions used:

log infectious dose 50 = (log dilution >50%) + (proportional distance× log dilution factor)

The reciprocal of this number is used to express the titer in infectious units per unitvolume.

An example an ID50 calculation using the Reed-Muench method is shown inFigure 15G.1.5.



15G.1.18


Figure 15G.1.5 Example ID50 calculation using the Reed-Muench method.

REAGENTS AND SOLUTIONSUse deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Antibiotic-supplemented phosphate-buffered saline containing potassium

Supplement 100 ml PBS containing potassium (KPBS; APPENDIX 2A) with:1 ml penicillin-streptomycin stock (100 U/ml penicillin G and 100 µg/ml strepto-

mycin)0.2 ml 50 mg/ml gentamicin stock (10 µg/ml)Filter sterilize with a 0.2-µm membraneStore up to 2 months at 4◦C

Complete Dulbecco’s modified Eagle medium (cDMEM)/7.5% BSA

Supplement 465 ml DMEM with:5 ml penicillin-streptomycin stock (100 U/ml penicillin G and 100 µg/ml strepto-

mycin)5 ml L-glutamine (2 mM)12.5 ml 7.5% bovine serum albumin solution (0.2% BSA)12.5 ml HEPES buffer (25 mM)Filter sterilize with a 0.2-µm membraneStore <2 months at 4◦C

Influenza virus growth medium

Supplement 500 ml cDMEM/7.5% BSA with 0.5 ml TPCK-trypsin working stockfor a final concentration of 2 µg/ml. Store up to 2 months at 4◦C.

This medium is for use with MDCK cells only.

Influenza virus plaque assay medium, 2×Supplement 455 ml 2× DMEM with:10 ml penicillin-streptomycin stock (200 U/ml penicillin G and 200 µg/ml strep-

tomycin)10 ml L-glutamine (4 mM)25 ml HEPES buffer (50 mM)Filter sterilize with a 0.2-µm membraneStore <2 months at 4◦C

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15G.1.19


Influenza virus plaque assay wash medium

Supplement 490 ml DMEM with 5 ml of penicillin-streptomycin stock (100 U/mlpenicillin G and 100 µg/ml streptomycin). Filter sterilize with a 0.2-µm membrane.Store up to 2 months at 4◦C.

L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin workingstock, 2 mg/ml

10 ml DMEM20 mg trypsin, TPCK-treated, type XIII from bovine pancreas (Sigma)Filter sterilize for up to 0.2-µm membraneDispense into aliquotsStore aliquots at −70◦C until reagents expire

Dispose of unused thawed TPCK-trypsin, do not refreeze, as the TPCK-trypsin is not stableafter thawing.

MDCK growth medium

Supplement 440 ml DMEM with:5 ml penicillin-streptomycin stock (100 U/ml penicillin G and 100 µg/ml strepto-

mycin)5 ml L-glutamine (2 mM)50 ml heat-inactivated (inactivated 30 min at 56◦C) fetal bovine serum (10% FBS)Filter sterilize with a 0.2-µm membraneStore for up to 2 months at 4◦C

Tryptose phosphate broth

Dissolve 29.5 g tryptose phosphate broth in 1 liter ddH2OSterilize by autoclaveStore up to 3 months at 4◦CJust prior to use, add 2% gentamicin 50 mg/ml to broth

COMMENTARY

Background InformationInfluenza A and B virus genomes con-

sist of eight gene segments encoding at leastten proteins (Palese, 1977; Lamb and Krug,2001). The major surface glycoproteins are thehemagglutinin (HA) and the neuraminidase(NA), which form the basis of multiple sero-logically distinct influenza A virus subtypes.There are 16 HA and 9 NA subtypes of in-fluenza A viruses known to currently circu-late in nature (Kilbourne, 1975; Webster et al.,1992; Rohm et al., 1996; Fouchier et al., 2005).Wild water birds are the natural reservoir forall influenza A viruses (Webster et al., 1992).In the last century, influenza A viruses bear-ing one of three HA (H1, H2, and H3) andtwo NA (N1 and N2) subtypes underwent sus-tained circulation and caused widespread dis-ease in humans (Wright and Webster, 2001).Since 1977, influenza viruses of the H1 and H3and N1 and N2 subtypes as well as influenzaB viruses have caused widespread, seasonaldisease in humans.

The influenza A virus undergoes two kindsof antigenic variation, antigenic drift, and anti-genic shift. Antigenic drift is the result ofaccumulation of point mutations in the HAand/or NA genes caused by transcriptional er-rors of the viral RNA polymerases that lackproofreading mechanisms (Scholtissek et al.,1993). Influenza variants with amino acid sub-stitutions in antigenic sites are able to escapeneutralization by existing host antibodies andemerge from this selective pressure to becomethe predominant viral population (Wright andWebster, 2001). Antigenic drift variants are re-sponsible for annual epidemics of influenza Aviruses (Wright and Webster, 2001). Alterna-tively, influenza A viruses can undergo anti-genic shift, which occurs when there is re-assortment of gene segments between viruses,resulting in the appearance of a new subtype ofinfluenza A virus containing a novel HA withor without a novel NA (Wright and Webster,2001). Such a novel strain can circulate inan immunologically naı̈ve human population



15G.1.20


and can potentially result in an influenza pan-demic if able to spread efficiently (Wright andWebster, 2001).

Human influenza A viruses are transmitteddirectly between individuals via aerosolizeddroplets generated by coughing and sneezingor indirectly through contact with fomites oncontaminated surfaces (Alford et al., 1966;Lidwell, 1974; Bean et al., 1982). Infectionbegins in the nasal and tracheal passageways,and rapidly spreads throughout the upper andlower respiratory tract. Apical shedding ofvirus from respiratory epithelial cells gen-erally limits viral infection to the respira-tory tract. Clinical symptoms of an acute in-fluenza A virus infection can range from mildto severe and typically include fever, cough,headache, malaise, and anorexia. Each year inthe United States, ∼36,000 people, on average,die from complications of influenza virus in-fection, with 90% of these deaths being in theelderly population aged >65 years (Thompsonet al., 2003).

Virus isolation is a highly sensitive and use-ful technique for the diagnosis of influenzavirus infection when used with clinical spec-imens of good quality. In fact, isolation of avirus in cell culture along with subsequentidentification by immunologic or genetic tech-niques or by electron microscopy are standardmethods for virus diagnosis. One importantadvantage of virus isolation is that this methodamplifies the virus from the original specimenand makes it available for further antigenicand genetic characterization, and also for drug-susceptibility testing if required. The propa-gation or use of laboratory-adapted strains orinfluenza strains of animal origin in laborato-ries also attempting isolation of viruses fromhuman clinical specimens for diagnostic or re-search purposes is not recommended, sincethere is a risk of contaminating the diagnosticspecimens with laboratory or animal strainswith high growth phenotypes.

In the past, human influenza viruses oftenwere isolated in embryonated eggs; however,today the majority of laboratories use cell cul-ture. Since vaccine candidate viruses must beisolated in eggs, the trend to use cell cultureshas decreased the availability of suitable vac-cine viruses. For this reason, laboratories thathave the capability to isolate influenza in eggsare encouraged to continue. However, somestrains of influenza, and in particular, recenthuman field isolates, are difficult to isolate andgrow in eggs, even after amniotic inoculation.For this and other reasons, MDCK cells arethe preferred cell line for isolation of humaninfluenza viruses from clinical specimens.

Critical Parameters andTroubleshooting

Viruses must be kept on ice/4◦C at all timesafter thawing. Do not freeze-thaw viruses morethan once, a substantial reduction in virus in-fectivity will result. Specimens for virus iso-lation should be refrigerated immediately af-ter collection and inoculated into susceptiblecell cultures as soon as possible. If the spec-imen cannot be processed within 48 to 72 hr,the specimen should be kept frozen at or be-low −70◦C. If a working stock with a de-fined infectivity is desired, virus should be har-vested, aliquoted, and frozen and the EID50 orTCID50 should be determined on a vial that hasbeen frozen and then thawed once. For routinepassage of laboratory stocks, virus inoculumshould be diluted in the range of at least 1:100,since inoculation of large amounts of virusmay lead to formation of defective interfer-ing (D.I.) virus particles, which can lower theoverall infectivity of a virus stock (Azzi et al.,1993). D.I. particles are virus particles that arelacking most of their genome. Because of thesedeletions in their genome, D.I. particles cannotsustain an infection by themselves. Instead,they depend on co-infection with a suitablehelper virus, which provides the gene func-tions that are absent from the D.I. particles.

The cell-based assays described herein arevery dependent on the quality of MDCK cells.Over a number of passages, MDCK cells mightlose their susceptibility to influenza viruses.For this reason, the laboratory should keep astock of frozen cells at a low passage leveland return to this stock to refresh the workinglaboratory stock at regular intervals, e.g., after20 to 30 passages. Cell lines should be free ofmycoplasma contamination (APPENDIX 3B) andshould be routinely tested. Optimal conditionsfor virus growth require the MDCK cells tobe confluent and in exponential growth phase.If the monolayer is overgrown, it is less sen-sitive to virus infection. In the plaque assay,if MDCK cells are not confluent, the resultingplaques will be diffuse and difficult to count.In contrast, if the MDCK cell monolayer isovergrown, patches of the monolayer may beremoved with the agarose plug after the 72-hrincubation, resulting in the inability to visual-ize and count plaques.

Although many mammalian cell types maybe infected with human influenza viruses, fewsupport productive infection, i.e., the releaseof infectious progeny virus into the culturesupernatant. This is due, in part, to the re-quirement for proteolytic cleavage of the HAmolecule into two subunits, HA1 and HA2.This enables the HA molecule to undergo

Animal RNAViruses

15G.1.21


a conformational change essential for mem-brane fusion in the endosome and the releaseof the nucleic acid allowing for replication toproceed. Although MDCK cells lack such anendogenous protease, addition of exogenoustrypsin to the medium at a typical concentra-tion of 1 to 2 µg/ml is sufficient for prote-olytic cleavage of the HA and the generation ofinfectious progeny. The trypsin used to prop-agate the virus is heat stable L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin. This should not be confusedwith the trypsin-EDTA solution used to re-move cell monolayer from flasks during cellpassage, which is not suitable as a source oftrypsin for virus propagation. Each new work-ing stock of TPCK-trypsin should be titeredfor optimal activity and lack of toxicity to thecell monolayer. The optimal concentration oftrypsin will produce maximal amounts of virusin infected culture supernatants but will notresult in destruction of uninfected monolayersover the period of incubation. If the trypsinconcentration is too high, cells will roundup and detach from the plastic, i.e., presentwith morphology similar to the cytopathic ef-fect observed in virus-infected cultures. Fetalbovine serum reduces the virus infectivity andmay also inhibit the activity of trypsin. There-fore, removal of all traces of serum containedin growth medium by gently rinsing monolay-ers multiple times is an important prerequisitefor virus inoculation.

Ten- to eleven-day-old embryonated eggsare optimal for growth of influenza A and Bviruses. It is generally convenient to purchasefreshly fertilized eggs from a supplier and toincubate them in the laboratory for the requiredtime. The use of an egg incubator that auto-matically turns the eggs several times a day,and maintains constant humidity and tempera-ture is optimal, but a humidified CO2 incubatorthat has the CO2 turned off to avoid asphyxi-ating the embryos may be used successfullyif eggs are turned manually several times aday. From 4 to 5 days post-fertilization, theembryos should be clearly visible. A low per-centage of eggs should be expected to be non-viable and should not be used for inoculation.The percent of nonviable eggs may increase inthe summer months in warmer climates. Via-bility can be determined prior to inoculationby candling eggs.

The methods described here provide asource of a quantified infectious influenzavirus stock for use in many laboratory pro-cedures. However, if large quantities of viralantigens are required, e.g., for use in ELISA or

in vivo vaccine studies, virus will need to beconcentrated and purified from a large volume(typically, ≥1 liter) of infected allantoic fluidor cell culture supernatant. General methodsfor concentration and purification of influenzavirus have been described elsewhere (Aroraet al., 1985).

Avian RBCs are generally used to detect in-fluenza virus agglutination. Turkey RBCs areoptimal for agglutination of recent human in-fluenza viruses and can also be used for detec-tion of common laboratory adapted strains, butmutations in or around the receptor-bindingsite of a virus may influence the efficiency withwhich a given virus binds to a given species ofRBC. In some cases, guinea pig RBC or hu-man type “O” cells can be more sensitive thanavian RBC for detecting human strains of in-fluenza. Upon passage in tissue culture, moststrains will adapt to avian RBC agglutination.Blood should be received in Alsever’s solutionand can only be stored for 5 to 7 days, afterwhich hemolysis will occur and substantiallyreduce the accuracy of the hemagglutinationassay. Accurate determination of the RBC con-centration is necessary as low concentrationswill overestimate and high concentrations willunderestimate the concentration of virus. Thehemagglutination assay must be read promptlyas the viral NA will eventually cleave the sialicacid from the RBC and release the HA fromthe cell. Some viruses may have heightenedNA activity. In such cases, the hemagglutina-tion assay should be carried out at 4◦C.

Anticipated ResultsThe extent of influenza virus growth in

either MDCK cells or embryonated eggsis strain-dependent. Human viruses isolatedfrom primary clinical specimens may growpoorly in either eggs or tissue culture (titersof 4 to 16 HAU per 50 µl and titers of 107

ID50/ml or 106 pfu/ml) until the virus hasbeen adapted to grow in vitro. In contrast,many laboratory strains that have been pas-saged numerous times in eggs may achievetiters of 256 to >1024 HAU per 50 µl or 108

to 109 ID50/ml or 107 to 108 pfu/ml. For anti-genic characterization, hemagglutination unitsshould be ≥8 HAU. In tissue culture, virus re-lease into the supernatant may be readily mon-itored at regular intervals by removing a smallamount to detect rising hemagglutinationactivity.

Adaptation of human influenza virusesto grow in eggs may select for variantsthat possess amino acid substitutions in theHA molecule that can also affect biological



15G.1.22


properties (Katz et al., 1990). The acquisi-tion of amino acid substitutions in the HA inviruses repeatedly passaged in MDCK cellshas also been reported (Mochalova et al. 2003).The potential for such changes should be con-sidered when repeatedly passaging influenzaviruses in eggs or tissue culture and may beidentified by sequence analysis.

Time ConsiderationsHuman influenza A viruses must be incu-

bated for 48 hr, whereas influenza B virusesrequire a 72-hr incubation period. Since clini-cal material should be blind-passaged at leasttwice, it may take up to 14 days before theresults of virus diagnosis can be stated to be“virus not recovered.” Optimal infectivity ofa virus stock will be achieved when the virusis kept on ice/4◦C during harvest, clarifica-tion of fluid and estimation of virus HAU, andthen frozen immediately at or below −70◦C.Viruses may be stored for several years at−70◦C, but prolonged storage at this temper-ature will cause a reduction in virus viabil-ity. Incubation times for the hemagglutinationassay must be followed to produce accurateresults; 30 min for avian RBC and 1 hr formammalian RBC at room temperature is rec-ommended. The plaque assay requires 4 days;a half day to set up, a 72-hr incubation, and ahalf day to process and read plates. During theplaque assay setup, work quickly after mixingthe warm 2× plaque assay medium with the1.6% agarose solution, to ensure that the me-dia mixture does not solidify prior to applyingto 6-well tissue culture plates, but is not toohot for the MDCK cell monolayer. The opti-mal temperature of the overlay media shouldbe in the range of 37◦C to 42◦C.

Literature CitedAlford, R.H., Kasel, J.A., Gerone, P.J., and Knight,

V. 1966. Human influenza resulting from aerosolinhalation. Proc. Soc. Exp. Biol. Med. 122:800-804.

Arora, D.J., Tremplay, P., Bourgault, R., andBoileau, S. 1985. Concentration and purifica-tion of influenza virus from allantoic fluid. Anal.Biochem. 144:189-192.

Azzi, A., Bartolomei-Corsi, O., Zakrzewska, K.,Corcoran, T., Newman, R., Robertson, J.S.,Yates, P., and Oxford, J.S. 1993. The haemagglu-tinins of influenza A (H1N1) viruses in the ‘O’or ‘D’ phases exhibit biological and antigenicdifferences. Epidemiol. Infect. 111:135-142.

Bean, B., Moore, B.M., Sterner, B., Peterson, L.R.,Gerding, D.N., and Balfour, H.H. Jr. 1982. Sur-vival of influenza viruses on environmental sur-faces. J. Infect. Dis. 146:47-51.

Fouchier, R.A., Munster, V., Wallensten, A.,Bestebroer, T.M., Herfst, S., Smith, D.,Rimmelzwaan, G.F., Olsen, B., and Osterhaus,A.D. 2005. Characterization of a novel influenzaa virus hemagglutinin subtype (H16) obtainedfrom black-headed gulls. J. Virol. 79:2814-2822.

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Contributed by Kristy J. Szretter, AmandaL. Balish, and Jacqueline M. Katz

Influenza Branch, Centers for DiseaseControl and Prevention

Atlanta, Georgia

influenza: propagation, quantification, and storage

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