of Measles Virus in Cell Culture
for Disease Control and Prevention
November 29, 2001
National Center for Infectious Diseases
Division of Viral and Rickettsial Diseases
Respiratory and Enteric Viruses Branch
Measles Virus Section
American Health Organization
Pan American Sanitary Bureau
Regional Office of the World Health Organization
Division of Vaccines and Immunization
and Identification of Measles Virus in Cell Culture
1: Measles CPE in B95a cells
of a sensitive cell line (B95a) for isolation of measles virus from clinical
specimens and establishment of automated DNA sequencing techniques have
allowed for rapid genetic characterization of a large number of wild-type
strains of measles virus. This database of sequence information now makes
it possible to use molecular epidemiologic techniques to identify the
source of wild-type viruses and to differentiate between wild-type and
vaccine strains (1-4).
is made toward elimination of measles in many regions of the world, it
will be critical to examine virus isolates from as many outbreaks and
isolated cases as possible in order to identify the source of the virus.
The World Health Organization (WHO) held a meeting in May 1998 to standardize
the protocols for the genetic characterization of wild-type measles viruses
and to establish a consistent system for describing the genotypes (5).
Collection of measles specimens will help to determine which outbreaks
may be related and to monitor patterns of virus transmission. The ability
to determine the effectiveness of measles elimination programs will also
be enhanced by continued characterization of viruses from sporadic outbreaks
of measles (1).
and genetic characterization can take several weeks to complete. Therefore,
laboratory diagnosis of measles should always be based on detection of
measles-specific IgM in serum. IgM enzyme immunoassay (EIA) can be completed
in 1 day, and assay kits are available from several commercial sources.
Specimens (urine, throat, or nasal) for virus isolation should be obtained
at the same time that serum is drawn, since a delay in collection will
reduce the chance of isolating the virus. However, urine or nasal specimens
should not be substituted for serum specimens for measles diagnosis.
virus-transformed, marmoset B lymphoblastoid cell line, B95a, is the preferred
cell line for primary isolation of measles virus (5). These cells are
as much as 10,000 times more sensitive for isolation of measles virus
from clinical specimens than other commonly used cell lines, such as Vero
and PMK. B95a cells are relatively easy to maintain in the laboratory,
and the cytopathic effect (CPE) from measles infection is readily observed.
However, laboratorians should note that this cell line does produce Epstein-Barr
virus and should be handled as infectious material (Biosafety Level 2)
at all times.
are available from the American Type Culture Collection (# CRL 1612).
When cultured in Dulbecco's modified Eagle medium (DMEM) supplemented
with fetal bovine serum (FBS), these cells will adhere to the surface
of the culture vessel; the adherent cells are referred to as B95a. Cell
growth is sustained in medium containing 8-10% FBS. FBS is used at a 2%
concentration for cell maintenance during viral isolation. Optimal growth
conditions for the cells are obtained by incubating the culture in a 5%
CO2 incubator at 37 C. (See Note in section
II) Cell stocks can be prepared by using standard cryoprotection medium
modified Eagle medium (DMEM):
with 4,500 mg/L D-glucose (high glucose)
without sodium pyruvate
10,000 units/ml penicillin G
10,000 ug/ml streptomycin sulfate
in 0.85% saline
0.53 mM EDTA
in Hanks' balanced salt solution (HBSS) without Ca++ and Mg++
bovine serum (FBS)
culture vessels (T-25)
microscope, preferably with phase contrast optics
Minimum essential medium (MEM) may be substituted when CO2 incubator
is not available, however DMEM has been used with good results with
the addition of 1% Hepes buffer and 1% NaCO3 to improve buffering
of trade names and commercial sources in this manual does not imply
endorsement by the Centers for Disease Control and Prevention.
of B95a cell line
all procedures under an appropriate biosafety cabinet. Because the
B95a cell line contains potentially infectious Epstein-Barr virus,
all discarded medium should be placed into a beaker containing 10%
DMEM and trypsin solution to warm to room temperature. It may be convenient
to prepare small amounts of DMEM mixed with FBS.
medium from T-25 flask and wash cells with either 3-5 ml of DMEM or
trypsin by gently swirling the flask for about 10-20 seconds, then
remove wash solution.
1-2 ml of trypsin to the T-25 flask, screw the cap on tight, and rock
the flask side to side. The cells will quickly become detached from
the vessel surface in about 30-60 seconds. Dislodge the cells by rapping
the flask with one hand. The cells should knock off easily.
add 5 ml of DMEM to the flask (total volume 6 ml) to collect the
cells and transfer the fluid to a small tube to mix.
The cells from one flask can be split 1:2, 1:3, or 1:4. Do not
passage B95a cells at subcultivation ratios higher than 1:4. After
1:2 and 1:3 splits, the cells are usually ready to infect within
the desired number of flasks, adding the appropriate volume of cell
suspension to the flasks containing DMEM with10% FBS. Place the flasks
in an incubator at 37 C. A CO2 incubator is optimal for cell growth,
but if one is not available, the substitution of minimum essential
medium (MEM) for DMEM is recommended or DMEM with the addition of
hepes buffer and sodium bicarbonate (NaCO3) may improve buffering
To set up 3 T-25 flasks from 1 T-25 flask, add 5 ml of DMEM to the
trypsinized cells (total volume 6 ml). Vortex gently or pipet up and
down. Distribute 2 ml (1:3 split) to each T-25 flask containing 10
ml of DMEM with 10% FBS. Cells should be ready to infect (75-85% confluent)
in about 2-3 days.
of stock (frozen) B95a cells
It is extremely
important to prepare multiple frozen stocks of B95a cells as soon as they
are available in the laboratory. These cells will gradually lose their
ability to form smooth, uniform monolayers, making detection of measles
virus more difficult. It is recommended that B95a cells be passaged no
more than 30-35 times.
Cells can be frozen using any standard cryopreservation technique. Commercial
freezing medium is available or the reagents described below should be
a solution of 90% FBS and 10% DMSO (or glycerol) and store on ice. Make
enough to allow for 1 ml for each vial of cells. For a 150-cm2 flask,
10 vials of cells can be prepared. Decant the medium from a confluent
150-cm2 flask of B95a cells, saving 10-20 ml of the medium in a centrifuge
tube. Cells should be removed from the flask using a small amount of trypsin-EDTA.
Add the cells to the decanted medium in the centrifuge tube and centrifuge
the cell suspension at 400 x g for 5 minutes. Pour off the supernatant
and add 10 ml of the required volume of cryoprotectant solution. Pipet
gently up and down to mix and dispense 1 ml into each of 10 plastic cryovials.
If available, the vials should be cooled using a programmed cell freezer
or a commercial product designed for gradual temperature reduction. Store
vials in gas-phase liquid nitrogen.
of growing B95a cultures
can be transported at room temperature in a 25-cm2 tissue culture
flask. For shipment, flasks should be filled to the top with medium containing
DMEM/2% FBS, and securely sealed. The package should include secondary
watertight packaging required for infectious perishable material and have
the appropriate labels on the outside. Additional information on shipping
is given in section VII.
of shipped cells, examine the cell sheet. If many cells are free-floating,
centrifugation (400 x g, 5 minutes) of the medium will recover the cells,
which can be added back to the flask (or to another flask for passage).
For a 25-cm2 flask, add 10-15 ml of the medium back to the
flask for cultivation.
specimen is logged in, a laboratory identification number and information
for the patient and specimen should be recorded in the log book or spreadsheet.
The specimen information may be helpful in identifying problems that may
contribute to loss of virus and inability to make isolations. Problems
in shipment or with the samples should be reported to the sender.
data to record:
(urine/throat swab/nasal washing/blood)
of collection of sample
(temp. upon arrival)
taken (centrifugation, storage location)
nasal, or nasopharyngeal swabs or aspirates: If the specimen contains
2-3 ml of viral transport medium or phosphate-buffered saline (PBS), it
can be either frozen at -70 C or stored at 4 C. If the original swab tube
is sent, add 2 ml of DMEM, vortex to collect swab material, and drain
the swab as much as possible against the side of the tube. If debris is
heavy, centrifuge to remove.
a centrifuge is available, transfer the urine to tube(s) to collect the
sediment for 5-10 minutes at 400 x g. (A refrigerated centrifuge is recommended,
but otherwise start with urine that has been chilled at 4 C). Resuspend
sediment in 2 ml of cold DMEM. If a centrifuge is not available, transfer
the urine to sterile, leak-proof containers and refrigerate.
Heparinized blood: Use a product designed to separate lymphocytes from
peripheral blood during centrifugation.
Important: It is recommended that the sample be divided in case
contamination is a problem. Do not routinely filter clinical specimens
before inoculation. However, if a culture is found to be contaminated
on the first isolation attempt, the remaining specimen can be filtered.
(To filter the specimen, bring the volume to 1-2 ml with DMEM, then filter
contents through a syringe filter ( 0.45 um) and collect in a sterile
INOCULATION OF SPECIMENS FOR MEASLES ISOLATION
should be at 75-85% confluency when specimens are inoculated.
initial infection of B95a cells (T-25 flask), decant medium and add
specimen (in total volume 1.5 ml, sufficient to keep monolayer from
drying out). Clinical specimens usually consist of urine sediment,
throat swab fluid, or centrifuged nasal specimens that have been re-suspended
in a small volume (1.0 - 2.0 ml) of tissue culture medium. See section
IV regarding the processing of specimens.
at 37 C for 1 hour to allow the virus to adsorb. After the adsorption
period, observe the cells under the microscope to determine whether
the sample was toxic to the cells (rounding of cells, cells floating).
The cells will usually recover but may need a change of medium. This
is virus passage #1.
10 ml of DMEM containing 2% FBS, with antibiotics.
of specimens with antibiotics: At the time of inoculation, penicillin/streptomycin
may be added to the media. When supplied as 10,000 units penicillin
and 10,000 ug per ml streptomycin, add at 1:50-1:100 so the final
concentration in the medium will be 100-200 units penicillin and
100-200 ug/ml streptomycin.
following day, check the cells. Tiny holes often can be seen as early
as 1 day after infection by observing the cell layer against a light
If holes in the monolayer are observed, examine by microscope to see
if fused cells (syncytia) are visible. Sometimes infected foci detach
from the monolayer and float into the medium. Be aware that some holes
in the monolayer may appear which are not caused by measles. Examine
the cell sheet for signs of bacterial contamination. Discard the flask
if yeast or bacteria is present.
the cell medium if it appears too acidic (orange-yellow). Centrifuge
to collect cells if they have detached from the cell sheet.
cells when monolayer is confluent, usually 2 days after inoculation.
Transfer medium to a tube and add the trypsinized cells, and centrifuge
(400 x g for 5-10 minutes). Resuspend cells in a small amount of medium
(6 ml) and then gently mix or vortex. Following the initial infection,
it is recommended to use all of the cells for the next cell passage,
distributing half of the cell mixture to each of 2 T-25 flasks (virus
passage #2). Add DMEM with 2% FBS.
the flasks daily. It may be necessary to split the cells again within
1-2 days. For passage #3, use a 1:2 split and discard the remaining
cells. If no CPE is observed after cultivation of the passage #3 cells,
is observed, continue to grow the cells (change the medium, if necessary)
until the CPE becomes extensive. It may be necessary to re-distribute/passage
the cells 1-2 times to allow the infection to spread before cells
become overgrown. When CPE has spread to at least 50-75% of the cell
layer, the virus has reached a suitable titer for viral stock.
prepare a viral stock, scrape the cells into the medium with a cell
scraper. Mix and distribute to 4-6 vials at -70 C.
typical measles CPE is observed, confirmation of measles virus should
not be necessary if the clinical symptoms were consistent for a measles
of typical measles cytopathic effects (CPE)- large syncytia and multinucleated
giant cells- in the B95a cell line suggests isolation of measles virus
with reasonable certainty. Other respiratory viruses, with the exception
of the mumps virus, are not known to produce such cytopathic effect in
this cell line. However, it may be necessary to confirm the presence of
measles virus when using other cell lines.
immunofluorescence assays (IFA), type-specific antibodies labelled with
fluorescein isothiocyanate (FITC) are reacted with virus-infected cells
fixed to a microscope slide. Both direct and indirect IFA kits are commercially
available from a number of sources. It is also possible to configure an
indirect IFA without using a commercial kit. in the IFA procedure described
below. In particular, Mab:KK2 is available from the WHO measles reagent
bank maintained by Dr. Fabian Wild in Lyon, France.
antibodies to the nucleoprotein will perform well. Monoclonal antibodies
directed against other viral proteins, such as the hemagglutinin and fusion
proteins, may recognize conformational epitopes that are not stable after
AND SHIPPING OF CLINICAL SPECIMENS
for virus isolation should be obtained as soon as possible when measles
infection is suspected, preferably at the onset of rash when the serum
sample is collected. Urine and respiratory samples are both good clinical
specimens for viral isolation. With very young patients, a respiratory
sample (throat swab) may be easier to obtain than urine. Other types of
respiratory samples may be more readily obtained in a clinic or hospital
where the equipment is available and it is recommended to take advantage
of the opportunity. Protocols are described below.
ml aliquots of viral transport medium (VTM; sterile PBS or suitable
isotonic solution such as HBSS, containing antibiotics:100 units/ml
penicillin, 100 ug/ml streptomycin) and either 2% fetal bovine serum
or 0.5% gelatin in 15 ml polycarbonate or polystyrene centrifuge tubes
ml plastic syringes
aspirators or 30 ml syringe
to obtain the sample as soon as possible after onset of rash. Virus
is most frequently recovered within the first 3 days following rash
(but up to 7 days after rash onset is acceptable).
nasal wash (nasopharyngeal aspirate) can be obtained by using a syringe
attached to a small piece of plastic tubing. After placing about 3-5
ml of saline in the nose, aspirate as much of the material as possible
and add to the centrifuge tube containing the VTM. (In a clinic or
hospital setting, if available, a vacuum may increase the recovery
of fluid.) Rinse the syringe and collection tubing into the VTM.
sterile swabs can be used to obtain throat and naspharyngeal specimens.
A throat swab is taken by rubbing the posterior nasal passages with
a dry sterile cotton swab. Place swab in a tube containing 2-3 ml
of VTM. The swab can be broken off into the tube of VTM
respiratory specimens at 4 C and ship to an appropriate laboratory
with cold packs.
Refrigeration of samples is adequate if cold shipment can be arranged
within about 48 hours. If there is a delay, and it is possible, freeze
the samples at -40 to -70 C and ship frozen on dry ice.
collection cups, preferably with lid.
polystyrene screw-cap centrifuge tubes.
be collected within 7 days of rash onset (within 1-3 days if possible).
First- morning voided specimens are ideal, but any urine collection is
adequate. Collect 10-50 ml of urine in a urine specimen container.
Centrifuge the urine specimen as soon as possible after collection. After
collection, keep the specimen cold (refrigerator or wet ice). Transfer
the urine specimen to a 50-ml plastic conical centrifuge tube and centrifuge
at 400 x g for 5-10 minutes at 4 C to collect the sediment. Resuspend
the sediment in 2-3 ml of VTM (above) or any cell culture medium (DMEM,
EMEM, RPMI plus antibiotics). Preferably, specimens that have been centrifuged
and resuspended should be frozen at -70 C and shipped on dry ice. If dry
ice is not available, however, they can be stored at 4 C and shipped on
wet ice or cold packs.
If centrifugation is not available, do not freeze the urine sample. The
entire urine specimen should be stored at 4 C, and shipped to the lab
on wet ice. It is best to have the specimen shipped to a viral laboratory
within 48 hours so that it can be processed and frozen at -70 C for optimal
virus recovery. Seal the specimen container tightly to prevent leakage.
also be isolated from lymphocytes. If it is possible to collect several
milliliters of heparinized blood, the lymphocytes will be a good source
of virus. The whole blood should be stored at 4 C and transported to the
laboratory within 48 hours of collection.
Shipping of clinical specimens and viral isolates
It is recommended
to use containers made specifically for shipping infectious substances,
for example, the Saf-T-Pak system.
For shipping of viral isolates in cell culture, it is best to use a plastic
25-cm2 tissue culture flask. Cells should be infected 1-2 days
before shipping. Before shipment, fill the vessel to the top with DMEM
(plus antibiotics and 2% FBS). Screw the top on tightly and seal with
plastic film or tape. Place the flask in a leak-proof container, such
as a zip-lock plastic bag with absorbent material, and ship at room temperature.
Infected cells can be pelleted, resuspended in a small volume of DMEM,
and frozen at -70 C before shipping on dry ice.
CDC is a
designated WHO measles strain bank and will accept viral isolates or clinical
specimens from cases of measles for genetic characterization. It is important
to notify the Measles Virus Section before shipping, particularly for
instructions for shipping will be provided. Viral isolates and clinical
specimens arriving from outside of the USA will require a CDC import permit,
available upon request (a faxed copy is acceptable). For international
shipments, a copy of the import permit must accompany the shipping documents.
A copy of the label must be affixed to the outer shipping container. Please
call, fax or e-mail to obtain a valid permit if the date has expired or
if the permit has been lost
Dr. William J. Bellini
Measles Virus Section, C-22
DASH Group #81
Centers for Disease Control and Prevention
1600 Clifton Rd.
Atlanta, GA 30333 USA
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