Growth of Semliki Forest Virus
Semliki Forest virus (SFV) is an enveloped RNA
virus, with a genome of positive polarity that belongs
to the Alphavirus group of the family Togaviridae. It
readily infects a variety of mammalian and insect cells
and can be grown to high titres in tissue culture. Upon
infection, host cell-specific synthesis of macromolecules
is suppressed within a few hours, structural viral
components are made, and new virus particles bud out
from the plasma membrane of the infected cell. The
SFV particle is spherical with a diameter of approximately
65 nm (molecular mass ≈ 42 × 103
kDa). It consists
of a nucleocapsid (NC), a single copy of the RNA
genome packed together with 240 copies of a capsid
(C) protein (33kDa), that is surrounded by a lipid
membrane in which 80 glycoprotein complexes, the
viral spikes, are anchored. The viral spikes are trimeric
associations of a protein complex: two membranespanning
proteins, E1 and E2 (49 and 52kDa), and a
peripheral protein, E3 (10kDa) (Garoff et al.
Strauss and Strauss, 1994). This article provides protocols
to grow SFV in intermediate scale (up to ≈1.5 mg;
protocol A) and small scale (35
II. MATERIALS AND
Culture medium Glasgow minimum essential
medium (MEM) (BHK-21) (Cat. No. 21710), foetal
bovine serum (FBS) (Cat. No. 10106), tryptose phosphate broth (Cat. No. 18050), 1M HEPES (Cat. No.
(100X) (Cat. No. 25030),
penicillin-streptomycin (Cat. No. 15140), MEM
(Cat. No. 21090-022), bovine albumin fraction V
solution 7.5% (BSA; Cat. No. 15260-037), and
phosphate-buffered saline (PBS) Dulbecco's with Ca2+
(Cat. No. 14040) are from GIBCO BRL.
Sea-plaque agarose (Cat. No. 50100) is from FMC Bio
S]methionine (Cat. No. AG 1094)
is from Amersham Biosciences. Sucrose (Cat. No. 0335)
is from Amresco. Tris (Cat. No. 146861) is from Angus.
Sodium chloride (NaCl) (Cat. No. 106404), HCl
(Cat. No. M317), and Titriplex III (EDTA; Cat.
No. 108418) are from Merck. NaOH (Cat. No. 05-
400201) is from EKA Nobel AB (Tamro). Cholesterol
(Cat. No. C3045), neutral red (Cat. No. N6634), and
methionine-free MEM (Cat. No. 31900-012) are from
Sigma-Aldrich. The density gradient fractionator
(Model 185) is from Instrumentation Specialities
Company (ISCO). Filter papers No. 1 (Cat. No. 1001
090) are from Whatman. The 75-cm2
flasks (Cat. No.
3375) and 162-cm2
flasks (Cat. No. 3150) are from
Corning Life Sciences. Sixty-millimeter tissue culture
plates (Cat. No. 50288) are from Nunc. BHK-21 cells C-
13 (Cat. No. CRL-8544) are from American Type Culture
Collection. Cotton-tipped applicators are from Solon
manufacturing company. Fifty-milliliter Nalgene tubes
(Cat. No. 3139-0050) are from Nalge Incorporated. SW
28 tubes (Cat. No. 344058), SW 40 tubes (Cat. No.
331374), and 6ml-scintillation vials (Cat. No. 566831)
are from Beckman. Eppendorf tubes, 1.5 ml (Cat. No.
0030 102.002) and 2.0ml (Cat. No. 0030 120.094), are
from Eppendorf-Netheler-Hinz GmbH. Emulsifier
Safe is from Packard Instrument Co. Inc.
A. Growth of SFV
- Complete BHK-21 medium: To make 585 ml, add 50 ml
of tryptose phosphate broth, 25 ml of FBS, 5 ml of
1M HEPES, and 5 ml of 200mM glutamine to 500
ml of Glasgow MEM (BHK-21). Store at 4°C.
- Complete BHK-21 medium + cholesterol (optional): To
make 250ml, add 0.5 ml 10mg/ml cholesterol stock
solution (dissolve at 37°C prior to use) to 250ml
complete BHK medium.
- 10mg/ml cholesterol stock solution (optional): Add
50mg cholesterol to 5 ml 99.5% ethanol. Dissolve.
Aliquot and store at -20°C.
- Supplemented MEM: To make 529ml, add 14ml of
7.5% BSA, 5 ml of 1M HEPES, 5 ml of 200 mM glutamine,
5 ml of 10,000 U/ml penicillin/10,000 µg/ml
streptomycin to 500 ml of MEM. Store at 4°C.
- TN: 50mM Tris-HCl, pH 7.4, 100mM NaCl. To
make 500 ml, add 3.0 g of Tris and 2.9 g NaCl to distilled
water, adjust pH to 7.4 by adding 1M HCl,
and complete the volume to 500ml. Autoclave.
Store at room temperature.
- 0.25M EDTA pH 8.0 stock solution: To make 50ml,
add 4.65 g of Titriplex III to 30ml of distilled water.
Adjust the pH to 8.0 by adding 1M NaOH and complete
the volume to 50ml. Autoclave. Store at room
- TNE: 50 mM Tris-HCl, pH 7.4, 100 mM NaCl,
0.5 mM EDTA. To make 100 ml, add 200 µl 0.25M EDTA, pH 8.0, stock solution to 100ml TN. Store at
- 200 g/kg sucrose solution: To make 100 g, weigh 20 g of
sucrose and adjust to 100 g with TNE. Store at -20°C.
B. Growth of 35S-Methionine-Labelled SFV
- Grow BHK-21 cells to 100% confluency in a 162-
cm 2 tissue culture bottle in complete BHK medium
with or without cholesterol (≈2.2 × 107 cells/
- Dilute the virus to a concentration of 1.1 × 106 pfu/ml in supplemented MEM.
- Remove the medium and wash the cells with 10 ml
of PBS (with Ca2+ and Mg2+). Add 2.0ml of the
diluted virus to the cells. Incubate for 60min at
37°C and 5% CO2. Tilt the bottle every 20 minute
to ensure even distribution of virus particles.
- Remove virus solution and rinse with 10ml PBS
(with Ca2+ and Mg2+). Add 30ml complete BHK
medium with or without cholesterol and incubate
for 18h at 37°C and 5% CO2.
- Transfer the virus containing medium to a 50-
ml Nalgene tube (30ml/tube) and centrifuge at
26,500g and 4°C for 10 min in a J2-21 Beckman centrifuge
equipped with a JS 13.1 rotor (13,000rpm).
- Pipette the medium to a fresh tube, without disturbing
the pellet, and centrifuge as in step 5.
- Repeat step 6.
- Transfer 20ml of the clarified medium to an SW
28-tube. Layer 4.0ml 200g/kg sucrose in TNE
under the sample. Add the remaining 8-10ml of
clarified medium to the top of the tube and centrifuge
at 112,000g and 4°C for 90min in a L8-M
Beckman centrifuge equipped with a SW 28 rotor
- Aspirate off the supernatant. Pour the last 0.5 ml
into the pipette by tilting the tube. Wipe off the last
drops of supernatant from the inside of the tube
with a sterile, cotton-tipped applicator.
- Add 200 µl TNE, cover the tube with Parafilm, and
leave on ice for 15 h.
- Pass the virus suspension slowly up and down in a
Gilson P-200 pipette to resuspend the virus. Transfer
the suspension to a 1.5-ml Eppendorf tube, rinse
the SW 28 tube with 100 µl TNE, and pool.
- Mix 10µl of the virus suspension with 90µl TNE
(dilution factor, D = 10-1) and measure the optical
density at 260 and 280nm, e.g., in a Pharmacia
Ultrospec plus spectrophotometer equipped with
a 50-µl cuvette with a 10-mm path length. Calculate
the ratio R = A260/A280 (R = 1.4 ± 0.1 for pure
SFV particles). Estimate the virus concentration
(CSFV) if the preparation is sufficiently pure; CSFV = A260/(D × 8) [mg/ml].
- Analyse the virus preparation by SDS-PAGE
under nonreducing conditions and visualise the
bands by Coomassie brilliant blue staining.
- Aliquot the virus suspension in smaller portions,
quick freeze in dry ice/ethanol, and store at -70°C.
- Starvation medium: To make 103ml, add 1 ml of
200mM glutamine, 1 ml of 1 M HEPES, and 1 ml of
10,000 U / ml penicillin/10,000 µg / ml streptomycin
to 100ml of methionine-free MEM. Store at 4°C.
- Labelling medium: To make 7.5 ml, add 50 µl Redivue
[35S]methionine (370MBq/ml) to 7.5ml starvation
- 550g/kg sucrose: To make 50 g, weigh 27.5 g sucrose
and adjust to 50 g with TNE. Store at -20°C.
- Complete BHK medium, supplemented MEM, and
200g/kg sucrose: see Section III,A.
C. Quantitation of Infectious Virus Particles
by Plaque Titration
- Grow BHK-21 cells to 100% confluency in a 75-cm2 tissue culture bottle in complete BHK medium
(≈ 1 × 107 cells/bottle).
- Dilute the virus to a concentration of 1 × 108 plaque-forming units (pfu)/ml in supplemented
- Remove the medium and wash the cells with
10ml of PBS (with Ca2+ and Mg2+). Add 1.0ml
of the diluted virus to the cells. Incubate for
60min at 37°C and 5% CO2. Tilt the bottle every
20min to ensure even distribution of virus
- Remove virus solution, add 15ml complete BHK
medium, and incubate for 3.5h at 37°C and 5%
- Rinse the cells two times with 10 ml PBS (with Ca2+ and Mg2+). Add 15ml of starvation medium, and
incubate for 30min at 37°C and 5% CO2.
- Remove the starvation medium, add 7.5ml of
labelling medium, and continue incubation at 37°C and 5% CO2 for 15-16 h.
- Harvest the labelling medium and dispense in four
2-ml Eppendorf tubes.
- Centrifuge at 12,400g and 4°C for 5min in an
Eppendorf 5416 centrifuge equipped with a 16
F24-11 rotor (11,000rpm).
- Transfer the supernatants into new tubes, without
disturbing the pellet, and repeat centrifugation as
in step 8.
- Repeat step 9.
- Pool the clarified medium in an SW 40 tube. Layer
4.5ml of 200g/kg sucrose under the radioactive
medium and 1 ml 550g/kg sucrose under the
- Centrifuge at 143,000g and 4°C for 2h in an L8-M
Beckman centrifuge equipped with a SW 40 rotor
- Connect the ISCO density gradient fractionator to
a peristaltic pump (Pharmacia Pump P1, inner
diameter of tubing = 1.0 mm, speed setting = 2× 10) and a Gilson FC 203B fraction collector. Clamp
the SW 40 tube in the fraction collector, perforate
the tube from the bottom, and collect 20 fractions
of five drops each.
- To measure the radioactivity in each fraction,
pipette 50µl H2O followed by 2µl of the fraction
(delivered into the water droplet) and 3ml
Emulsifier-Safe into twenty 6-ml scintillation vials.
Mix and count using the 35S window in a liquid
- Pool the peak fractions (usually fractions 5-9) and
analyse the virus preparation by SDS-PAGE under nonreducing conditions. Aliquot 25-µl portions
in 1.5-ml Eppendorf tubes, quick freeze in
dry ice/ethanol, and store at -70°C.
- Complete BHK medium and supplemented MEM: see
- Agarose stock solution: To make 100ml, add 1.9g of
Seaplaque, low melting point agarose to 100ml
MEM. Autoclave and store at 4°C.
- BHK-medium + 2x additives: To make 136ml, add
20ml of tryptose phosphate broth, 10ml of FBS,
2ml of 1M HEPES, 2 ml of 200mM glutamine, and
2ml of 10,000U/ml penicillin/10,000 µg/ml
streptomycin to 100 ml of Glasgow-MEM (BHK-21).
Store at 4°C.
- Neutral red (2% stock solution): To make 50ml, add 1.0
g neutral red to 50 ml H2O. Filter through a Whatman
No. 1 paper and store at room temperature.
- Neutral red stain: To make 100 ml, add 3 ml of neutral
red, 2% stock solution to 99 ml of PBS (with Ca2+ and
Mg2+). Use fresh.
- Grow BHK-21 cells to ≈90% confluency on 60-
mm tissue culture plates in complete BHK medium
(≈ 3.4 x 106 cells per plate). Prepare 10 plates per virus
preparation to be titrated and 2 extra plates to be used
as negative and positive controls.
- To make a serial dilution of the virus preparation,
label ten 2.0-ml Eppendorf tubes (1-10) and pipette 445
µl supplemented MEM to the first tube and 1.35ml
supplemented MEM to the following nine tubes. Add
5 µl of the virus preparation to the first tube, mix thoroughly,
and transfer 150 µl of the mixture to the second
tube. Mix the contents of the second tube and transfer
150 µl to the third tube using a fresh pipette tip. Continue
in the same fashion with the last seven tubes.
Make two parallel dilution series for each virus preparation
to be titrated.
- Melt the agarose stock solution in a microwave
oven. Mix 35 ml of the agarose stock solution and 35
ml of the BHK-medium + 2× additives to complete the
overlay solution. Keep in a 37°C water bath until use.
- Remove the medium from the cells and wash
with 2 ml of PBS (with Ca2+ and Mg2+).
- Add 1.0ml of diluted virus (use tubes 6-10 from
the two dilution series; these correspond to dilution
factors 10-7 through 10-11). Use supplemented MEM as a negative control and a suitable dilution of a
known virus stock (if available) as a positive
- Incubate for 60min 37°C and 5% CO2. Tilt the
plates every 20 min to ensure even distribution of virus
- Remove the virus solution from the cells, rinse
with 2 ml of PBS (with Ca2+ and Mg2+), and add 4 ml of
overlay solution (keep the bottle in a beaker filled with
37~ water). Leave the plates at room temperature
until the agarose solidifies.
- Incubate the plates for 48 h at 37°C and 5% CO2.
- Add 3 ml of neutral red stain and incubate for 3
h at 37°C (5% CO2 is optional).
- Score the number of plaques (diffuse, clear areas
on a dark red background) on each plate. To calculate
the virus titre as plaque-forming units per milliliter,
divide the number of plaques per plate by the appropriate
Cells used for SFV infections in this protocol (old
BHK cells) are BHK-21 cells that with time in culture
have transformed further. In doing so, they have lost
the extended form of normal BHK-21 cells (freshly
obtained from ATCC) and appear more like penta- or
hexagons. The SFV strain used [SFV4 (Liljestr6m et al.
1991)] had undergone an unknown number of passages
in the old BHK cells (Glasgow et al.
, 1991) before
it was cloned. At present the specific titre (i.e., the
number of infectious virus particles divided by the
total number of virus particles produced) is approximately
10 times higher when a virus preparation is
titrated on old BHK cells as compared to normal BHK-
21 cells. This is also the case when the virus is produced
in normal BHK-21 cells and most likely reflects
an adaptive change in SFV4 that facilitates entry into
the old BHK cells.
The expected yield of SFV particles is approximately
of confluent BHK cells. When larger
amounts (mg) of SFV are desirable, the amount of
complete BHK medium used in the production step
(Section III,A, step 4) can be reduced down to 20 ml per
An alternative method to estimate the amount of
SFV in a preparation is to use CBB-stained SDS-PAGE
gels and compare the intensity of the capsid protein
band to that of known amounts of BSA ran under
reducing conditions on the same gel (five wells
with 0.15, 0.3, 0.6, 1.2, and 2.4µg, respectively, is
sufficient). The amount of C protein (mc
) in a band is half the amount of BSA in a band of equal intensity.
The amount of SFV (mSFV
) is calculated as mSFV =
(3 × mc)/2.
The stability of the produced SFV is improved if
the producer cells are supplied with cholesterol in
the growth media. This procedure is indicated as
optional in the protocol and is not necessary for the
production of stock virus intended for infection of new
The crude virus preparation obtained in Section
III,A, step 11 can be purified by isopycnic tartrate gradient
centrifugation as described by Haag and collegues
(2002). To this end, cholesterol should be used
during virus production and the TNE used in step 10
should be replaced by TNM
(50mM Tris-HCl, 50mM
, pH 7.4) for improved virus
The E1 and E2 proteins of SFV comigrate upon
SDS-PAGE under reducing conditions. Without
reduction, E1 and E2 are separated readily, with
E2 showing a higher apparent molecular mass than
Intact SFV particles contains 88% (w/w) of protein
and 12% (w/w) of RNA (Garoff et al.
, 1982). This is
equivalent to an A260
ratio of 1.4, provided that A260
of pure RNA equals 2.0 (Glaser, 1995; Manchester,
1995). Deviations from this figure (A260
1.4 ± 0.1) imply that the SFV preparation contains
impurities and/or defective particles.
To maintain high virus quality in successive SFV
preparations, it is important to use a low multiplicity
of infection (MOI). The use of MOI - 0.1 (i.e., 0.1 infectious
particle per cell) or less ensures that the initial
infection is caused by a single virus particle. In this
case, virus particles that carry deletions or other deleterious
mutations in their genomes cannot be rescued
by multiple infection with functional virus particles. If
a high MOI is used in a series of successive infections,
the number of so-called defective interfering (DI) particles
will increase dramatically (Stark and Kennedy,
1978). The presence of high numbers of DI particles
may express itself by low specific infectivity of
the newly produced virus. In single round infections,
such as radiolabelling experiments, a MOI of 5 to 10
can be advantageous as this will produce a synchronised
burst of SFV production in the shortest possible
Avoid repeated freeze/thaw cycles, as this will
reduce virus infectivity.
If SFV infection is carried out in serum-containing
medium, e.g., complete BHK medium, the infectivity
of the particles is reduced dramatically. Without interference,
at maximum 30% complete BHK medium may
be present during infection.
Efficient clarification of the virus containing
medium (Section III,A, steps 5-7) is important. Cell
debris present during virus pelletation (step 8) will
glue the virus particles together and make resuspension
To preserve the three-dimensional structure of the
virus particles, it is important to allow sufficient time
for resuspension. Do not decrease the time that the
virus is left on ice (Section III,A, step 10).
When trace amounts of SFV proteins are separated
on SDS-PAGE, the C protein tends to smear over the
lane. This can be avoided if a small volume of BHK cell
lysate is included in the sample buffer prior to heating
(add 1 µl BHK cell lysate for every 10µl of SDS-PAGE
sample buffer). To make BHK cell lysate, grow BHK-
21 cells to 100% confluency in a 35-mm tissue culture
plate, lyse in 300µl 1× lysis buffer, and remove cell
nuclei by low-speed centrifugation. Store the BHK cell
lysate at -20°C. The addition of cell lysate is not necessary
when the amount of virus protein in the gel is sufficient for Coomassie brilliant blue staining. Silver
staining is not recommended.
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