Growth of Semliki Forest Virus

I. INTRODUCTION
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 × 10³kDa). 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., 1982; Strauss and Strauss, 1994). This article provides protocols to grow SFV in intermediate scale (up to ≈1.5 mg; protocol A) and small scale (³⁵S-methionine labelled; protocol B).

II. MATERIALS AND INSTRUMENTATION
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. 15630), L-glutamine 200mM (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⁺ and Mg²⁺ (Cat. No. 14040) are from GIBCO BRL. Sea-plaque agarose (Cat. No. 50100) is from FMC Bio Products. Redivue-[³⁵S]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.


III. PROCEDURES
A. Growth of SFV
Solutions

1. 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.
2. 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.
3. 10mg/ml cholesterol stock solution (optional): Add 50mg cholesterol to 5 ml 99.5% ethanol. Dissolve. Aliquot and store at -20°C.
4. 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.
5. 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.
6. 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 temperature.
7. 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 room temperature.
8. 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.

Steps

1. 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/ bottle).
2. Dilute the virus to a concentration of 1.1 × 10⁶ pfu/ml in supplemented MEM.
3. 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.
4. 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.
5. 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).
6. Pipette the medium to a fresh tube, without disturbing the pellet, and centrifuge as in step 5.
7. Repeat step 6.
8. 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 (25,000 rpm).
9. 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.
10. Add 200 µl TNE, cover the tube with Parafilm, and leave on ice for 15 h.
11. 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.
12. 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 (C_SFV) if the preparation is sufficiently pure; C_SFV = A₂₆₀/(D × 8) [mg/ml].
13. Analyse the virus preparation by SDS-PAGE under nonreducing conditions and visualise the bands by Coomassie brilliant blue staining.
14. Aliquot the virus suspension in smaller portions, quick freeze in dry ice/ethanol, and store at -70°C.

B. Growth of ³⁵S-Methionine-Labelled SFV
Solutions

1. 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.
2. Labelling medium: To make 7.5 ml, add 50 µl Redivue [³⁵S]methionine (370MBq/ml) to 7.5ml starvation medium.
3. 550g/kg sucrose: To make 50 g, weigh 27.5 g sucrose and adjust to 50 g with TNE. Store at -20°C.
4. Complete BHK medium, supplemented MEM, and 200g/kg sucrose: see Section III,A.

Steps

1. Grow BHK-21 cells to 100% confluency in a 75-cm² tissue culture bottle in complete BHK medium (≈ 1 × 10⁷ cells/bottle).
2. Dilute the virus to a concentration of 1 × 10⁸ plaque-forming units (pfu)/ml in supplemented MEM.
3. 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 particles.
4. Remove virus solution, add 15ml complete BHK medium, and incubate for 3.5h at 37°C and 5% CO₂.
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.
6. Remove the starvation medium, add 7.5ml of labelling medium, and continue incubation at 37°C and 5% CO₂ for 15-16 h.
7. Harvest the labelling medium and dispense in four 2-ml Eppendorf tubes.
8. Centrifuge at 12,400g and 4°C for 5min in an Eppendorf 5416 centrifuge equipped with a 16 F24-11 rotor (11,000rpm).
9. Transfer the supernatants into new tubes, without disturbing the pellet, and repeat centrifugation as in step 8.
10. Repeat step 9.
11. 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 lighter sucrose.
12. Centrifuge at 143,000g and 4°C for 2h in an L8-M Beckman centrifuge equipped with a SW 40 rotor (30,000 rpm).
13. 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.
14. 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 scintillation counter.
15. 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.

C. Quantitation of Infectious Virus Particles by Plaque Titration
Solutions

1. Complete BHK medium and supplemented MEM: see Section III,A.
2. Agarose stock solution: To make 100ml, add 1.9g of Seaplaque, low melting point agarose to 100ml MEM. Autoclave and store at 4°C.
3. 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.
4. 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.
5. 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.

Steps

1. 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.
2. 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.
3. 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.
4. Remove the medium from the cells and wash with 2 ml of PBS (with Ca²⁺ and Mg²⁺).
5. 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 control.
6. Incubate for 60min 37°C and 5% CO₂. Tilt the plates every 20 min to ensure even distribution of virus particles.
7. 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.
8. Incubate the plates for 48 h at 37°C and 5% CO₂.
9. Add 3 ml of neutral red stain and incubate for 3 h at 37°C (5% CO₂ is optional).
10. 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 dilution factor.

IV. COMMENTS
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 1 µg/cm² 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 162-cm² bottle.

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 (m_c) in a band is half the amount of BSA in a band of equal intensity. The amount of SFV (m_SFV) 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 cells.

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 NaCl, 10mM MgCl₂, pH 7.4) for improved virus stability.

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 El.

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/A₂₈₀ ratio of 1.4, provided that A₂₆₀/A₂₈₀ of pure RNA equals 2.0 (Glaser, 1995; Manchester, 1995). Deviations from this figure (A₂₆₀/A₂₈₀= 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 time.

V. PITFALLS
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 difficult.

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.

References
Garoff, H., Kondor-Koch, C., and Riedel, H. (1982). Structure and assembly of alphaviruses. Curr. Top. Microbiol. Immunol. 99, 1-50.

Glaser, J. A. (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. Biotechniques 18, 62-63.

Glasgow, G. M., Sheahan, B. J., Atkins, G. J., Wahlberg, J. M., Salminen, A., and Liljestr6m, P. (1991). Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice. Virology 185, 741-748.

Haag, L., Garoff, H., Xing, L., Hammar, L., Kan, S.-T., and Cheng, R. H. (2002). Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode. EMBO J. 21, 255-264.

Liljestr6m, P., Lusa, S., Huylebroeck, D., and Garoff, H. (1991). In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: The 6000-molecular-weight membrane protein modulates virus release. J. Virol. 65, 4107-4113.

Manchester, K. L. (1995). Value of A₂₆₀/A₂₈₀ ratios for measurement of purity of nucleic acid. Biotechniques 19, 208-210.

Stark, C., and Kennedy, S. I. T. (1978). The generation and propagation of defective-interfering particles of Semliki Forest virus in different cell types. Virology 89, 285-299.

Strauss, J. H., and Strauss, E. G. (1994). The alpha viruses: Gene expression, replication and evolution. Microbiol. Rev. 58, 491-562.