The Propagation, Quantification, and Storage of Vesicular Stomatitis (VSV)

Abstract

Vesicular stomatitis virus (VSV) is the prototypical member of the Rhabdoviridae family of negative sense single-stranded RNA viruses. This virus has been used as a powerful model system for decades and is currently being used as a vaccine platform and an oncolytic agent. Here we present methods to propagate, quantitate, and store VSV. We also review the proper safety protocol for the handling of VSV, which is classified as a Biosafety Level 2 pathogen by the United States Centers for Disease Control and Prevention.

INTRODUCTION

Vesicular stomatitis virus (VSV) is an enveloped, non-segmented, negative-sense RNA virus belonging to the Rhabdoviridae family. Its genome (~11-kB in length) encodes five proteins: the surface glycoprotein (G), the matrix protein (M), the nucleocapsid protein (N), the phosphoprotein (P), and the RNA-dependent RNA polymerase (L) (Wagner & Rose, 1996). The virus was first isolated in 1925 and has a broad host range that includes horses, cattle, swine, sand flies, grasshoppers, rodents, and humans. Infected horses, cattle and pigs can develop oral vesicular epithelial lesions (Simon, van Rooijen, & Rose, 2010). VSV has served as a model system and research with this virus has shed light on the infectious cycle of negative-stranded, non-segmented RNA viruses, virus-host interaction, interferon (IFN) susceptibility (Matveeva & Chumakov, 2018), and viral evolution (Wagner & Rose, 1996). It has also served as a tool to illustrate fundamental principles in evolutionary biology and population genetics (Suder, Furuyama, Feldmann, Marzi, & de Wit, 2018).

There is significant interest in developing VSV as a vaccine platform (Medaglini, Harandi, Ottenhoff, Siegrist, & Consortium, 2015). Its success for this application is highlighted by the Ebola rVSV-ZEBOV vaccine, and can be attributed to several factors (Regules et al., 2017). An infectious cDNA clone of this virus is available (Whelan, Ball, Barr, & Wertz, 1995), allowing facile genetic manipulation of the genome to express foreign antigens (Mundell et al., 2015). Human contact with VSV is rare therefore humans lack pre-existing immunity to the virus (Li, Liu, Han, Tang, & Ma, 2020). If a person does become infected, they typically develop mild flu-like symptoms or remain asymptomatic; therefore VSV is considered fairly safe for use in humans (Suder et al., 2018). Finally, VSV is easily propagated in a wide variety of mammalian cell lines on a large-scale basis. VSV is being developed as an anti-HIV therapy, and a recombinant vaccine platform (Monath et al., 2019; Regules et al., 2017).

Many of these factors also make VSV a promising oncolytic agent (Fernandez, Porosnicu, Markovic, & Barber, 2002). Key to this application is the finding that the majority of human tumor cells lack functioning antiviral pathways compared to normal cells (Zhao et al., 2014). This defect leaves cancer cells susceptible to infection with oncolytic viruses like VSV, while healthy cells are spared because they contain functional antiviral responses that limit viral replication (Hastie & Grdzelishvili, 2012).

Here we describe methods required for the generation, purification and storage of VSV virus stocks (Basic Protocol 1) and quantification of VSV by plaque assay (Basic Protocol 2). The protocols described here require proficiency in cell culture techniques (Support Protocols 1).

CAUTION: Vesicular stomatitis virus Indiana strain is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. We recommend that all procedures with virus be performed in a certified biological safety cabinet (BSC). All potential aerosol generating procedures should always be performed in a BSC. Permission for working with the Indiana strain of vesicular stomatitis virus should be obtained. See Burnett et al., 2009 for more information.

NOTE: We recommend taking extra precautions with glass pipette tips and other sharp objects that could puncture the skin through laboratory gloves. A biohazard bin, which should be autoclaved when full, should be placed in the hood for disposal of pipette tips and glass pipettes used with VSV. We also recommend that workers wear a second layer of gloves when working with VSV. The second pair is to be removed prior to exiting the BSC. 70% ethanol can be used as a disinfectant prior to and after working with cell culture and virus. A small amount of bleach should be added to the liquid waste container to inactivate any living cells and to inactivate virus.

BASIC PROTOCOL 1: GENERATION, PURIFICATION, AND STORAGE OF VSV VIRUS STOCKS

VSV can be easily propagated in different mammalian cell lines (e.g., Vero, BHK-21). Here we describe propagation using the Vero cell line; however, this protocol can be modified to suit the use of other cell lines. Various strains of VSV can be purchased from the ATCC website (http://www.atcc.org/en.aspx). The authors recommend titration of any initial VSV stock prior to experimentation (Basic Protocol 2). Once the titer is known, proceed to propagation. Briefly, a confluent monolayer of Vero cells is infected at a low multiplicity of infection (MOI) to reduce the number of defective viral particles (Gélinas, Kiesslich, Gilbert, & Kamen, 2020). Supernatant is collected once the cells are dead, debris removed, and the virus is aliquoted before long-term storage at -80°C.

Materials

• Vero cell line (ATCC Cat. no. CCL-81; see Support Protocol 1)

• Vero culture medium (see recipe in Reagents and Solutions)

• Eagle's Minimum Essential Medium (EMEM), (ATCC, Cat. no 302003)

• DPBS (See recipe in Reagents and Solutions) can be used instead of EMEM alone

• VSV Indiana strain (ATCC, Cat. no VR-1238)

• Cell culture incubator set to 37°C and 5% CO2

• 37°C water bath

• 150 cm² tissue culture flasks

• 15 ml and 50 ml conical tubes

• 2.0 ml cryotubes with O-ring

• Centrifuge

Sample and monolayer preparation

1. Place a bottle of EMEM, and Vero culture medium to warm in a 37°C water bath. Thaw VSV stock solution to be used for infection. All solutions that come into contact with the cells should be previously warmed to 37°C.

2. Begin with six 150-cm² tissue culture flasks that are 90% to 95% confluent (see Support Protocol 1).

Number and size of flasks used may vary based on personal needs.

• 3. Use one of the 150-cm² flasks for cell count (Support Protocol 1).

• 4. Using an MOI of 0.0001 and the calculated cell count, calculate the volume of virus inoculum required:

$$\text{Amount of virus}=\frac{(\#\text{of cells on T}-150)(0.0001)}{\text{titer}}$$

Select a previously titered VSV stock solution. Insert the plaque forming units per ml (PFU/ml) of this stock solution into the equation above, and then solve for volume of inoculum. A low MOI helps limit the development of defective interfering particles.

• 5. Remove and discard growth media from the flasks, and rinse the flasks with 5 to 10 ml DPBS.

Do not apply the DPBS with high pressure directly to the cells as this may cause the monolayer to disassociate from the culture flask. Gently add the DPBS down the neck of the flask and rock backward and forward and left to right for 1 to 3 min to aid in the removal of cellular debris and excess media.

• 6. Remove and discard the wash solution.

Infection of the monolayer

• 7. Bring the volume of each flask to 10.5 ml with EMEM without fetal bovine serum (FBS) after adding the volume of virus stock solution required (step 4).

• 8. Incubate the flask at 37°C and 5% CO₂ for 1 hour. Rock the flasks gently every 15 minutes to spread the viral inoculum evenly across the monolayer.

• 9. After the 1-hour adsorption period, add 21 ml of Vero culture medium to each flask.

• 10. Incubate the flask at 37°C and 5% CO2 for ~48–72 hours. Stop incubation once cytopathic effect is observed (cell rounding that is associated with the induction of apoptosis) and all the cells are dead.

Harvesting VSV

• 11. After the incubation period transfer the media to 50 ml conical tubes, and centrifuge 10 minutes at 2,900 × g at 4°C.

• 12. Remove and pool the supernatant; mix thoroughly. Set aside 200 μl for determination of viral titer by plaque titration assay (Basic Protocol 2).

• 13. Aliquot pooled supernatant in 1 ml increments into 2.0 ml cryotubes, or in 50 ml tubes.

• Do not fill the tubes completely as the liquid will expand upon freezing.

• 14. Store samples at −80°C until further use or proceed to virus purification if require (Hastie et al., 2013).

BASIC PROTOCOL 2: QUANTIFICATION OF VSV BY PLAQUE ASSAY

The plaque assay is the gold standard for quantification of viral stock solutions and virus containing samples (Baer & Kehn-Hall, 2014). The assay described here is applicable to determining the titer of VSV stocks or to quantitate the amount of infectious virus in the supernatant of infected cells (e.g. growth curve samples). This protocol utilizes an agarose overlay that is added to the infected cell monolayers after adsorption, which should be prepared before experimentation.

Materials

• Vero cell line (ATCC, Cat. no. CCL-81; see Support Protocol 1)

• Vero culture medium (see recipe in Reagents and Solutions)

• Eagle's Minimum Essential Medium (EMEM), (ATCC, Cat. no 302003)

• Dulbecco’s phosphate-buffered saline (DPBS; see recipe in Reagents and Solutions)

• FBS (ATCC, Cat. no 302003)

• 4% agarose (see recipe in Reagents and Solutions)

• Agarose/EMEM overlay (see recipe in Reagents and Solutions)

• VSV stock samples to be assayed

• Methanol:acetic acid fix solution (see recipe in Reagents and Solutions)

• Crystal violet stain (see recipe in Reagents and Solutions)

• Cell culture incubator set to 37°C and 5% CO2

• 65°C and 48°C water baths

• Microwave oven

• 6-well tissue culture plates

• Sterile microfuge tubes

• 50 ml conical tubes

• Sink strainer

Seeding well plates and sample preparation

1. If the plaque assay is to be done the next day, seed ~8.5 x 10^5 Vero cells per well of a 6-well plate. We routinely use 2, 6 well plates (12 wells total) per virus stock to be titered. Do not swirl the plates, otherwise cell density will be lower in the center of the well. We recommend gently rocking the plates left and right and front and back instead. Allow cells to grow for 24 hours.

The cell plating density may need to be adjusted. For example, fewer cells should be passed into each well if the plaque assay will be done two or three days after cell passage. The goal is to obtain 90–95% confluency but not an overgrown monolayer of cells by the time of infection.

• 2. If quantitating a VSV stock, a titer of approximately 10⁹ PFU/ml is expected and we recommend the serial dilution series shown in Table 1. Label 7 sterile microcentrifuge tubes, and set up the dilution series suggested in Table 1 below. Make sure to mix tubes well and use a new pipette tip for each transfer.

Table 1:

Suggested dilutions series for a VSV stock with an estimated titer of 10⁹ PFU/ml.

Dilution #	μl virus from previous dilution	μl media	Total dilution	Expected # plaques
1	10 μl of virus stk	990	1X10−2
2	10 μl from tube 1	990	1X10−4
3	50 μl from tube 2	450	1X10−5	1000
4	50 μl from tube 3	450	1X10−6	100
5	250 μl from tube 4	250	5X10−8	50
6	250 μl from tube 5	250	2.5X10−8	25
7	250 μl from tube 6	250	1.25X10−8	13

See Anticipated Results for recommendations when using unknown concentrations of VSV harvested directly from infected cell culture lysates. Never make more than a 100-fold dilution and always pipette at least 10 µl of virus or solution to reduce pipetting error.

• 3. Remove medium in the 6-well plates using a sterile Pasteur pipette.

Steps 3 through 5 should be done quickly to make sure the cells do not dry out.

Infection of the monolayer

• 4. Add 100 μl EMEM to the first top and first bottom well of each plate as an uninfected control or mock (See Fig. 1).

• 5. Add 100 μl of the indicated dilution dropwise to each well.

Figure 1.

Plaque assay 6-well plate setup.

See Figure 1 for suggested plate setup. This setup saves time and serological pipet tips when working from highest dilution factor to lowest. Work quickly to avoid the cells from drying out.

• 6. Adsorption: incubate the plates at 37°C for 1 hour to allow the virus to infect the cells. Rock the plates every 15 minutes to ensure optimal distribution of the virus and cell coverage.

• 7. During adsorption, prepare the agarose/EMEM overlay (see Reagents and Solutions).

Addition of the overlay

• 8. Remove adsorption mix and add 2 ml of the agarose/EMEM overlay to each well, pipetting carefully down the side of the well.

Be careful to use a new pipette to remove the adsorption from the mock wells. DO NOT swirl after addition of agarose to well and avoid bubbles.

• 9. Allow the agarose to solidify for 15 minutes in the biosafety cabinet or on the lab bench.

• 10. Move the plates to the incubator and incubate for 24–48 hours at 37°C

Prior to staining or fixing the cells, plaques should be counted once to ensure accuracy of number of plaques after staining as the cells can come off while removing the agarose overlay (See Critical Parameters and Troubleshooting).

Fixing and Staining

• 11. To fix cells through the agarose overlay, add 0.5 ml per well of Methanol:Acetic Acid fix solution.

The agarose should turn yellow.

• 12. Incubate at room temperature for 30 minutes.

At this point, the cells are fixed and can be handled outside of a biosafety hood.

• 13. To remove the agarose overlay, use gently flowing tap warm water and direct the stream to the side of the well while tilting the plate. Collect the overlays into a sink strainer to prevent them from going down the drain and discard safely.

• 14. Invert the plates and allow the monolayers to dry for approximately 5 minutes.

• 15. Once dry, add 100 μl of the crystal violet stain to each well. Stain for 30 minutes on an orbital shaker.

• 16. After staining, gently rinse each well with water. Invert to allow the monolayers to dry.

Estimation of viral titer

• 17. Select the dilution that produced 10–100 plaques and re-count the number of plaques for each replicate.

See Figure 2 for an example of the results of plaque assay performed using the suggested dilutions in Table 1.

Figure 2:

Image of a plaque assay showing the dilutions giving rise to different numbers of plaques. Note: These plaques are large because they were allowed to develop for 3 days to ensure that they were visible in the figure.

• 18. For each sample, calculate the average number of plaques for that dilution.

• 19. Calculate the plaque forming units per ml (PFU/ml) for each sample:

$$\text{virus tier}(\text{PF}/\text{ml})=\frac{\text{average number of plaques}}{\left(\text{dilution factor of well}\right)\left(\text{volume of inoculum per plate}\right)}$$

• 20. Optional: Take photos of the results and store the plates in the dark at room temperature.

SUPPORT PROTOCOL 1: PROPAGATION OF VERO CELLS

Originally derived from kidney cells of the African green monkey (Cercopithecus aethiops), Vero cells are widely used as host cells for growing viruses and creation of viral stocks. After reaching confluency, Vero cells stop growing due to contact inhibition (Agbulos, Barelli, Giordano, & Hunter, 2016). Thus, daily monitoring of cell development and subculturing before the cells reach 100% confluency is critical for keeping healthy Vero cells. Subculturing can be done every 3 to 4 days. After dissociation, cell suspensions are either transferred to a new flask or seeded into n-well plates based on the experiment plan, keeping record of the passage number. We typically discard cells once they have been passaged 10 times. For cryopreservation, a complete culture medium consisting of Eagle’s Minimum Essential Medium (EMEM) with 10% Fetal Bovine Serum (FBS) is supplemented with 5% (v/v) Dimethyl Sulfoxide (DMSO).

Materials:

• Vero cell line (ATCC Cat. no CCL81)

• DPBS (see recipe in Reagents and Solutions)

• Vero culture medium (see recipe in Reagents and Solutions)

• 0.25% trypsin/EDTA (e.g., Invitrogen, cat. no. 25200-072)

• Cell culture incubator set to 37°C and 5% CO₂

• 37°C water bath

• 75-cm² tissue culture-treated flasks (sizes vary with personal needs)

Media and reagent preparation

1. Begin with a 75-cm² flask that is 80% to 90% confluent.

2. Warm DPBS, Vero culture medium, and 0.25% trypsin/EDTA to 37°C in a water bath.

All solutions that come into contact with the cells should be previously warmed to 37°C.

• 3. Remove cultural media and rinse the flask with 5 to 10 ml of DPBS.

DPBS should be applied gently down the flask neck to avoid dissociation of the monolayer from the flask.

Cell dissociation

• 4. Add 2 to 3 ml of 0.25% Trypsin/EDTA directly to the monolayer.

Gently rock the flask backward and forward and left to right so that the trypsin covers the entire monolayer.

• 5. Incubate the flask at 37°C and 5% CO₂ for 2 to 4 minutes.

Do not incubate for longer than 5 minutes.

• 6. After incubation, vigorously rock the flask from side to side.

Check to see if the monolayer has been removed from the surface of the flask; the bottom of the flask should no longer be opaque.

Cell resuspension and subculturing

• 7. Once the cells have disassociated from the flask immediately add Vero culture media up to a total of 10 ml into the flask.

• 8. Pipet up and down to vigorously dispense the cell-media solution where the monolayer used to be in order to dislodge any remaining cells.

• 9. For a 1:10 dilution add 1 ml of the cell suspension to a new 75-cm² flask.

A 1:10 dilution should take 3 to 4 days to reach 80% to 90% confluence in a 75-cm² flask.

• 10. Add 10 to 15 ml of warmed Vero culture media to the culture flask.

• 11. Incubate the culture flask at 37°C and 5% CO₂.

• 12. Monitor cell growth daily.

• 13. When cells reach an 80% to 90% confluent monolayer proceed to step 2.

REAGENTS AND SOLUTIONS:

Use deionized, distilled water (dH₂O) in all recipes and protocol steps.

Vero culture medium (EMEM + 10% FBS)

• 50 ml fetal bovine serum (FBS) (ATCC Cat. no. 30-2020)

• 450 ml Eagle's Minimal Essential Medium (EMEM) (e.g., ATCC, Cat no. 30-2003)

• Store at 4°C

Note: We do not routinely add antibiotics to our medium; however, they can be added based on personal preference. For example penicillin/streptomycin/L-glutamine (e.g., ThermoFisher, cat. no. 10378016).

Dulbecco’s phosphate-buffered saline (DPBS)

• Obtain a 1x DPBS solution (see below or purchase). Filter sterilize (0.2 μm) and autoclave at 121°C for 20 minutes. Store at room temperature for up to 6 months.

To make a 20x DPBS solution:

• Add to a 1L beaker: 160 g NaCl, 4 g KCl, 4 g KH₂PO₄, and 23 g Na₂HPO₄.

• Add to the beaker 800 ml of deionized water. Stir until dissolved.

• Make sure the pH is 7.4 and adjust with HCl or NaOH.

4% Agarose solution

• Weigh out 20g of powdered agarose and bring up to 500 ml dH₂O in a glass bottle.Autoclave the solution at 121°C for 20 minutes.

Note: Agarose can burn in the autoclave, so do not leave the agarose solution in a hot autoclave overnight.

• Agarose may be stored on the shelf at room temperature or used immediately after equilibrating in a 65°C water bath.

• We typically store 100 ml aliquots of solidified agarose, which can be melted in the microwave for about 1 minute.

Agarose/EMEM overlay (0.8X EMEM, 2% serum, 0.5% agarose)

• Prepare the following in a sterile 50 ml conical tube. This is enough to add a 2 ml overlay to two, 6-well plates (or 12 wells total):

• 23.9 ml EMEM

• 0.6 ml FBS (ATCC Cat. no. 30-2020)

• Equilibrate to 48°C

• Melt the 4% agarose solution in a microwave

• Once equilibrated to 65°C, add 3.5 ml of the 4% agarose to the EMEM with FBS prepared above.

• Mix well and keep this solution at 48°C to prevent solidification until use.

Methanol:Acetic acid fix solution (3:1)

• Add 30 ml absolute methanol into a bottle

• Carefully 10 ml glacial acetic acid into the methanol

• Store in a flammable cabinet at room temperature

Crystal violet stain (0.2% crystal violet in 20% ethanol and dH2O)

• Start with 0.1 g of crystal violet powder (Sigma-Aldrich)

• Add 40 ml of distilled water to dissolve the powder in

• Add 10 ml of methanol

• The solution should be stored in the dark at room temperature and used within 2 months.

COMMENTARY

Background Information:

Propagation of VSV virus in vitro

VSV can be easily propagated in many mammalian cell lines; however, Vero and BHK-21 (Hastie et al., 2013) are often used. Propagation of virus in cell culture is performed to increase the viral titer and to maintain adequate volumes of low passage stock solutions.

Quantification of VSV by plaque assay

The plaque assay is considered the gold standard for detection and quantification of a wide variety of viruses. Modifications of the assay we describe here are possible based on personal choice, and may include the choice of culture medium, final FBS concentration, final percent agarose in the overlay, and the addition of antibiotics or antifungal components to the growth medium. The authors prefer to use an agarose overlay during the plaque formation incubation period; however, this method does have limitations. If the liquid agarose overlay is too hot when added, it can damage the cells. Conversely, if the liquid agarose temperature drops below 35°C, it will begin to solidify. In our hands, however, the use of well-monitored water baths and timely addition of the overlay eliminates these issues. Due to these drawbacks, many researchers prefer to use a carboxymethylcellulose (CMC)-based solution to overlay the infected cell monolayers instead of agarose. CMC is semi-solid at room temperature therefore this method avoids the temperature constraints described above when agarose is used. We find that the plaques generated with the CMC overlay are less well-defined and are therefore more difficult to count. Also, the plaques can appear smeared or comet-like if the overlay is disturbed during the plaque formation incubation period. Baer and Kehn-Hall compare different plaque assay overlays in their article (Baer & Kehn-Hall, 2014).

Critical Parameters and Troubleshooting:

We have found that VSV is easily propagated in the laboratory using standard cell culture practices. Here we describe the protocols we have developed that yield optimal results.

Generation and purification of VSV stocks

The generation of high-quality virus stocks is dependent on the cells used for propagation and the virus itself. It is important to infect cells that are close to confluent (>90%). It is extremely important to use a low (0.005 to 0.0001) MOI to reduce the presence of defective interfering particles and ensure generation of infective virus. Use of cells that have undergone multiple passages does not appear to affect viral propagation. It is crucial to use low-passage virus stocks when generating a new virus stock because the use of virus that has been serially passaged can result in the accumulation of genetic mutations. Therefore it is recommended to keep a single parental virus stock that is used to create subsequent virus stocks.

Quantification of VSV by plaque assay

Care must be taken to prevent damaging the cell monolayer during this assay. For example, it is important not to touch the monolayer with the pipette when removing solutions from cells. The authors recommend tilting the plate at a 30° angle when removing media and wash solutions. Likewise, it is important that addition of solutions to the cells be done gently and not directly onto the monolayer by directing the pipette to the side of the well/flask.

Another common error that can occur during this assay is leaving the cells uncovered (without media or DPBS) for too long. This can result in the cells drying out and dying. This is particularly common when multiple plates are treated at once. To avoid this error, it is important to work quickly and to break the plates down into groups of 4 to avoid drying out the monolayer. The authors also suggest minimizing the amount of time the lid is open to reduce cell exposure.

Researchers must also optimize the time post infection to ensure that the plaques are large enough to visualize, yet not so large that they merge together - making them difficult to count. Usually the plaques generated by VSV under the conditions described herein are a good size to be counted. It is possible that certain mutant strains result in smaller or larger plaques, therefore the optimal time post infection may vary. We do not recommend incubation periods of longer than 3 days for optimal plaque counts as Vero cells are contact-inhibited and the monolayer will start to break down.

The authors recommend counting the plaques before the cells are fixed and stained, in case the monolayer is damaged when the agarose overlay is removed. If the plaques are difficult to see, we recommend holding the plates up at a 45° angle into a light source. We place a dot on the bottom of the plate to keep track of each plaque as it is counted. We recommend writing the number of plaques counted on the bottom of the well. The plaques are then recounted after the cells are fixed and stained, and any plaques there were not initially counted are added to the initial tally.

We recommend the use of warm tap water and medium water pressure to remove the agarose overlay and staining solution. These conditions do not mechanically damage the fixed monolayer or cause cells to dissociate with the plate.

Statistical Analysis:

If 6-well plates are used, it is recommended to count wells containing ~10-100 plaques. We recommend doing each dilution in duplicates so that the average number of plaques can be calculated and used to determine the titer.

Anticipated Results:

Generation and purification of VSV stocks

Typically, non-purified VSV stocks generated on Vero cells will reach titers of 10⁹ PFU/ml.

Quantification of VSV by plaque assay

Basic Protocol 2 is an efficient way to determine the titer (PFU/ml) of a VSV stock. Plaques can usually be observed as early as 1 day post infection but will be quite small. For best results, we recommend a post-infection incubation period of no longer than 3 days.

The serial dilution series shown in Table 1 was designed to quantitate VSV stocks with an estimated titer of 10⁹ PFU/ml. The virus is diluted 1:2 in dilutions 5, 6 and 7 such that several wells should be in the countable range (10-100 plaques). If quantitating unknown concentrations of virus harvested from infected cell lysates (e.g. growth step experiment lysates) the titer of the virus may be much lower, therefore it may be necessary to modify the serial dilution series. It is possible to use 1:10 or 1:100 serial dilutions to determine an estimated titer. The plaque assay can then be repeated using a narrower range of dilutions to generate a more accurate titer.

Time Considerations

Generation and purification of VSV stocks

Preparation time for Basic Protocol 1 can take 3 to 4 days because 90% to 95% confluent flasks are required. Cytopathic effect is generally observed 40 to 48 hours post-infection; depending on the MOI used.

Quantification of VSV by plaque assay

This plaque assay takes a total of 2 to 3 days, not including passage of the cells before the assay. Well plates are to be seeded 1 to 3 days before Basic Protocol 2 is initiated so the cells have time to adhere to the well plates and replicate to the desired confluency.

A number of solutions and reagents should be prepared in advance. For example, the 4% agarose solution should be prepared in advance as it must be autoclaved and aliquoted before use.

Preparation time for Basic Protocol 2 on the day plaque assay is performed can take 1 to 2 hours. Therefore, it is important to schedule sufficient time to prepare the virus dilutions, label the wells, and equilibrate the media and melted agarose to the proper temperature.

Minimal preparation is required for the day of fixation and staining. We recommend that the wells be stained with the crystal violet stain for at least 30 minutes. Turning the lights off reduces degradation of the crystal violet stain. After the crystal violet stain has been removed and rinsed in the sink, invert the plates and dry for 4 to 6 hours or overnight.

Propagation of Vero cells

Typically Vero cells require two to three passages to reach their regular growth rate after being thawed. Preparation time for Support Protocol 1 can take 30 to 60 minutes. Vero cells should be passaged every 3 to 4 days or before reaching 80% to 90% confluency.