This is a ready to use virus preparation of the Western Reserve strain of Vaccinia Virus. The virus encodes bacteriophage T7 RNA polymerase for rescue of recombinant viruses from plasmid DNA encoding viral genes driven by a T7 RNA polymerase promoter¹.

The virus titer is functional infectious virus, not total virus particles, in purified supernatant. The titer is determined using an end-point dilution assay that measures the amount of virus required to produce cytopathic effects in 50% of infected cells (tissue culture infective dose per mL).

Note: A USDA permit is required for shipping Vaccinia Virus. Click here to fill out Application Form VS 16-3 to obtain a USDA APHIS VS 16-6 or 16-6A permit.

References:
¹Fuerst et al., PNAS. 1986, 83(21): 8122-8126.

This virus should be handled in a BSL2 facility  by trained personnel following proper biocontainment practices.

Basic protocol
(All volumes are given for a T75 flask; increase or decrease as needed.)
1. Seed producer cells (e.g. Vero or BHK) in complete medium at an appropriate density to achieve 80-90% confluency at the time of infection and incubate in an appropriate incubator.
2. Thaw the virus stock on ice.
3. In a microcentrifuge tube, prepare virus at a MOI of 0.02  in 3 mL total serum free media.
4. Remove culture medium from cells and replace with prepared virus. Return cells to incubator.
5. After 2-3 hours, remove virus innoculum and replace with 12 mL complete medium. Return cells to incubator.
6. Harvest supernatant when 80-90% of cell monolayer is showing cytopathic effects (CPE); usually 2-3 days post-infection.
7. Spin supernatant for 10 minutes at 2000 RPM to remove cell debris. Aliquot virus supernatant and store at -80C. Determine virus titer using an appropriate method.

Cytopathic Effect

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A: Mock-infected BHK-21 cells 48 h post infection.

B: Vaccinia vTF7-3-infected (MOI = 0.05) BHK-21 cells 48 h post infection.

 

Virus Stability

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A stock of Vaccinia vTF7-3 was subjected to repeated freeze (-80^oC) and thaw cycles and the titer after various thaws was determined by TCID₅₀ endpoint dilution assay. The virus was stable for up to 10 freeze-thaw cycles.

 

VSV Rescue

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Vaccinia vTF7-3-infected BHK-21 cells were used to rescue a GFP-expressing VSV. Cell photos were taken 48 h after transfection at 100X magnification.