VV_(Li)-RlucGFP-mTyr-GusA is a ready to use oncolytic virus preparation of the Lister strain of Vaccinia Virus. The virus encodes a Renilla luciferase (Rluc)-green fluorescent protein (GFP) fusion, murine tyrosinase (mTYR), and β-glucuronidase (GusA). These additional transcription units are inserted into, and disrupt the function of, the viral F14.5L, J2R (thymidine kinase) and A56R (hemagglutinin) genes, respectively (see diagram below). The purchased virus is ready to use for generation of new viral stocks.

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.

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) 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 0.1  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 at 48 hours post-infection.
7. Freeze-thaw or sonicate the supernatant and cells for 3 cycles.
8. Clarify the supernatant by low-speed centrifugation and purify through a  sucrose cushion.
9. Resuspend the viral pellet in 1mM TrisCl, pH 9. Determine virus titer using appropriate methods. Store at -80C.

Infectivity

Vero cells were mock-infected (A-C) or infected with VV(Li)RlucGFP-mTYR-GusA (MOI 0.1) (D-F) and imaged at 48 h.p.i for fluorescence (A-E) or β-glucuronidase (C&F).

Tyrosinase Expression

Vero cells were mock-infected or infected with VV(Li)-RlucGFP-mTYR-GusA at the indicated MOI. After 48 hours, tyrosinase activity was assayed with addition of 50µl of 2mM L-DOPA. Absorbance was measured using a micro plate reader.

Luciferase Expression

 Vero cells were mock-infected or infected with VV(Li)-RlucGFP-mTYR-GusA at an MOI of 0.1 or 1. After 48 hours, 1.25 µg of coelenterazine was added to each of the wells and luminescence (RLU) was immediately measured using a microplate reader.