This is a ready to use oncolytic virus preparation of the 17+ strain of Herpes Simplex Virus Type-1 (HSV-1). The virus encodes green fluorescent protein (GFP) and Sodium Iodide Symporter (NIS). 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 clarified 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).

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 0.01  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 shows cytophathic effects (CPE); usually 2-3 days post-infection.
7. Freeze-thaw the cells and supernatant for 3 cycles.
8. Centrifuge the supernatant at 14,000 x g for 1 h at 4°C.
9. Resuspend in DPBS, aliquot, and store at -80°C. Determine virus titer using an appropriate method.

Infectivity

Vero cells were mock-infected (A&B) or infected with HSV-GFP-NIS (MOI 0.1) (C&D). Photos taken at 72 h.p.i.

NIS Expression

The functionality of the hNIS gene was assessed using an uptake assay; uptake of I-125 by HSV-GFP-NIS infected BHK-21 cells in 6-well plates was measured in the presence or absence of KClO₄, an inhibitor of NIS-mediated I-125 uptake.