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Viral Titering-TCID50 Assay Protocol

Viral quantitation determines the number of viruses in a specific volume of fluid. Its utility is most apparent in the production of recombinant proteins or viral vaccines that use viral vectors as a manner for cellular entry or propagation. There are several different approaches to virus quantification; the most noteworthy is the plaque assay, where viral infection causes cell lysis. Then, surrounding cells will become infected and eventually a larger area of the cell monolayer will have been denuded. These areas, known as plaques, can be counted to calculate a plaque forming unit, which is one measure of virus quantity. Although plaque assay is an extremely useful approach for determining viral titers, however, there are several virus types which do not form plaques in culture. Alternative procedures such as TCID50, LD50, EID50 assays are being used to determine the infectious titer of any such virus types, which can produce cytopathic effects (CPE) in tissue culture over a period of 5 to 20 days while cells in culture remain viable.

However, because not all virus types cause CPE in tissue culture, and therefore cell line and virus must be carefully matched in order to see a cytopathic effect. TCID50 is the tissue culture infectious dose defined as that dilution of virus required to infect 50% of the cell monolayers. There are several statistical approaches for analyzing the data generated (such as Probit formula or Spearman-Karber analysis). A TCID50 was found of greatest value when lack of experimental material or costs of analysis precludes extensive replicate titer determinations but good estimates of titers and treatment differences are essential. Relative errors associated with the plaque assay have been shown to be 10–100%, while TCID50 has about a 35% error. As such TCID50 are being widely used for plethora of application both in experimental and diagnostic virology including HIV-1, influenza and human herpes virus.

The workflow of TCID50 assay as follows:

1. Seed culture plate with host cells

Seed 7 x 104 cells per ml in growth media on each well of 48-well plates. Alternatively, another cell density could be required, based on the cell line required for viral growth. Gently rock plates back and forth and from side to side so that cells are distributed evenly. Once cells have been seeded, allow the cells to grow all-night. Next day, cells are visualized under a light microscope to confirm that cells are evenly distributed and reached over 80% confluency.

2. Prepare serial dilutions of viruses

Make a series of dilutions at 1:10 of the original virus sample. Fill the first tube in series with 2.0 ml of PBS, and fill the remaining 6 tubes in series with 1.8 ml of PBS. Make virus sample vortex and then transfer 20 μl of virus to the first tube. Briefly mix original virus stock and make a 1:100 dilution by transferring 20 μl of virus stock to first tubes of each column containing 1.8 ml of medium. Then transfer 200 μl of the diluted virus to the second tubes in the series. Repeat to make a serial 1:10 dilution of the virus, e.g. such as 10-3 through 10-7.

3. Infect monolayer cells

Label lid of 48-well dish by drawing grid lines to define quadruplicates and number each grid to correspond to the virus sample and label the rows of the plate for the dilution that will be plated. Include four negative wells on each plate that will not be infected. Carefully remove all but 0.1 ml of media from each well. Mildly add 0.1 ml of virus dilution per well, infecting 4 wells per dilution, proceeding backwards through the dilutions. Allow virus to adsorb to cells at 37°C for 2 hours (some virus types grow better at 34°C) followed by adding 0.5 ml infection medium to each well and place plates back to the CO2 incubator at 37°C or 34°C for monitoring CPE for one to four week.

4. Visualization and calculation of TCID50

The endpoint is determined when the CPE or immunofluorescence assay (IFA) read-out appear the same per dilution for 3 separate readings. The titer is calculated using the method of Muench and Reed. A titer expressed as 10(3.0) TCID50/0.2 mL in 3 days in XXXX cell line may be translated as: 0.2 mL of virus diluted at 1:1000 will infect 50% of the cells in 3 days when using XXXX cell line.

The TCID50 can be converted to plaque forming units (PFU) through the Poisson distribution. This conversion is an estimate based on the rationale that the limiting dilution, which would infect 50% of the cell layers challenged, would be expected to produce a single plaque in a cell monolayer.

For research use only. Not intended for any clinical use.

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