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Virus Titration

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Introduction
Tissue culture, is the removal of cells from an animal or a plant, and growing the cells in a favourable artificial condition (Wiley, J.M et al, 2013). There are three types of cell culture: primary, this is cultivating the cells away from the parental tissue, secondary, which is when a primary culture is sub-cultured and cell lines, which can be continuous or finite depending on the life span of the culture. Virus titrations are used to estimate the virus concentration; it is a viral quantification technique. When detecting the virus, the cytopathic effect is looked at, whether there is lysis of the cells, vacuolation, formation of syncytia and the presence of inclusion bodies. TCID50, is the measure of the infectious titre. The end point dilution assay quantifies the amount of virus that is required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells (Kumar P, 2013). The purpose of the virus titration within tissue culture is to isolate and identify viruses within clinical samples, to carry out research on the viral structure, replication, genetics of the virus and the effect on the host cells, and also to prepare viruses for vaccine production.

Results
Table 1 Data used to determine the 50% endpoint using the Reed-Muench method Log of virus dilution | Infected test units | Cumulative infected (A) | Cumulative non-infected (B) | Ratio A/(A+B) | Percentage infected (%) | -1 | 5/5 | 37 | 0 | 38/38 | 100 | -2 | 5/5 | 32 | 0 | 33/33 | 100 | -3 | 5/5 | 27 | 0 | 28/28 | 100 | -4 | 5/5 | 22 | 0 | 23/23 | 100 | -5 | 5/5 | 17 | 0 | 18/18 | 100 | -6 | 4/5 | 12 | 1 | 12/13 | 92 | -7 | 3/5 | 8 | 3 | 8/11 | 72 | -8 | 2/5 | 5 | 6 | 5/11 | 45 | -9 | 2/5 | 3 | 9 | 3/12 | 25 | -10 | 2/5 | 1 | 13 | 1/14 | 7 |

Figure 1 The percentage of infected cells by the virus. As the log of virus dilation decreases the less cells are infected. The dashed line that is on the graph represents the 50% endpoint. The log of virus dilution that infected 50% of inoculated cell cultures. ID50 = 10-7.8

The Reed Muench method was used to obtain the results. The Reed-Muench method was used to determine the 50% endpoint in this experiment. From Table 1 the 50% end point can be calculated, quantifying the concentration of the virus required to produce a cytopathic effect in 50% of inoculated tissue culture cells.
Table 1 shows that the 50% end point lies between the log virus dilutions 10-7 and 10-8. From the results obtained, it can be seen that the 50% endpoint lies between the log virus dilutions 10-7 and 10-8. From this, the proportionate distance was able to be calculated. It was calculated by: (% positive above 50% -50%)/ (% positive above 50%) (% positive above 50%), the result obtained from this was 0.814 as the proportionate distance. This result, allowed the logID50 to be calculated, which was 10-7.8. This is the infective dose, which is the dose of the virus that will infect 50% of the cultures inoculated. The results in table 1 show that with the highest virus dilution, infected 100% of the test units, from the log virus dilutions 10-1 to 10-5, 100% of test units were infected. However, as the virus concentration decreased, the percentage of infected cells also decreased. As with the lowest log virus dilution of 10-10, the percentage of infected test units was 7%. Figure 1, suggests that the lower log virus dilution the smaller the number of inoculated cells are infected. A cell count was carried out before the culture media was incubated. From looking at the haemocytometer under the electron microscope, calculations were carried out: Total cells/ml = Total cells counted x (dilution factor/number of squares) x 10,000 cells/ml Total cells/ml = 15 x (0.1/4) x 10,000 = 3750 cells/ml Discussion
One other technique that can be used instead of the end point dilution assay method, is a the hemagglutination assay. This is an indirect way of counting viruses. When there is a large enough virus to cell ratio, the virus particles join together i.e. they agglutinate. When carried out, red blood cells are mixed with diluted samples of the virus, and then each mixture is examined (Wiley, J.M et al, 2013). The advantages of the hemagglutination assay is that it is an accurate method for determining the relative quantity of viruses, it is also a quick method as it can be performed within 30 minutes, unlike the virus titration which takes days for results to show. Hemagglutination assays are very inexpensive to carry out, the equipment used is readily available, and it is also a simple process to carry out. However, the end point dilution assay method, there is a higher risk of contamination, as the assay is left for longer than the hemagglutination assay which results can be seen within 30 minutes (Kumar P, 2013). Also the determination of the cytopathic effect is subjective, as the infected cells can be difficult to visualise, where as the hemagglutination assay the results are quite conclusive (Bradley C. Long et al, 2004).

References

Bradley C. Long, Tony L. Goldberg, Sabrina L. Swenson, Gene Erickson, Gail Scherba (2004) Adaptation and limitations of established hemagglutination inhibition assays for the detection of porcine anti–swine influenza virus H1N2 antibodies

Kumar, P. (2013) Methods for Rapid Virus Identification and Quantification

Smither SJ, Lear-Rooney C, Biggins J, Pettitt J, Lever MS, Olinger GG Jr. (2013) Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity.

Willey, J.M., Sherwood, L. and Woolverton, C. (2014) Prescott’s microbiology. 9th edition. New York: McGraw Hill Higher Education

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