Animal virus virulence assay
Animal virus virulence assay
Determination of viral infectivity is one of the commonly used methods for assessing its virulence, which is usually done by determining the half lethal dose (50% lethel dose, LD50) in experimental animals and by determining the half tissue (cell) culture infectious dose (50% tissue culture infectious dose, TCID50) in cell cultures to assess the viral infectivity (virulence). Determination of TCID50 in cell cultures is simpler and more economical than LD50 in experimental animals, and it is easy to control the wheeling conditions. In addition, the homogeneity of the cell lines is high, and the sensitivity is more consistent, without genetic differences between individuals. Therefore, this experiment introduces the method of TCID50. Source: Laboratory Microbiology (3rd edition).
Operation method
Method for the determination of TCID50
Principle
The virulence of cytolytic viruses is directly related to their ability to cause cytopathic lesions, and can be determined by inoculating sensitive host cell cultures with different levels of viral fluids. The virulence of a virus in an experiment is determined by the degree of its cytopathic effect (CPE), i.e., the highest and lowest amounts of virus that are observed to cause cytopathic lesions, with half of the cells lesioned as the dose of virus infection. However, what can be observed in the experiment is the qualitative results of cytopathic effect of different dilutions of the virus, and the results need to be statistically processed and calculated using the Reed-Muench formula in order to obtain the relative quantification (titer or potency) of the viral TCID50. For ease of understanding, the calculation of the Reed-Muench formula is presented as an observation of a viral cytopathic lesion. First, the observations in Table 1 are obtained: Table 1 Viral CPE Observations As can be seen from the table, the TCID50 of the virus is between 10-3 and 10-4 dilutions. According to the Reed-Muench formula TCID50 = logarithm of the dilution of the virus above 50% CPE percentage + specific distance × logarithm of the dilution factor (1) in the equation, the specific distance = (above 50% CPE percentage - below 50)/(above 50% CPE percentage - below 50% CPE percentage) (2), the value of Table 1 will be substituted for the (2) in the equation, then the specific distance = ((91.6-50)/(91.5-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) / (91.6-50) 91.6-50)/(91.6-40) =0.8. Then substitute the value of the ratio into (1), lg10-3+0.8×lg10-1=-3.8, then lg TCID50=-3.8, i.e., TCID50=10-3.8. Check the number of objections to get 6310, i.e., 0.1 ml of a 6310-fold dilution of the virus is equal to 1 TCID50.
Materials and Instruments
Influenza virus A Transmissible Canine Kidney Cell Line MDCK (Canis familiaris) Move 1. MDCK cell culture MDCK cells frozen in liquid nitrogen were routinely resuscitated, inoculated into culture flasks, added with 7-10 ml of DMEM culture medium, mixed thoroughly, and incubated at 37 ℃ for 2-3d, until the cells formed a dense monolayer. 2. Cell suspension preparation One bottle of MDCK monolayer cells, discard the supernatant, add 0.25% trypsin 1 ml, digest for 2~5 min, add 3 ml DMEM culture medium after the cells are completely detached from the wall, fully dispersed cells were sampled for microscopic counting, and adjust the concentration of the cells to be 2×105~5×105/ml. Trypsin digestion time should not be too long, otherwise it will cause damage to the fine holders. Beginners can put the cell culture flask under the microscope to observe, the fine run rounded off the wall can be. 3. Cell inoculation Take a piece of 96-well plastic cell culture plate and add 200 ul of cell suspension to each well with a spiker. 4. Cell culture and proliferation The cell culture plate should be incubated at 37℃ with 5% CO2 for 24 h. The cells should grow rapidly and form a monolayer of about 70%, which can be used for virus inoculation. 5. Virus Dilution Make 10-fold serial dilutions of the virus solution in 5 ml sterile test tubes, i.e., use a 1 ml pipette to draw up 0.2 ml of the virus solution and add it to the first small test tube containing 1.8 ml of PBS ( 10-1 ), mix thoroughly, change the pipette and draw up 0.2 ml and add it to the second tube, and then continue to do the same operation, continue to the third dilution, and so on, dilute to the sixth tube. Always add a small amount of trypsin (2.5 ug/ml) to the virus solution to enhance virus adsorption. 6. Virus Infection Take out the 96-well cell plate from the CO2 incubator, discard the supernatant, wash it twice with PBS, add 100 ul of different dilutions of virus to each well, and repeat 8 wells for each dilution. In the control group, 100 ul of PBS was used instead of virus solution, and then 100 ul of fresh DMEM culture medium was added to each well, the total volume was 200 ul. 7. 7. Observe the cell culture plate at 37℃, 5% CO2 incubator for further incubation. Observe the cell lesions with an inverted microscope day by day for at least one week. For more product details, please visit Aladdin Scientific website.
DMEM cell culture medium Trypsin solution Phosphate buffer
96-well cell culture plates 10 to 200 ul Adjustable sampler Sterile test tubes Blood cell counter Inverted microscope General microscope CO2 incubator