After viral infection of a cell, the released virus can only expand from the initially infected cell to the periphery due to the limitations of the solid medium. After several cycles of proliferation, a confined area of diseased cells is formed, which is known as a viral plaque. Theoretically, an eclosion is formed by a single viral particle from the initially infected cell, and thus the technique is commonly used for counting viral particles and isolating viral clones. However, in practice, several virus particles often infect a cell at the same time, affecting the accuracy of the titration and the purity of the clone, for this reason, the inoculated virus solution should be fully dispersed and diluted. For cell-bound viruses, such as MDV, a monolayer of cells is required; for cell-released viruses, either cells suspended in solid-phase medium or monolayers of cells can be used, but the latter need to be covered with a solid medium such as agar on the cells to prevent the release of the virus from flowing in the liquid medium. The concentration of the solid medium is determined by the size of the virus, with a lower concentration medium for large viruses and a higher concentration medium for small viruses in order to keep the growth rate of the etchings within a suitable range. Small etchings need to be observed with a microscope, 1-10mm large etchings can be counted with the naked eye. In order to facilitate visual observation, dyes such as neutral red are commonly used for staining. Because the lesion cells do not absorb neutral red, the lesion cell area shows colorless etchings.
Appliance
Viral biological purification; etch reduction neutralization assay
Operation method
Titration or purification of Akabane disease virus (AKV)
Materials and Instruments
[Materials] Virus, target cells.
[Reagent] Agar, neutral red dye
[Instruments] MicroscopeMove
(1) Green monkey kidney cells (Vero) were cultured in sterilized plastic dishes with a diameter of 55 mm to form a monolayer. The cell culture time was about 3-4 days, and the inoculum volume was about 2,000,000 cells/ml. Select a dish that is fully covered with monolayers of cells, leaving no empty space, for the test.(2) Dilute the AKV virus 10-fold to 10-7 with the cell culture solution and store at 4℃.(3) Inoculate 3 dishes with each dilution of virus solution.Caveat
The titer of the virus suspension is expressed in units of etch formation per milliliter (PFU/ml). For example, if the average etch of 3 cell vials is 58, the inoculum volume is 0.2 ml, and the dilution of the virus is 2.5 × 103 , the titer of the viral stock solution is:58 ÷ 0.2 × 2.5 × 103 = 7.25 × 105 (PFU/ml)
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