Have been running an experiment to grow high titre virus that I have repeated twice using the same virus batch, and also repeated the experiment with a different batch of virus i.e. stocks that originate from different sources. I have consistently found that at the point of virus harvesting the cells have detached following the third virus growth passage. This affect also only seems to happen when the virus is grown to concentrate the viral titre by reducing the cell culture media volume from 50ml to 20ml and only after the third passage i.e. the first and second innoculations are fine at 20ml volumes, but the third innoculation results in the above phenomenon of cell detachment. Control flasks are fine at these volumes.
I was wondering if the MOI could theoretically become too high and literally the cells no longer able to bare the viral burden?
Any ideas would be appreciated.
Kindly,
Stephen
Stephen, Did you calculate the virus titer after first and second passage? How long did you incubate the virus-infected cells after using virus from the third passage? Lastly, what was the virus volume did you use for infecting your cell?
Hi Abdou,
Thanks for your response. To answer your first question, No. We are in the process of titrating each passage to see if there has been concentration of virus. We simply followed long standing protocols to do this work with the aim of titrating after the third growth phase. Obviously didn't expect the current CPE!. Each pasage had the same length of incubation, which was four days (standard). Finally the virus volume was 1ml on a T75 flask, which is also consistent with SOP.
Thanks again,
Stephen
Hello Stephen,
I'm cultivating and doing assay with non-virulent virus at least to mammalian cells. It will kill cells if you use high MOI. The mechanism is more because of apoptosis or other immune response rather than cell lysis, because of many cellular responses. Even if the virus contained in culture medium is inactivated (UV), the medium will cause cell death if you passage it at a pretty high concentration to healthy cell. At least I observe it with the virus and cells I'm using.
Thanks for that information it is interesting and not the first time this has been mentioned as a possibility for this cytotoxicity.
Kindly,
Stephen
You could try banding your virus on a gradient or pelleting it with a sucrose cushion (sucrose would be quick) to remove the apoptotic factors you get from serially passaging the virus-containing supernatant.
Hi Stephen, a possibility that has not been mentioned so far is the selection for cytotoxic viruses. By passaging your virus stock on cells, you might be selecting for the viruses that have gained the ability to kill cells owing to their high replication fitness. Secondly, I don't understand the logic for passaging virus 3 times to obtain high titer stocks. Passaging essentially leads to the selection for various mutants, resulting in a mixed virus population. Why not to infect cells with your original stock at a low multiplicity of infection (for example, 0.01), and harvest the medium when all of your cells are infected? As a pilot experiment, you can infect a small number of cells with virus at moi of 0.01, and do IF or flow to determine the time when almost all cells are positive, and then use that time point in your actual experiment to harvest the viruses.
..The first I would do is to (let) check for mycoplasm contamination which can be enriched with your protocol. An indication for contamination may be that the culture mediums gets acidic (unusal yellow culture medium). A general remark: Freqwuently questions do not inform about cells and organisms used. Since universal answers are difficult to give, more Information would be helpful to spead up the process.
All good answers - I would also add that interferon proteins are often produced by cells following exposure to virus. This can be MOI-dependent in both a positive or negative manner depending upon the cells (responsiveness to IFNs) and the virus (and if it produces its own anti-interferon products). If you could provide more info on the cell type and virus/strain I would be glad to provide further advice
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