Why do my cells not show a cytopathic effect?

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Garglaes's picture
Why do my cells not show a cytopathic effect?

Currently in my lab we use a few cell lines ( Vero 76, MDCK, LLCMK2, HEP2 ) for diagnostics and research of respiratory viruses. We have noticed that out cells are not as receptive towards our respiratory virus samples as they do not show CPE for any of our samples. We performed a test with our viral controls to see if our cells would still demonstrate CPE with known viral titres as well as knowing which cell line was receptive to which virus and therefore know what type of CPE we should be observing. Our results demonstrated that our HEP2 cells infected with RSV started to detach around day 6 and when we performed immunocytochemistry, no fluorescence was noted. For our influenza samples, our MDCK cells demonstrated 3+ CPE around day 9, while previously they were harvested at day 3 with 3+ CPE, and the immunocytochemistry was low around 1-2+ CPE.

Also, for Influenza A we usually see grape-like group of cells as a positive CPE followed by total destruction of the cell monolayer, however, now we see CPE as the cells forming darker cell membranes and slowly losing the inside of the cell, while still remaining in monolayer or sometimes a monolayer with small holes of cells missing. It would seem as if the CPE remains stunted, one can tell the cells are unhappy and you see the darkening of the membranes as small granules if you go on higher magnification, yet they remain stunted and do not have a degradation of the cell monolayer as we usually observe when MDCK cells are infected with Flu A.

We are thawing the same frozen lot of cells at the same passage as we have previously when we infected cells and had no problem with CPE, the same media and the same viral supernatant is used with the same protocol. We thought the problem could be the media and its variables, we use 10% FBS ( same lot as when cells demonstrated CPE ), E - MEM ( again, same lot ), 1% Antibiotic-mycotic, 1% non-essential amino acids, 1% sodium pyruvate. We borrowed some powdered media and prepared it from scratch to see if the media was the variable affecting the cells, as well as obtained a new set of non-essencial amino acids from a different company. Any ideas as to why we have little/different CPE in our cell lines ?

Nikail Collins
Nikail Collins's picture

Is there any way the cell lines are not what you think they are?  If for some reason, the cells have been contaminated with cell lines that aren’t affected by the pathogens you are testing, you wouldn't see the normal CPE. If this is a possibility, there are kits to test your cell lines for "correctness."