Hallo all,
I am engaged in a project which involves growing the SARS-CoV-2 virus in cell cultures.
We use Vero E6 cells to amplify SARS-CoV-2. The idea is to produce a collection of viral isolates from local patients.
At present we have only one viral isolate, which was produced locally, obtained from a local patient. The nature of the virus (that it is SARS-CoV-2) was confirmed by PCR on RNA isolated from cell culture supernatants. This cannot be a false positive because 4 different primer pairs were used, and all 4 primer pairs produced expected products whereas negative controls were truly negative.
My question is: why this virus does not kill all cells in a monolayer?
I mean, we see CPE in infected cultures, which looks like a fraction of cells (~20%) die and detach from support, but the majority of cells still remain attached and continue growing. Infected cultures continue to proliferate and eventually become overconfluent.
We change media over the infected cultures every day to fresh media.
The medium is DMEM High Glucose + 10% АИЫ + Pen/Strep+Vitamins.
Each day I observe the infected cultures, and I see that a fraction of cells have died and is freely floating, however a number of live cells also increased, as is confluence.
This is very different from CPE that is published in papers. Would anyone explain why our isolate behaves differently?
I would prefer that the virus kill cells completely – this would simplify our work on collecting clinical isolates.
Any suggestions? Can I reduce serum (FBS) content in the medium to increase the CPE?
I meant my medium is DMEM High Glucose + 10% FBS + Pen/Strep+Vitamins.
Alexandr V Shustov Cells cultured in monolayer are likely not as permissive to viral infection. I would be interested if you repeated these experiments where the cells were grown on a 3D biomimetic scaffold. It is likely that you would see massive increases in infection kinetics. We produce a scaffold being used in this application by multiple virology groups right now. Please email me and i can provide you some samples.
There can be two things, first the virus that you use is not virulent enough to infect all the Vero cell. Second, it might be possible that the cells produce interferon alpha resulting in antiviral effect for neighboring cells.
To: Fadel Muhammad Garishah
Fadel, Vero cells are believed to be defective in secreting alpha-IFN and beta-IFN.
Anyhow, a significant number of published papers from other groups uses Vero E6 to support propagation of SARS-CoV-2, and the reported CPE is quite extensive. The majority of cells die from the CPE in their hands. I just want to know how can I increase the CPE in our infceted cultures.
What is the MOI you used for infecting the Vero cells? May be the initial viral inoculum is not adequate to infect the monolayer on larger extent.
To: Vishal Kavathekar.
I don't know about MOI when we inoculate clinical samples. It is impractical to us to make PCR-based titer determination in a clinical sample before inoculating the latter in a cell culture.
However, the virus, if propagates, infects other cells, so in principle the majority of cells should become infected to the end of 5-days observation period, right? I know that cells are difficult to infect in overconfluent monolayers. We infect at a cell density ~50-80% so the cells have a place to grow.
This is because the virus cannot analyze the basal membrane of cells and only can bud and exit cells infected with new copies of the virus.
Muhsin Hamad Edham yes. 3D culture systems can restore normal/native cellular polarisation resulting in biomimetic viral- host infection kinetics. Alexandr V Shustov we can provide you with scaffolds that may remedy your observed phenomenon and result in near native viral- host infection kinetics (In vero cells at least). [email protected]
I do not think this virus is inherently very cytotoxic. Try altering the amount of glucose. do one run with very low amounts and one run with very high amounts. please let me know of the results. Also, this virus behaves significantly different with age and so i would also consider adding thyroxine to the mix in small amounts to see if this has an impact. I honestly would not expect the cells to die so quickly because the incubation time can be really long. The average time is 5.1 days but the assumption that "all cells" would die within that time is not necessarily realistic. furthermore, do you know how much ace2 these cells are expressing? I am sorry I don't have a better answer but everything I have read about this virus suggests the real problem is that it is NOT very cytotoxic. If it were, an earlier recruitment of the adaptive arm would occur, i think. The lack of early damage causes the virus to be able to spread significantly. it may also be that there is signalling between the cells to remove ace2 from the membranes, that would make it harder to infect the other cells. So I would definitely look at ace2 expression if you can.
Could you please reply with a reference showing that the cells in the monolayer SHOULD all die within 5 days? In essence, why do you think that your observation is abnormal? Most children get infected and have little to no symptoms.
your experiment is very interesting to me and I would be happy to talk about it.
Probably you can try making a colony of cells using hanging drop assay( Article Hanging-drop multicellular spheroids as a model of tumor angiogenesis
)by this method you can have a tissue not mono-layer of cells and then try your method on such tissue.
Dear all!
During a debate about the results of in vitro experiments, the whole organism often goes to the second. Conditions in a whole organism in vivo when modeling processes that are the result of many interrelated events of different organs and systems cannot be studied in vitro.
In this case, it is advisable to choose one of the fragments of a complex process at the level of the whole organism and to model in detail in vitro. But then again go to the organismal level in vivo and verify the obtained data in vitro.
Is the meaning of what has been said is clear?
To: Marc-Andre Rousseau
I apologise for not making all goals of experiments clear. We need to: 1. collect viral isolates from local patients with COVID-19. 2. Set up an assay to titer virus- neutralizing antibodies.
I intendeded to do a simple PRNT assay with a cytotoxic SARS-CoV-2 isolate. To the very surprise, one our isolate does not give clear plaques under agar coating. I see accumulation of dead cells in forms like "foci", however staining with crystal violet is not informative because many cells in a monolayer remain live and attached even within the "focus".
I will try varying glucose and add tyroxine as you suggested. Will report the results. Thank you!
Try another cell type, we had problems with our VERO E6 and plaque assays and TCID50 on SARS-COV-2. Our observations are that plaques do form, but the vero E6 grows over these plaques, making it hard to visualise even when maintained in MEM with low/no FBS.
Even when maintained in cultures over weeks, sometimes we don't see over 30% CPE with VEROs, but culture supernatants are PCR positive and can be passaged.
Eventually we overcame this by testing a few other cell lines, some are better for plaque assay, Vero E6s however, were good for virus propagation, in our hands at least. Also instead of plaque assays, it is possible to run qPCR and immunofluorescent assays.
Hope this helps & all the best!
we should consider that the virus need to find the interest cells that could produce stable virus progeny and these cells profoundly stay alive. so, may your lab has worked constantly on a same stock of the virus and probably on a same cell type and the virus has been established on the cells (or virus gets to used it).
Probable remedy would be a new sorce of virus along with a new batch of cell from another lab.
Best
What is your MOI? How are you suspending the virus isolate before innoculating of the media and adding to the culture? If the virions are not adequately dispersed, you will get clumping and then focal CPE
This is fascinating and although it is frustrating you may have found something important.
The cells that are resistant quite possibly are molecularly variant on a quantitative scale- compared to those that die.
It is possible signaling between cells bioelectrically can be happening incredibly swiftly and upregulating warning pathways such as the interferon signaling system. It is also conceivable that subtle cell variances exist since with each cell division the cytosolic compartment is never divided precisely in half so sometime one cell receives many more mitochondria and other cells far less. Probably not the answer you want to hear but the cells that fail to die have possibly got some “magic” that may help us understand this virus better.
In fact, it is not expected that all the cells will die at the same time. Successfully infected cells will display lysis from 1-2 days. the virions can also faild to infect all the cells at the same time, its possible. However, if the cell lysis is less than 80% during 5days culture it means the virion you have used is no typical. otherwise, if the lysis is more than 80%, it means your experiment well done.
Alexandr V Shustov Did you really Isolate and Purify the virus from a sample?
Best Regards.
There could be a defense mechanism that the cells deploy when neighboring cells are affected just like plant cells do sometimes. Remember neighboring cells are connected and communicate with each other.
It could be due to impurities in culture media or also due to the resistance shonwn by the cells to the virus.
There are many possibilities for that, you should observe those cells which are remaining in culture media and try to look what are the similarities and dissimilarities in those cells.
May be then you could get better results @Alexander V Shustov
Please, be careful of all substances that can alter viral activity:
Article A Panel of Broad-Spectrum Antivirals in Topical Ophthalmic M...
Hi Art. We see the same thing with the WA isolate. We‘ve gone a step further and determined they the majority of surviving cells seem to be infected based on IFA staining with anti-SARS NP antibody staining, yet resistant to killing for some reason.
We use reduced serum to propagate virus. Post infection, we use 2-5% FBS. You may also try TPCK trypsin to enhance infection with SARS2. Our methodology is published here: Article Isolation, Sequence, Infectivity, and Replication Kinetics o...
Good luck!
Andrew Herbert's observation that the surviving cells were infected but resistant to killing is most interesting . Is it possible to have this pursued?
Alexandr V Shustov I have no experience with the coronaviruses, but I can share my experience with Vero cells while cultivation rabies virus. In practice, the culture of Vero cells has stronger adhesive properties in comparison with other sensitive cultures (Neuro-2a, BHK) and a greater resistance to culturing the virus in them. However, this culture provides a higher yield of virions. The cells can actively produce the virus for 2-3 weeks without dying. I think this is due to the way the virus exits the cell. The rabies virus, like the coronavirus, has a supercapsid and leaves the cell by exocytosis. Apparently, this is a low-traumatic process for the cell and it can withstand the infection until the synthetic apparatus is depleted. It is possible that different isolates have different virulence, which led to different results for different researchers. I do not pretend that my opinion is correct, but I can only make such assumptions.
I may be incorrect but perhaps the different vulnerability of individual cells help us understand viral mechanisms and different gender susceptibility?
Curiously the article* by Sakuma et al may provide possible reasons. They identified the Vero cells as female in origin. Hence the cells would automatically be a mosaic regarding X chromosome inactivation. If all the cells that die have the same X chromosome inactivated this may assist in identifying genetic modifiers. The cells are immortal so some may be older and others more aged and this could be relevant- possible telomere variances. They mention SERV and transposable element differences between cells may fine tune cell susceptibility.
It seems this virus is going to be with us for a long time to come and hence my concern we are missing a potentially important clue. My guess clarifying this would involve single cell omics. I have no capacity to investigate this . On the other hand there may be many variables that i have not imagined and I am being naive. However I suspect that the purpose of this forum is to introduce alternative ideas as well as expert answers.
*Sakuma C, Sekizuka T, Kuroda M, et al. Novel endogenous simian retroviral integrations in Vero cells: implications for quality control of a human vaccine cell substrate. Sci Rep. 2018;8(1):644. Published 2018 Jan 12. doi:10.1038/s41598-017-18934-2
Why are you so concerned on the cells deth? Maby if cell culture living longer with infection it can produce more virions. I think it is necessary to control the activity of the virus in suspension, but not to focus on the cytopathic effect.
Dear Dr Shustov,
Could you pls apply some more info to your project?
Enclosed.
Please contact me if you find the material interesting. I've got much more to share.
Honestly, I think it is just paracrine signaling that makes the neighboring cells less likely to be either infected or killed