Hi, everyone.
I am currently doing infectious clone enterovirus work.
I have tried to use Mirus transfection kit to transfect my in vitro transcribed RNA into Rhabdomysarcoma cells. after 24 hours, I can clearly observe CPE in the cells, which transfected in duplicate with 2 different virus clones RNA. however, after i harvest the supernatant by freeze thaw twice, i re-infect again with about 1ml of the supernatant into another flask of cells. I couldnt observe any CPE even now is already day 4 (the cells just grow healthily).
Anyone know what is the reason behind that? is that means that the clone is not successfully made? or the clone virus is not infectious? or should i incubate it even longer?
It shouldnt be the CPE that I observed in transfection is due to transfection kit reagent toxicity since I also did a control with the reagents (just without the RNA) for transfection.
Hope any experts can answer me about this and guiding me please. Thanks.
Are you sure that there isn't infection in your second round? Unless your virus is known to be lytic, maybe there is infection without cell death in your second round? Have you looked by immunostaining or qRT-PCR?
If there is no infection in your second round, then I think you have to go step-by-step through the viral infection cycle to see where the problem is. First, is there replication in your transfected cells (e.g. immunostaining if you have an antibody, qRT-PCR if you don't). If there is replication, are you in fact assembling/secreting virus into the supernatant? and so on...
Hi Andrew,
Thanks for your kind suggestion. I will try first to do the immunofluorescence assay with the transfected cells. Let's assuming first if this is positive, would you expect there is some failure in successfully harvest the virus? or could be due to other factors?
Antonio,
It is entirely possible that there was not initial infection in your flask. I don't think there is something wrong with the passaging. However, if you have introduced a less stable capsid mutation, you could be losing a large component of your titers. Working with coronaviruses in the past, we have seen CPE in cells that have been electroplated with transcribed genetic RNA but did not progress to full infection. This is typically due to generation of viral proteins associated with cell hijacking or disruption of cell events whereas there remains a problem with packaging or assembly of the virions.
If it were me, I would repeat the virus recovery and before disrupting the monolayer by freeze-thaw, take a sample for a Western for enterovirus proteins. This would give you a quick indication of whether virions are being packaged and released.
Hope this helps and best of luck.
Chris
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