I have provided with a stock of RSV A2 (tested on mice) by my collaborator. I propagated it successfully with a quite impressive titre. But when I infected my mice with this stock they were not showing any symptoms that I've seen before with the collaborator's one. I tried several times, but never could manage to see symptoms in Balb/c mice. I propagated the virus in Hep 2 cell line using the same protocol that my collaborator group provided. I'm very confused that how could the virus changed it's virulence with only one passage! Can anyone give me any idea why it might happen?
In short, my protocol for propagation is-
Day-1: Seeding the cells in T175 flask; using DMEM+10% FCS
Day-2: Infect cell line with RSV (MOI-1:10)
Day-3: Changing media to reduce % of FCS (2%)
Then incubate until 50% cell death. (Few hour to day; with this stock I had 50% dead cells after around 10 hours of changing media.)
Then collect, sonicate for 2 min, centrifuge and snap freeze the aliquotes in dry ice/ liquid N2.
Hi Khandaker,
Are you able to infect other ''in vitro'' cell lines with your viral stock ? or Are you able to blot an RSV protein from your Hep2 cells?
Hello, why did you change the media after infection? You might have lost the virus in the media.
Hi Nicolas,
Yes, I can. But Hep-2 is the most cited cell line in the papers.
Blotting the antibody is also possible, but it is not my plan of project. My main target at this moment is to prepare a nice infectious viral stock to have persistent disease symptom in mice.
What I'm thinking now, I might have a large number of defective viral particle in my stock that is interfering the infection process. I found a protocol for preparing stocks without or less particle, but it is pretty long(may be will take one month). It would be great if anyone has a working, reproducible and simpler protocol to do the propagation.
Hi Shubhada,
It is the protocol. Assuming most of the virus is inside the cells.
From 30 ml media (DMEM+10%FCS) I remove 25 ml, centrifuge it, discard the supernatant, resuspend it in 25 ml DMEM and add back to the flask. then the flask is incubated again until the development of cytopathic effect.
Hi Shadia,
I am not experienced with the RSV A2, so firstly how did you titrate the virus in vitro? I mean you say you successfully propagated the virus, and had a good titre. So in which cell line did you titrate the virus? I think you should dilute the virus 2logs10 and infect in case you fear the formation of DI particles. How long is your incubation time - till the CPE appears? If you have a Hep-2 monolayer, surely you will have some infected cells which are in the medium when you change after 24hrs. I think the process is not necessary. Also for infection (after adsorbtion -that is after you incubate the monolayer with your virus) you should use 2% or maximum 5%. And there is no need to change after infection after 24 hours.
Hi Shubhada,
I use Hep-2 for propagation and titration. According to the protocol and most of the published papers MOI should be 0.1, but I tried 0.2 as well.
You are right about the media changing issue and the % of FCS. I also thought about it, the cells should be under stress during the process of adsorption.
The time of development of cytopathic effect was unexpectedly quicker with this new stock comparing to my previous experience with RSV propagation. It took only 10 hours to develop the CPE and reached 50% cell death. I'm really surprised with that though the collaborator group said it might happen to a very virulent virus!!
Hi,
First of all, I think you should be assured of your stock titer. Also, it is important to check for contamination with mycoplasma. Sometimes, the rapid cell death that you perceive as a CPE is because of contamination. Then, I also suggest not changing the media after infecting the cells with RSV.
Good luck
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