Hello all,
our group is studying the differences in host innate immunity against RSV and EVD68 using primary human epithelial cells. After a pioneer experiment we found that RSV needs a higher MOI (5) to replicate as efficiently as D68 does at a lower MOI (2). So my advisor asked me to infect the cells with RSV, MOI =5, 37 degrees Celsius, and EVD68, MOI = 2, 33 degrees Celsius (this virus grows better at a lower temperature, at least in our lab).
It is a little weird to me; I thought the lower growth rate of RSV in this cell is its nature, and might contribute to the immune responses it induces. Just like that D68 grows worse at a higher temperature is its nature. Do we really need to artificially make these "optimizations" ?
I counseled a professor, who is in collaboration with our lab on another project, after a joint meeting. She's on the same page with me: we don't need to make these two viruses replicate equally. The professor suggested me to infect cells with these viruses with exact the same condition (starting MOI and temperature). And I should infect cells with different MOIs to see if there's a dose-dependent response.
I'd like to know is there a guideline and what are the factors we need to take into consideration when design, or "optimize", a experiment like this. Thank everyone for reading this.
In order to answer your question I need more information. How are you measuring the "immune response"?
Hi Karen, we analyze cytokine/chemokine mRNA and protein level as immune response parameters. Expression of pattern-recognition receptors is also considered. We only collect samples (lysate and supernatant) of the first two days of infection.
Thank Florian and Randy for very detailed explanations.
I'm not completely against my advisor, and I am aware of keeping as many variants constant as possible. That's why I got confused that when we are using different viruses, different MOI, different temperatures, are we seeing what we are asking and what's really happening in this system? Maybe either way is correct; I just got stuck.
All these discussions are helpful and clear my thoughts. Thank you.
I totally agree with the previous comments. These are two viruses from different families, and the optimal replication/infection conditions for one virus may not necessarily apply for the other. To compare the cytokine/chemokine response profile, I suggest to apply the optimal infection conditions for each virus, and to use a range of MOI from very low to very high.
I agree with Wassim. Not sure that is what the others have said. I come from the vaccine industry and when we evaluate the immune response, such testing is conducted in animals. We grow each virus to its maximum, determining the MOI for each one independently, then prepare our vaccines. That would be my approach.
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