I want to study the release dynamics of influenza-like virus in cells at different time points. I was wondering which is the best MOI to use to get good results. Should I use low MOI (0.1 or 1.0) or high (like 10)?
Hi,
I am not a influenza specialist, but if you want to see differrences and be precise, you should start with low MOI in order to not reach the plateau to early.
Thanks Frank, I have done it cumulatively by assessing the viral RNA in the cells and supernatant at different time point using MOI 1.0 and 0.1. Now if I want to study the single-cycle infection which is the best MOI?
which MOI gave you the best results? are results similar? at what time point do you start to observe RNA replication? By analysis the data you already have and by looking at the litterature you will have the beginning of your answer, the rest will come with the experimentation.
MOI 1.0 gave the best results (supernatant and in the cells), for both replications starts at 3 hpi. Thank you.
An MOI of 1 is not a good starting point for an experiment aimed at investigating single cycle kinetics as the 1 infectious virus particle per cell will not distribute evenly across the culture. Assuming your titre and cell counts are perfect and the cells are all equally infectable, you'll only infect around 60% of the cells. So if the system is such that infectious virus will be produced by the cells infected at the start (not necessarily true for flu without trypsin in the culture), there are uninfected cells waiting for a second round of infection.
Of course the assumptions over titre, cell count etc will not be true and there are all sorts of other potential confounders waiting like virus particle:infectvity ratios and IFN responses in the culture, but nevertheless, avoid an MOI of 1 unless you have no choice because the virus stock titre is too low to go higher. It probably doesn't matter too much if all you're doing is asking how fast your WT virus replicates in a particular cell line, but the moment you start trying to make comparisons between mutant viruses (or mutant cells for that matter), the inevitable small errors in titre determination and cell counting will destroy the validity of the experiment. For example - your titres and cell counts vary two-fold around the actual number (which would be pretty good for our systems), so a set of wells infected at a nominal MOI of 1 could actually lie between MOIs of ~ 0.25 and ~ 2. The number of infected cells would accordingly vary between ~ 20% and 85%. Unless you carefully measure the number of infected cells and control for this, any comparisons of what's going on in the pooled populations will be horribly flawed.
The solution is simple - use an MOI of 5 or higher for single cycle kinetic studies. Small errors in cell counts etc will have much less of an affect.
Read the Wikipedia page on the Poisson formula and how to use it for virology. There are plenty of online calculators too.
Paul
https://en.wikipedia.org/wiki/Multiplicity_of_infection
Thank you very much Paul Digard for the useful information
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