Influenza virus - (Dec/09/2013 )
I am going to infect A549 cells with H1N1 virus at various time points ( 1 day, 2 day and 3 day) to know exactly at which of these time points, the virus show higher replication ability or higher infection status.
SO my Question
If I infect these cells with the virus, How can I measure this infectivity i.e what is the most suitable parameter in Influenza virus on which I can build my idea to come up with a conclusion that at this time point the A5s9 cells is highly infected or highly affected with virus.
Thanks In advance
A couple measurements can be used.
The best measurement for what you are doing is virus titer. You can calculate this by doing a plaque assay http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
You can accompany this data with qRT-PCR. Here you are measuring the amount of viral RNA (by amplifying cDNA).
Nice answer, Thanks. but Can I do Plague assay in the A549 cells ?
Also another question, I will perform q-PCR for the cell pellets but for which Influenza virus gene ? ( Influenza has 8 genes) i.e. which one of them is indicative more for the infectivity ?
I have never worked with influenza/A549 cells. But, if influenza produces a cytopathic effect in these cells, then the plaque assay will work just fine.
For the qRT-PCR, you need to isolate total RNA, you can use TRIZOL reagent for this (most commonly used reagent for RNA isolation). You use this RNA as the starting material for the RT-PCR. As for which gene you want to use, that varies for each virus. You should look into the literature and see what others have used for influenza. You will be able to find examples in other researchers papers and they will even provide the primer sequences and probe sequence as well. For the virus I work with, we usually use RT-PCR with primers for a capsid gene. But, again this can vary from study to study.