I have been working on infecting A549 cells with Influenza A (H1N1)pdm09 virus. I seed the cells in a 96-well plate with 10% FBS overnight. After washing the cells three times with 1xPBS, I infect them with infectious media (MOI:1/MOI:5) without FBS for 1 hour (37C). During this time, I utilize TPCK-treated trypsin at concentrations of 0.25ug/ml and 0.5ug/ml. Then, I remove the virus after 1 hour and add media containing TPCK-treated trypsin (with 1% FBS, also without FBS) for 5 days. After the incubation period, I harvest both cells and supernatant for qRT-PCR analysis, but I am unable to detect viral replication (although the virus itself can be detected). I have come across a few papers infecting cell in 35C without TPCK treated trypsin during infection and after infection. I have also tried this temperature, but I got no result. I have used DMEM and F12 as media. The virus stock can infect MDCK cells and A549 cells comes from two different resources. I am probably missing something here!
Let’s dive into the specifics of infecting A549 cells with Influenza A (H1N1) virus.
Remember, fine-tuning experimental conditions and rigorous controls are essential. Your dedication to scientific inquiry is commendable! 🌟 If you have further questions or need clarification, feel free to ask.
Thanks for your kind response. Do you add FBS to the media after infection (Cells can not survive without FBS when I add 05ug of TPCK)? Do you harvest cells or soup for qRTPCR? Also do you have experience with infecting HEK293T cells with H1N1?
Some of our non-adapted pdm09 strains are very picky about 2,6 sialic receptor density and are poor at infecting A549s, so it may be you've run into this problem. Our prototype strain is also poor in MDCKs though, so we always use MDCK-SIATs. We would usually grow pdm09 at < 37°C too. 33-35°C depending on incubator availability I think.
Also - as Omar says, 99% of flu strains (including all the pdm09 ones I know about) won't spread in the presence of serum. The serum will inactivate the trypsin and, depending on receptor specificity, can also "neutralise" infectious virus through giving it other things to stick to than cells. Most protocols for flu growth use 0.14% bovine serum albumin instead of serum, to provide enough protein to keep the cells happy.
It could also be the cells. Are you sure they're A549s? Have they been mycoplasma tested? What passage are they? Periodically ours become resistant to infection with even "easy" strains like PR8, for reasons which usually defy troubleshooting - so we throw them away and bring up fresh ones.
Good luck!
I had a discussion with colleagues at a conference on this issue - it's not always possible to infect cell cultures on glass, and in experiments with live mice (they used H1N1).
I've always wondered why the infection is carried out at cell temperatures of 35-37C. I hypothesized that this could be the problem. I thought that by lowering the cell temperature to 32C and below, we could achieve effective results. This is because the cellular immune response is significantly suppressed at low temperatures, allowing the virus to effectively penetrate cells and replicate. These are known data (Lowen et al and others). However, for some reason, no one changes the established procedures and continues to work according to them. I would be interested in knowing the results at low temperatures - after all, this is essentially what happens with cells in organisms during cold seasons when we observe the seasonality of the flu and so on.
Paul Digard I appreciate your kind response. I have purchased A549 from two different resources. I have ordered Mycoplasma test kit. I was wondering if you utilize
Alex N Ishmatov Thanks for sharing your insights. Yes, most of the papers mention 37C for infection and subsequent incubation. I will probably test 34C and 35C temperatures. if I get result, I will update this post.
Alireza - the lower temp throughout the infection. We find it especially helpful for longer duration experiments - low MOI growth curves/stock growth and plaque assays, which run for 2-3 days. If you're getting nothing at all though, I doubt this will prove to be the magic cure. For us, the lower temp helps - we get an increase in titre and the plaques no longer need to be counted down a binocular microscope, but it's not binary, from zero to something.
we use NCI-H1666 cell line for pdm09
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Mind that qRT-PCR are sensitive enough to detect low levels of viral RNA. And then considering TCID50 to quantify the virus titers again. consequently
use ELISA method, which measures the ability of the virus particles to infect and replicate. these tests help you understand in which part you may have problem like the inability of virus to even infect....