We have infected mosquitoes silenced for a specific protein with Zika virus . After a number of days we dissect the midgut, make a homogenate and try to check for virus replication by plaque assay.
Our plaque assay in the first dilution (1/10) shows complete lysis of the cells (and it doesn't look like plaques) in the second dilution (1/100) there is nothing. We have used just the midgut of non-infected mosquitoes to check if there was something in the midgut that would just kill the cells and nothing happened. We still have to make twofold dilutions... Thanks for thoughts on that!
10-fold dilution usually works fine for me.
How long did you incubate the plates after you inoculated the virus? What kind of overlay medium did you use? You might not be able to count the plaque if the plaque size is too big.
Have you ever tried the plaque assay protocol with the virus stock? If it woks with the virus stock then there might be a problem with your dilution.
we do the saem plaque Assay for virus titration and it works just fine....
Hi Carla,
What antibiotics and antimycotics are you using for your overlay? Moquitoes can be quite 'dirty' and frequently need a strong cocktail to prevent these wiping out the monolayer. This could easily explain the 1:10 result plus they tend to titrate out quicker than the infecting virus (although some may still grow in agar overlays).
Also, what's the titre of the blood meal and what day are you sampling? It is easy to imagine these factors could explain the lack of virus in your samples as too low a titre or sampling on the wrong day will dramatically impact results.
It might be worth including homogenates of the carcass next time to ensure you have the critical parameters needed.
All the best
Barry
The thing is: it does work for control mosquitoes... we do the same thing for infected non-silenced mosquitoes.
Are the 'non-infected' mosquitoes you describe in the OP silenced or non-silenced? If silenced non-infected mosquitoes do not produce this phenomenom, but silenced infected mosquitoes do, then it is puzzling! 1:2 dilutions sounds like the way forward - I'd also spend some time to see if you can confirm infectious ZIKV is present in low dilutions where the cell death is seen by passaging (no overlay) and monitoring for evidence of replication by qPCR or plaque assay at several time points. I'm guessing that the silenced protein should reduce ZIKV infectivity, but without knowing more it is difficult to work out what might be happening! It's understandable that you don't want to say more about this though!
Silenced! I think I have to go to the 1:2 dilutions.. but wanted to hear obter people’s thoughts! Thanks a lot!
Carla,
I guess you prepared the silenced and non-silenced mosquito the same way so it's strange that the control and experimental group did not have the same problem. I agree with Barry that the problem might come from contamination. Did you observed turbid overlay medium after incubation?
Also What is the infection titers in the control group and how many mosquito samples have you done with the plaque assay?
- Badges
- Science method