I'm actually doing plaque assays on DENV2 right now and I'm getting pretty good results. I use a semi-solid procedure though, I don't now how you are doing it. Basically, I plate 3 x 10^5 BHK cells/ well in a 12-well plate, incubate for 30min at 37c. Infect with DENV2 already diluted, incubate at 37c for 3hrs, and then add 1mL of 2% CMC overlay. I get optimal titers after 5 days. I can e-mail you the details on how to make the overlay. Good luck!

We quantified DENV2 by endpoint titration which is a very old but VERY easy and less subjective then plaque assay... Antiviral Res. 2011 Apr;90(1):80-6. You can search endpoint titration, find an old virologist who knows how, or I can email you a more detailed protocol.

Good luck!

Jodi

I think still lowest DeNV2 titer. For that reason, you must harvest on flask (for 2-3 times blind passage or until CPE occur) to get highest titer then you can do plaque assay.

Thank's

Hy, could you tell much more detail? sometimes plaque could be visible on well plate but couldn't see the CPE when observed under the microscope.

I also think the same with Kris. First of all you have to make sure that you could find CPE when your DeNV2 infected to your confluent monolayer cell lines, wait untill all the cell lysis/die. Then for checking the titer of ur virus, you can try to dilute 10^-1 untill 10^-5 of your virus. apply them to perform plaque assay, I recommend you to include the undiluted one as one of those well of assay, this is can be used as positive control. Also u hv to include the negative control I mean the uninfected one. Ussualy, at 24 hpi Strong CPE would possible to observed on the undiluted one even it covered by gel compared to control.

I am working with C6/36 cells and it was possible.

Good luck! -Wahyu-

The virus titer must be low and hence, the plaque assay did not respond. Pl follow the suggestion of Jodi Hedges. Lalitha kabilan

Hi Nagarjuna,

May be the virus titre is too low to produce CPE. Try to test the sample without any dilution or at low dilutions like 2 fold. I regularly do immuno-plaque assay for all four Dengue viruses and particularly DENV-2 is really good in plaque appearance as compared to other serotypes. DENV-2 usually produce CPE in about 6 days on Vero cells.

Good Luck.

I'm actually doing plaque assays on DENV2 right now and I'm getting pretty good results. I use a semi-solid procedure though, I don't now how you are doing it. Basically, I plate 3 x 10^5 BHK cells/ well in a 12-well plate, incubate for 30min at 37c. Infect with DENV2 already diluted, incubate at 37c for 3hrs, and then add 1mL of 2% CMC overlay. I get optimal titers after 5 days. I can e-mail you the details on how to make the overlay. Good luck!

hii.. I am trying plaque assays on DENV2 using vero cells using avicel overlay. they seemed to work at first. but the virus is not following the dilutions. can anyone mail me a protocol which works please.

Hi

Is it true that DENV does not form plaques with Vero cells? I plan to do plaque assay for DENV 1-4. If I can use Vero cells, could you tell me which clone to use? also any alternative cell line?

Hi Federico, I have the same problem with vero cells, so I am doing the plaque assay with BHK. How do you prepare the 2% CMC overlay? thanks!

You should try FFU, we had this problems in the past with DENV2. Or try another strain. We are currently working with a new isolate, and we get plaques when infecting Vero cells.

Yes, I am trying using a mouse antiDENV2 and a goat antimouse HRP conjugate and DAB, and see what happens.

Thank you

So Denv is the worst of all flavis for plaque assay. They loose pathogenicity really fast if you’ve passaged In mammalian cells. Deny for plaque assays should be cultured in insect cell lines like c6/36. Also, denv especially 2 & 3 have a lot of plaque variability between strain. I’ve used tons of different overlays and haven’t seen much of a difference. If you can immunostai, I’d try 1. Using virus cultured in insect cells. 2. Try a different strain. good luck