Recently obtained Zika virus strain PRVABC59, grew a new stock in Vero cells and want to titer it by plaque assay. If anyone could provide rough details or point to a publication with a Zika plaque assay protocol, I would greatly appreciate it.
Initially I tried to plaque it on Vero monolayer, 1 hr infection in DMEM, aspirate, add 2 mL of 1% CMC/10% FCS+DMEM (1:1). However, after 7 days still no signs of cytopathology. Could be related to the strain I suppose, but perhaps this will be fixed by a worked out protocol?
Hi Cory !
Your protocol seems to be good for me ! I think your issue come from cells ! there is several clones of vero and all or not appropriate for zika infection ! you need to use Vero E6.
Hi Cory,
My protocol is below. I've found for PRVABC59 4 days is enough time for plaques. They will be fairly large and turbid.
While Vero E6 are best since they have contact inhibition and won't grow on top of each other, Vero's will work, especially if you overlay with agarose.
Thanks Kristeene for the detailed protocol. Thanks as well Pauline for the tip.
I believe I am working with Vero 76 clone, I will give the protocol a go. If I keep having issues perhaps I'll just refresh my cell line from ATCC. Thanks again!
you are welcome ! Kristeene is right 4 or 5 days is enough if you don't get anything at day 7 that means the infection didn't work. I also went through these types of issues and I figured out it was from the cell clone. My other tip to get good titration quaity : Vero cells don't like to be too long without liquid on top (medium, PBS whatever) they dry quickly and if it happen you will get whole in the middle of your well with cells around. So my advice if you work with P24 well plates : manage your wells 2 lines by two lines. For the other plates just go quickly to manage the plate.
In my opinion CMC is better than agarose and allow you to get results quicker since you just have to get out CMC wash 3 times with PBS and fix with PAF 4% 20 min then crystal violet steps.
to conclude in my opinion your protocol is good, I have the same and it work, you just need to change cells !
Good luck !
I am also doing zika virus plaque assay in vero cell. during incubation period, plaque observed in microscope in 10-5 and 10-6 dilution. After fixation with 10% formaldehyde and 0.1% crystal violet, I didn't show clear plaque in my plate. what would be the reason for that. I am also attaching pic of plate for references.
thanks
I know it's been awhile since I looked at this thread, but seems like many people check this out on a weekly basis. So I just wanted to upload a protocol of mine that I put together that uses Vero cells and CMC as the overlay that works fairly well (at least for me).
It can be done in 6, 12, or 24 well plates though I prefer to use 6 or 12 wells since the plaques are bigger and easy to see by eye. I tried to overlay plates with 1% agarose as well, but CMC was quicker, less susceptible to disrupting the monolayer, and honestly had more clear plaques.
Good luck all!
HI Guillermo Galvez-Romero,
In Rota virus plaque assay , trypsin are used in agar media to induce the plaque formation.
Hello everybody! Sorry for invading your chat!
I have 2 strains of ZIKV: 275570 and 275569 (supernatant got from mosquitos).
After a differential RT-PCR I found out that they are coinfected with CHIKV.
In the 2 samples I saw the presence of both viruses with a strong Ct in both cases.
I performed a titration using PS cells and I got a title of 10^8 PFU/ML. The problem is that I'm not sure which virus I really title: is it the title of ZIKV or of CHIKV? I suppose it corresponds to ZIKV because it is really difficult to title CHIKV using PS cells, in general I do CHIKV titration using Vero cells because in PS cells titration doesn't work.
I would like to have the specific title of both viruses.
I then thought about performing the titration using the TCID50 technique, and I inoculated my strains in a 96 wells plaque prepared the day before with C636 cells.
Have you ever performed this technique using C636 cells to title ZIKV and CHIKV??
Thanks a lot!
Giulia
Btw I used to title ZIKV strains using PS cells in 24 wells-plate.
I do the lecture at day 4 and it works perfectly! If you need more details write me :)
Hi Giulia,
i am doing TCID50 assay for flavivirus. but we inoculated the vero cell in 96 well plate in same day while we are performing TCID50 assay.
So, if I well understand, do you perform TCID50 assay using vero cells? Can you explain me exactly the protocol?
Thanks a lot!
Giulia
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