I am working on Zika virus propagation and have conducted this process multiple times. Each time, I increased the viral input to the cells, but unfortunately, I consistently obtained low virus concentrations. I will explain the last propagation I performed and include some images. Hopefully, I can find someone who can help me identify the issue.

The protocol

1- T75 flask of Vero cell reaches 90 to 100% of confluency on the day of infection

2- discarded the media and wash with BPS

3- add 2% DMEM media, then 300ul of very infective virus (I have taken from lab mate )

4- incubate 1:30

5- remove the infection media, and add a new 2% FBS media

6- incubate till see the CPE

7- I harvested at day 3, I scraped cells and transferred them to a 15 ml tube

8- then centrifuge the tub for 10 minutes at 2000g

9 - transfer the supernatant into the new tube then aliquot into a 2 ml tube, then store at -80

In the attached images, the first shows the Vero cells at day 2 of infection, where the media color was slightly yellow. The second image displays the Vero cells at day 3, which exhibited significant morphological changes: the cells were all detached, and the media color had turned very yellow.

I have followed the same propagation steps multiple times, starting with an inoculum of 50 µL, then 100 µL, and finally 300 µL. In each case, I harvested on day 3 due to the dramatic morphological changes I observed.

I’m considering two options to address this issue:

Thanks in advance to anyone who could help with advice