In my study of Zika virus pathogenesis, I propagated the virus by inoculating 100 µL of virus stock into 80% confluent Vero cells. Cytopathic effect (CPE) was observed on day three post-inoculation image of CPE upload, after which I harvested the cells. The harvesting process involved scraping the cells and then centrifuging them at 2000 g for 10 minutes at room temperature.

Subsequently, I conducted plaque assays and TCID50 assays to determine the viral titer of the stock. I performed a series of 10-fold dilutions; however, the observed CPE was very low see the image. Then i performed four dilutions, and the results remained unsatisfactory. For reference, the TCID50 assay showed that at a dilution of 0, the cells were completely dead, while at a dilution of 4^-1 image upload, a significant number of live cells remained, When I stained with crystal violet, all the wells exhibited a dark purple color, with no gradual change in color observed.

Additionally, no plaques were observed in the plaque assay.

Upon reviewing the protocol mentioned on page 25 , I noted that a purification step after harvesting is recommended to increase viral concentration. Unfortunately, I cannot access an ultracentrifuge, which prevented me from performing this purification step.

Question

Given the absence of an ultracentrifuge, what would be the optimal RPM, time, and temperature settings for centrifuging the viral supernatant after harvest to maximize viral concentration?

Thank you for any information that could help me enhance my virus stock concentration