Hi Leonardo,

There is no "typical" viraemia for alphas or flavis, viraemia can range from low/transient to extremely high depending on both the virus and person infected. Similarly, detectable viraemia may last a couple of days or be much longer. If you are looking at something like Zika virus, this typically has a very low viraemia (sometimes barely detectable by PCR even during symptom onset) which is cleared in 1-3 days, but something like chikungunya virus may reach astonishingly high viraemia (10e11 pfu/mL which is a difficult titre to achieve when growing the virus in the lab!).

In terms of your study, I would imagine spiking human blood with high, medium and low titres for all viruses of interest and monitoring decay rates would be pretty straight forward. While you are performing the study you can data mine clinical reports of various case studies and work out the likelihood of a donor having the titres that show significant persistance and draw conclusions from that. My guess would be that even low levels of virus will persist in blood stored at 4C for long periods and the risk of onward transmission would be omnipresent for any infected donation, but it would be nice to be proved wrong!

All the best

Barry

Hi Barry,

Thank you very much for the reply. It make all sense, bur I still struggling to decide what would be a high, medium and low titres for the viruses, since it depends on the virus itself. Any recommendation?

Hi Leonardo,

There is no correct answer here. If I was attempting this study, I would spike the blood with as high a titre as I could achieve for each virus using standard purification/concentration techniques to simulate the worst case scenario (even if this is not biologically realistic). Many viruses can be cultured to 5e11pfu/mL or more using standards methods, so you can easily spike blood at 5e10pfu/mL. Some viruses struggle to reach 5e7 in cell culture, so obviously your high titre will be much lower here.

In a perfect world you'd titrate your viral stocks 1:10 down to below 1pfu/mL and spike blood for each dilution, but obviously this may not be feasible if your studying multiple viruses in parallel and would be highly unrealistic if you are also looking at various storage times of the blood!! If this is the case, your low would be about 10 or 100 pfu/mL (in the blood), and the medium somewhere between this and your high titre. However, the more data points the more informative your results will be!

How do you intend to demonstrate viability - recovery of an infectious virus in cell culture?

Sounds like an interesting project - good luck

Thanks for the advise. To demonstrate viability at first we will attempt cell culture and real time pcr.

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