Hi Arturo,

May I suggest using copy-number RT-qPCR to measure ZIKV RNA genome equivalents in serum. Many groups use this as an alternative, as you are right - both active/intact complement and 56oC incubation can potentially affect infectivity of ZIKV.

It is possible to dilute the serum to be tested to such a degree that any complement present will have a negligible impact (making PFU determination feasible), however the lower limit of detection is thus necessarily quite high. Perhaps a combination of plaque titration and genome equivalent qPCR would work for you?

Best of luck,

Justin

Hi Arturo,

Many people HI serum used for plaque assay analysis including Zika virus, so there is no need to worry. Complement can be detrimental to virology assays so is often removed by heat inactivation, but the trade off for heat inactivating is the potential to also inactivate growth factors in the serum. It is always worthwhile testing growth rate of your cell line using both HI and non-HI serum whenever you get a new batch of serum in (and against your current batch of serum), but as long as you don't HI for too long or at the wrong temperature you shouldn't see a noticeable difference.

I would recommend using HI if possible, and would caution against looking at genome copies as these are frequently 1-3 logs higher than pfu (DI particle formation, inefficient packaging + several other factors) and are prone to fluctuations that have little to no impact of production of infectious virus which are easy to misinterpret. Genome copies are fine for a quick screen or to assess trends, but use techniques that assess production of infectious virus when you want to generate meaningful data unless there is an definitive reason not to.

Barry Atkinson - My interpretation was that Arturo was asking about HI of the serum harvested from the infected animal and containing the virus he wishes to measure, not the serum he wishes to add to the plaque assay overlay medium. HI of the serum to be measured for viremia would likely detrimentally impact infectivity of ZIKV. Just thought a clarification was worthwhile.

You are quite right that genome copy number is only a surrogate of infectious particle measurement, but it is a frequently used and published gold-standard in viremia measurement as it negates impact of both complement and any neutralizing antibodies present in the harvested serum. Of course this could be coupled with plaque assays, though as mentioned, these come with their own caveats.

Ahh, I really was barking up the wrong tree - an interesting (and overlooked) aspect of virology, but not the topic in hand. Sorry about that.

In that case, not much more to add to Justin’s comment, and I have used qPCR plenty of times in these studies to reduce workload but still tie in key data with plaque assay. I would recommend validating the system beforehand by comparing serum spiked with virus (one which you HI and one not)- this will tell you what effect HI has on the virus before you use precious samples. 56c might be too high (56c for 30 mins is standard inactivation protocol for many viruses!!), but not sure what the minimum temperature is to decomplement.

Justin A Roby , Barry Atkinson

I would like to measure viremia by focus assay and RT-qPCR. I tried to do it by the focus assay, but I do not find it reproducible when I use serum from mice, however when I use virus from cell culture this test is reproducible. I will continue trying to standardize the test. On the other hand, I will also standardize the test to evaluate viral load. When I have more results, I will let you know how it went.. Thanks a lot for your help and advice.