During my experiment, I couldn't account for all the virus in most of the purification processes, so I thought it might be getting inactivated during the process. Yield is measured by plaque formation. I am wondering if people have had same problem and found some solution for this. Thanks
Can you briefly how you purified your virus? Actually HSV1 is easily growth in vero cells, if you have problem to get the amount of virus you need, it might be easier to increase the amount of virus at the beginning.
I have a lot of experience with this with HSV-2. As asked by Yeankong Yong, more detail would be very helpful if not essential.
The biggest loss for me routinely would be from resuspending pelleted virus after ultracentrifugation and the resuspension protocol could make a big difference in infectivity at the end (from getting close to 100% of the starting infectivity down to 20%). We tried triturating the pellet with different gauges of flat-ended needles and that was far too harsh.
The biggest non-routine loss would be pulling or pipetting off banded virus from a ultracentrifuge gradient. Practically speaking, though, the loss was not really from handling but more from isolating a particular fraction (single virions) from the total since I measured infectivity, PFU yield, and protein yield throughout.
HSV-1 is very big virus with envelop; hence it needs a careful handling during purification. We normally resuspend pellets in MNT buffer ( 20 mM MES, 100 mM NaCl, 30 mM Tris, pH 7.4 ) gently using 1 ml pipette (just break the pellet into small pieces) and then keep for 6-10 h in fridge to expand pellets by their own followed by mild sonication of the pellets in sonication bath filled with ice coupled with resuspension using 1 ml pipette (30 sec sonication and 30 sec resuspension, 3 times each alternatively). Never use small pipettes for resuspension and keep the mixture always cool. If you are using glass pipette or tubes, pre-coat them with 5% BSA solution, which prevents losing the virus by binding to the surfaces.
Another factor is the time of harvesting. Always harvest the virus when 90-95% cells (Vero) are rounded and easy to detach. And also, when you are aliquoting and storing the vials, use snap freezing in liquid nitrogen which keeps the virus viable. I hope this information is helpful to you. Let me know if you have any further questions.
Thanks for all the answers. Yes, HSV are produced in vero cells. Our protocol doesn't involve centrifugation. I was talking about column purification. I agree with you all that its a sensitive during the process but was looking for biochemical properties which makes it sensitive. Thanks
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