I'm planning to produce a herpes simplex virus 1 stock (an enveloped DNA virus) for future infection experiments. HSV-1 production protocols seem to contain the cell lysis step with freezing/thawing and/or sonicating - however, wouldn't cell lysis release either naked HSV-1 nucleocapsids or enveloped HSV-1 virions inside cytoplasmic vesicles?
If cell lysis is nonetheless required, are there more options in addition to freezing/thawing and sonication?
Hi Satu,
As we’ve used to use recently while working with HSV-1, you will find ALL answers to your questions regarding HSV-1 production protocols containing the cell lysis, freezing/thawing, sonication, etc. in the paper of Peggy Marconi & Roberto Manservigi, entitled “Herpes Simplex Virus Growth, Preparation, and Assay”, published in “Protocol Herpes Simplex Virus”, Volume 1144 of the series “Methods in Molecular Biology”, pp. 19-29, 2014.
Best wishes,
Ilya
Hi Satu,
Freezing/thawing cycles for cell lysis will increase the titer of your virus prep. Nevertheless there are some things to consider:
1) If you make a couple of plates/flasks/dishes/whatever-you-are-using-for-cell-culture more, you will more or less get the same virus as if you freeze/thaw the cells
2) When you lyse the cells, you get intracellular virus, but also a lot of other stuff: DNA fragments, cellular protein complexes, membranes and so on. All these things will more or less end up in your virus prep and you will put a lot of junk on the cells once you infect them. Cells don't like it
3) HSV-1 grows to high titers and you basically don't need to lyse the cells to get more. However, if you do it, I'll suggest you to purify the virus over a sucrose gradient, or something similar. This will reduce the amount of junk coming from cell lysis
I hope this helps
Good luck
Antonio
Thank you, Ilya, Angela and Antonio, for all your answers and advice! I think I'll start the HSV-1 production simply without lysing the cells and see how much (relatively) junk-free virus I can get that way.
Hi Satu,
Following Antonio's suggestion, if you'd like to produce a stock of virus without cell lysis, you should use cell lines that secrete more virus that traditionally used Vero cells. We usually do it in HaCaT cells. You can find my protocol in the paper below.
This is more time consuming than simple cell lysis to get a 'dirty' stock. You should consider whether you really need a purified stock for your experiments.
Anna
Article Structural analysis of Herpes Simplex Virus by optical super...