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Releasing cell associated virus

Virus releasing cell-associanted

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#1 Scientist1

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Posted 13 December 2018 - 12:30 AM

Hi everybody, do you have any idea of how to release enveloped virus which is cell associated? The virus must remain infectious after releasing.

 

I tried sonication, but I was unsuccessful. Thank you.



#2 bob1

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Posted 13 December 2018 - 06:52 AM

Which virus? There are many many different types of viruses and due to receptor differences each has a different protocol.



#3 Scientist1

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Posted 13 December 2018 - 07:14 AM

I´m interested in bovine respiratory syncytial virus (BRSV).



#4 Scientist1

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Posted 15 January 2019 - 04:39 AM

Nowbody knows hot to do it? I tried also freeze-thaw cycles but the titre was lower and lower... Sonication seems not to be the best option too... Do somebody have any idea, please?



#5 bob1

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Posted 15 January 2019 - 07:35 AM

I don't work on BRSV, however, freeze-thaw is a common method for many viruses, and looks like it works for BRSV too (from a scan of the literature). Most methods of release of cell associated virus will result in lower titres due to the methods being quite harsh on the virus. It is expected that you will lose some titre with each freeze/thaw cycle.

 

The only thing you can do is to choose a point at which you are satisfied with the amount of virus released and the titre resulting from that, then stick with that point for the rest of your experiments.



#6 Scientist1

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Posted 17 January 2019 - 11:57 PM

Thank you for your answer. Yes, freeze-thaw is a commen method for virus relasing and from literature it should work also for BRSV. I will try to optimize this process.

 

I have just one idea? Do anyone use Dounce homogenizator?



#7 bob1

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Posted 18 January 2019 - 06:56 AM

I've not used it for virus release, and it will work to disrupt the cells fairly effectively - however there are a few things to be concerned about:

 

1) foaming - bubbles can destroy viruses in some cases (no idea about BRSV), due to the surface tension and drying effects.

2) shear forces - A Dounce might be the way to go, but you could also try Potter-Elvehjem and Ten-Broek, which are more grinding than shearing.

3) Will this be enough to release the virus from the protein attachment? No way to know without trying I guess.



#8 Scientist1

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Posted 23 January 2019 - 05:11 AM

Thank you for your answer, I will try it!

 

I think that major problem is that the BRSV is enveloped virus. Therefore, cells need to be lysed as gentle as possible.

 

Is there any way of enzymatic lysis of cells without disruption cell/virus membrane??? It sounds crazy, but maybe yes, I do not know...



#9 bob1

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Posted 23 January 2019 - 06:52 AM

It is possible - influenza virus (also enveloped) uses TPCK-trypsin for enhancing attachment and internalization, but not detachment. You could try a mild protease digestion, but you might well digest off the attachment proteins.



#10 Scientist1

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Posted 24 January 2019 - 05:13 AM

Hmm oki, I did not know it. On the other hand, the presence of trypsin in virus collection is not ideal for further use of virus...

 

I´ve found one enzyme, that solve this problem - dynamin (not dynamit :D ). But there is no way how to obtain it commercially :-(

 

Maybe it would go some way to promote its natural expression in cells???



#11 bob1

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Posted 24 January 2019 - 07:17 AM

You could try transfecting it with infection if you have a plasmid for it. Addgene has dynamin-1.



#12 Scientist1

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Posted 25 January 2019 - 01:44 AM

Thank you very much, it is interesting. There are three types of dynamins: I, II, III of which type II is expressed in most cell types.

 

So the path could be to express the dynamin in some bacteria, purify it and then use it for cells infected with BRSV to separate virus particles?



#13 bob1

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Posted 25 January 2019 - 06:52 AM

That would be one way - a short-cut could be to transfect a mammalian expression vector with the gene encoded and see if high expression in the cell would help at all.



#14 Scientist1

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Posted 30 January 2019 - 02:32 AM

Oki, firstly I will try to optimize freeze-thaw and Douce homogenization.

 

Do you have any idea what to use to protect virus and its envelope agains freeze-thaw damage? I studied literature and found that for example, MgSO4 in combination with HEPES can by used... But what else to try?



#15 bob1

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Posted 30 January 2019 - 06:48 AM

No idea sorry. You could potentially try using glass-state inducers, such as the DMSO that you would add to cells when freezing down.







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