I have done an experiment to study the persistence of avian influenza in lake water and in lake sediments. Do you know a good concentration method to use without the inactivation of the virus from water and sediments samples? I have to titrate the virus in MDCK cells.
Thank you very much,
Albert
Well since viruses are pretty small a stepwise filtration of your water samples should be feasible going down from 1.2 micrometer gradually to 0.22. In the end utlra-centrifugation should recover viruses. There is a very old fashioned way but I'm sure new types of filter exist for this purpose
https://aem.asm.org/content/aem/9/5/376.full.pdf
First, review the virus. https://viralzone.expasy.org/223
Article Structure and assembly of the influenza A virus ribonucleopr...
Influenza viruses are enveloped viruses. I would be surprised if you get anything that way. But, what I would do is be very careful with the sediment so you don't mechanically destroy any viruses that are there. Any compression or shearing forces on the sample will almost certainly destroy viruses there.
- So a basic question is, are these existing samples? If so, how were they harvested, how long were the stored and how? Etc..
In the lab, I would dilute the samples in normal saline to try to normalize the forces on the virus envelope. Natively, enveloped viruses are as saline as the cells that bud them off. This makes them absorb water, and causes osmotic bursting.
For sediment samples, i would take small aliquots and very gently agitate them to try to float the virus off. Then I would centrifuge at low speed and remove the supernatant. From there, you could use techniques for harvesting of enveloped particles. Here's some options.
Article Purification of influenza virus-like particles using sulfate...
https://www.biaseparations.com/en/featured/viruses
Anything you do is going to tend to wreck influenza viruses. I would suggest that you first culture an enveloped virus in eggs, and learn how to harvest that. You will need to have positive controls anyway. For those, you will need to inject your cultured virus into a sediment or water sample after first slowly adjusting your cultured virus from normal saline aliquot to the level of salinity of your sample.
Before I did all that, what I would do first is send samples out for sequencing. Perhaps you have a sequencing lab already in your university? If so, then do that. That will tell you if there is even any significant RNA influenza RNA in there or not. For that, you will want to use this buffer. https://www.thermofisher.com/us/en/home/brands/product-brand/rnalater.html You can make it yourself too. It will just mean digging around and some more time.
At the worst, you will spend 1 or 2 years learning how to do virus culture, purifications, DNA/RNA sample preparations, assays, and how to use sequencing assembly software.
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