I tried 5 different gene combinations out of which I could rescue only 2 viruses. What could be the probable reason apart from the genetic stability of virions?
Hi Sushant,
Which viruses are you trying to rescue and what protocol are you using?
In the case of influenza virus, polymerase subunit incompatibility can be a factor.
@Alice: We do Transfection by Lipfectamine2000 using 1ug of each plasmids. Some viruses were rescued but some couldnt.
@olivia: What could be the reason for polymerase subunit incompatibilty?
some mutations are difficult or impossible to rescue likely because of there deleterious effect on viral fittness.
Try using TransIT LT1 as your tranfection reagent, we find some viruses can only be rescued this way. Also, you dont mention whether you are using a pure 293T culture to rescue or a co-culture of 293Ts and MDCKs. I recommend using the coculture if you arent already.
@hassan: there is no mutation, infact only whole gene change (eg PB1)
@alice: We are doing tranfection in cocultured cells
If the problem is incompatible gene segments, it is possible to try rescue in only 293T cells to enhance the amount of virus that is initially being produced, and then put on to MDCKs where infectivity may be low but visible.
If the problem is an avian-tropic PB2 (627E for instance) then co-cultures with an avian fibroblast cell such as QT35s or DF1s has worked for us in the past.
Have you ruled out trivial reasons such as a duff plasmid? In these circumstance I tell my students (hello Alice!) to (a) check the plasmid is supercoiled, (b) the right size (they love the quick and simple Nanodrop spec but it doesn't tell you everything) and (b) to check that the plasmid expresses what it's meant to - either by western blotting and/or (for a P/NP gene) by minireplicon assay.
Hello Yang,
As far as mutant HA is concerned, there won't be any problem in rescuing the virus if the mutation is not detrimental to virus survivability. What protocol for virus rescue you are using?
If everything is working perfectly, then virus generation is taking place but you are not able to rescue virus. We normally go for 3-4 blind passages and after that we may say that virus is not there.
what backbone are you using? WSN or PR8?
We rescued WSN/33 (8 plasmid)/H1N1 solely in cell culture only. Try rescuing in cell culture as well as embryonated eggs?
May be the mutation you have incorporated in HA makes the virus less adapted to the host in which you are trying to rescue it. Try alternating your rescue methods.
Ultimately virus will be rescued only if it is fit to survival. Otherwise it may be lost.
Good luck!
Thank you, Sushant
We are using PR8 as the backbone. And we rescued H1N1 in embryonated eggs. We sometimes go one blind passage in embryonated eggs.
The adaption of virus to the host that you mentioned maybe a key point , and I would try 293T/MDCK co-culture or transfection in 293T/ passaging in MDCK separately.
Best wishes!
Dear Yang,
Passaging only once may be a reason that you are not able to rescue virus. If the virus particles are very low, you may not get detectable virus in the first passage. Try passaging blindly 3-4 times. Then only we can say that virus may not be there.
Good Luck!
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