Hello Hershna Patel,

I did not check the HA titer of supernatant yet. I've never done this technique before.

I've just purified Viral ARN from culture supernatant ,and then detected the M1 gene expression by cPCR. The band were so clear. 

I also checked the virus B propagation in culture Sup. at 2 specific times (0 time point and Day 2 post infection) on 3 cell lines ( MDCK, A549 and H292), and then purified the viral RNA and evaluated the copies number of M1 gene in each supernatant. Results, only culture Sup. from MDCk cells shown having virus propagation. Nothing changes in A549 and H292 cells. 

Thank you for your attention,

What culture conditions are you using?

Temperature

virus culture medium

Did you add TPCK-trypsin to the medium?  if so, what concentration?

Also, is this influenza B from a clinical sample or lab-grown virus?  Need to know the source of the virus and stain designation if you have it.

Thank you for your answer.

A549 cells are using DMEM medium plus  10% FBS, 1% P/S, 1% L-Glu

Virus Culture Medium: DMEM plus 2% FBS, 1% P/S, 1% L-Glu

TPCK- Trypsin concentration in the medium : 100 ng/ ml

Influenza B virus from Lab- Grown virus.

All suggestions are welcome !

Possible issues:

1. Do NOT use FBS in your virus culture medium.  It is inhibitory to virus propagation.  We use BSA (0.1 - 1% should work).

2. Increase your TPCK-trypsin concentration to 1 or 2 ug/ml

3. what is the virus name?

should help iron out some of your issues.  Also, if problems, switch to MDCK cells. 

If this does not help, there is a WHO influenza collaborating center in Tokyo http://www.niid.go.jp/.  You may be able to get some local help there as well.

Thanks for your recommendation.

The virus name: Influenza B virus  (Yamagata lineage)

I'd like to use the human host cells such as A549 or H292 cell line. MDCK cells is not human cells. That is the problem!

So, I will use BSA instead of FBS and increase in the TPCK- trypsin concentration. 

I hope so that will be better.

Thank you sir !

should not be a problem.  The changes should help increase your titer.  If the virus has already been passaged in cells, it will help tremendously.  The link I provided should give you some local contacts to help you as well.  They should have some protocols and a lot of experience with growing these viruses as well and in A549s.  If you have additional questions/problems, please e-mail me at [email protected]

Dear Mr. Thomas Rowe,

I will e-mail you if i have any problems or questions.

 Thank you very much !!

Tran, I believe influenza B is also best grown at 33 deg C instead of the typical 37 deg C for influenza A. Thomas's recommendations are also spot on. I have messaged you a protocol that I have used for propagation of influenza B. Joyce 

 Hello Joyce Sweeney,

Thank you very much for your sharing. 

I will try the protocol that you messaged. Could I email you if I have any question?

Absolutely! Thomas helped me out a lot also so he is great too!