Flu virus

Ruben Donis rdonis at UNLINFO.UNL.EDU
Thu Sep 30 15:18:27 EST 1993


Liang Xia writes:

>Dear netters:
>I am currently conducting an experiment which will use flu viruses
>(A/WSN/33). The first thing I had done was making virus stocks.
>Unfortunately, I was unable to make a high titer stock by using MDBK cells.
>Could someone out there give me some ideas about the proper way to make the
>flu virus stocks.

High titer (~8 log10 PFU/ml) can be routinely obtained by infecting 10
day-old embryonated chicken eggs 
by allantoic sac inoculation.  Harvesting allantoi fluind (5-7 ml/egg) is
done after 48 hour incubation at 35 C.  Titration of the harvested material
can be done by infectivity (plaque assay for example)  or by
hemagglutination. 

If you are not set up for egg work and/or must use cell culture you may try
to switch to MDCK cells and use the following overlay after infection:
MEM-E : 95% ;  Bovine serum albumin (GIBCO; 7% solution for tissue culture)
:5% and TPCK treated trypsin 1 mcg/ml. 
Reasons: even though WSN virus grows in the absence of trypsin, it will do
better with it.  If you want to have trypsin in the growth medium you must
omit Fetal Bovine Serum which has trypsin inhibitors.  
In addition, MDCK cells are more resistant to trypsin in the overlay than MDBK. 
Fetal Bovine Serum also has glycoproteins which may inhibit virus
attachment to target cells. 

Good Luck!

Ruben


Dr. Ruben Donis                                                 
Dept. of Veterinary and Biomedical Sciences      
202 VBS                                                                    
     
University of Nebraska,                                                  
Lincoln, NE 68583-0905                                   
Phone: 402-472-6063                                     
FAX to 402-472-9690                                     




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