Virus aggregation advice

[Keith Stuckly]

With all the hype and paranoia going on at the moment about the Ebola
outbreak (100 e-mail messages in my post box in 1 day!!), I just hope this
posting gets noticed! The arguments seem to be very circular..my virus is
more dangerous than yours, (AIDS vs Ebola) and  the government  & CDC are
deliberately misleading people cos thats what they always do and don't know
any better (how many times is that ridiculous film "Outbreak' cited). The
mis-informed paranoics have come out of the woodwork! 

Can anybody give me some help regarding the problem of aggregation in virus
preps. The methods of preparation of concentrated virus stocks are varied
but for Flaviviruses the simplest methods are either straight out
centrifugation of virus laden cell culture fluid (if you are not worried
about cell contaminants), or PEG precipitation. Both methods have a tendency
to create aggregated virus which is difficult to quantify without Electron
Microscopy (which I don't have access to).

A popular method of overcoming this is Ultrasonication. However there are 2
types, disintegrator and water bath type cleaner. As I understand it, the
main disadvantage of the disintegrator type is that the ultrasonicating
probe must contact the virus prep causing 'sterility' problems plus the
possibility of metal particles being shed by the probe. A search of the
literature reveals that ultrasonicators are extensively used but rarely do
researchers give sufficient details or rationales as to why they have chosen
the particular method, or more importantly length of sonication time. I have
access to a 40KHz water-bath type sonicator and have sonicated preps up to 7
mins without an increase or decrease in virus titre. To sonicate for longer
than this seems to be overkill ('kill' is that the correct term??)

OK my questions are thus:
1.Can anybody give me some advice or useful lit. refs on use of ultrasonication?

2. Some advice on ultrafiltration technique for concentrating virus (does
the virus stick to the membrane resulting in losses?)

3. I am desperately trying to grow the Pestivirus Bovine Viral Diarrheoa
Virus (BVDV) to high titre with limited success. I am currently using NADL
strain (I need a cytopathic strain) in BT cells. Best concentrated
(centrifugation) titre so far about 6.5 TCID50/ml. If anybody has had
experience growing BVDV I would really appreciate some advice or
suggestions. I have had reports of  C24V strain to higher titres but not
published as it seems to be commercially sensitive information.
Alternatively, our company is willing to purchase high titre cytopathic BVDV
(must be >7.5 TCID50/ml and straight forward to assay).

Sorry to distract everybody from the Ebola hype!
Thanks for your help in advance

Keith Stuckly