In article <2704251228101994.A66895.VENUS.118AE3190000*@MHS> PINTO at CCC.MEDCOLPA.EDU ("Angelo J. Pinto") writes:
>Can anyone give me any helpful suggestions on how to grow a
>high-titered HSV-2 pool that is pathogenic in mice?
>
The procedure is not, in itself, particularly difficult. Things
to remember are that mouse strains differ dramatically in their
susceptibilty to HSV, and also that HSV-2 strains rarely grow to titres
as high as HSV-1.
There are a number of HSV-2 strains you can use. HSV-2(333) has
worked well for me. Infect subconfluent Vero cells with a low MOI (say
0.01 - 0.1) - we usually started with, say, 1 x 10e8 or more cells (in our
case, 10 - 12 150mm tissue culture dishes). Incubate under standard
conditions for 2 - 3 days, untill CPE is advanced. You want to catch the
cells just before they start to lift off. Discard the medium, scrape the
cells off and spin them down; the vast majority of the virus is
cell-associated. Resuspend into 5- 10 ml medium. Freeze-thaw the cells,
then sonicate them - we did 50% power, 3 x 15 seconds or so, interspersed
with periods on ice. This should give you titres of around 5 x 10e8/ml.
Good luck.
Ian
--
Ian York (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-4328 Fax (617)-632-2627
