反应详情

Cells and viruses. BVDV-free MDBK cells (CCL 22) were obtained from the American Type Culture Collection and propagated in DMEM/F12 essential medium supplemented with penicillin (500 U/ml), streptomycin (500 U/ml), and 10% heat inactivated horse serum (Invitrogen). Cells were maintained in a humidified incubator at 37° C. with 5% CO2. BVDV (NADL strain). For infections, virus inoculum was added in complete medium and adsorbed for 1 hour at 37° C., the inoculum was then removed, the cells washed once with medium and fresh medium containing compounds added. Virus stocks were prepared by freeze-thawing the infected cells and culture supernatant three times followed by centrifugation at 1,000 g for 5 min. Stock titers were determined, and stocks were aliquoted and stored at −80° C. WNV was obtained from a cDNA clone of a human 2002 isolate from Texas; virus obtained from BHK cells electroporated with the in vitro synthesized RNA from this cDNA clone was passaged in Vero cells before use in antiviral assays. Dengue virus (DV) serotype 2 was a New Guinea C virus that had been passaged 28 times in suckling mouse brain, twice in Vero cells, and once in C6/36 mosquito cells.

1. customBVDV-free MDBK cells (CCL 22) were obtained from the American Type Culture Collection
2. temperatureheat inactivated horse serum (Invitrogen)
3. additionFor infections, virus inoculum was added in complete medium
4. customthe inoculum was then removed
5. washthe cells washed once with medium and fresh medium
6. additioncontaining compounds
7. additionadded
8. customVirus stocks were prepared by freeze-thawing the infected cells and culture supernatant three times
9. waitfollowed by centrifugation at 1,000 g for 5 min
10. customstored at −80° C
11. customWNV was obtained from a cDNA clone of a human 2002
12. customisolate from Texas