Fig 10 Viral Loads in Spleens and Brains

Three-week-old female CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA). The mice were intramuscularly infected with rSA14, rSA14-14-2, or one of their derivatives at a dose of one-tenth of its IM LD50 if between 1.5 and 1.5×105 PFU, a maximum dose of 1.5×105 PFU if greater than 1.5×105 PFU, or a minimum dose of 1.5 PFU if less than 1.5 PFU. Every 2 days after infection for 16 days, four infected mice were transcardially perfused with cold PBS, and their spleens and brains were harvested. The collected organs were homogenized in 1 ml of cold PBS using the TissueLyser II system (Qiagen, Valencia, CA) as recommended by the manufacturer, followed by centrifugation at 10,000 × g for 10 min at 4oC. Viral loads in the cleared homogenates were measured by plaque assays on BHK-21 cells, combined with immunostaining of viral plaques. Viral loads were determined as PFU/organ.