The virus suspension was diluted in α-MEM containing 2% FCS to give a concentration of 2 × 107 plaque-forming units/mL, and a volume of 1.6 μL of inoculum was infused over a period of 3 min by means of a microinjector (Narishige) fitted to the syringe. Alternatively, 1.6 μL of diluent (α-MEM

containing 2% FCS) was injected as a control. After the infusion, the CHIR-99021 cell line cannula was removed, and the burr hole was sealed with dental cement (Unifast III; GC, Tokyo, Japan). The incision was sutured with surgical silk (Natsume Seisakusho, Tokyo, Japan), and the mice were subjected to the following analyses. To accurately examine the mode of MPyV infection in mice after stereotaxic inoculation into the brain, the amounts of viral genomic DNA in the respective tissues were determined using quantitative real-time

PCR. The virus inoculum was infused into the brain parenchyma, as described above, and the mice were deeply anesthetized Ridaforolimus clinical trial with an intraperitoneal injection of sodium pentobarbital (100 mg/kg body weight). After the blood was collected, the mice were perfused transcardially with 50 mL of chilled PBS to remove intravascular blood, and then the brain, kidney, liver, and spleen were harvested and homogenized. Total DNA was extracted from the homogenates and blood by using a High Pure PCR Template Preparation Kit (Roche, Penzberg, Germany) following the manufacturer’s instructions. Two sets of primers and Taqman probes were designed

to detect the DNA sequences of MPyV VP2 and mouse β-actin genes (Table 1). The plasmid pPy-1 was used as a standard DNA for real-time PCR. For quantification of mouse β-actin DNA as an internal control, the standard DNA was amplified Dipeptidyl peptidase by conventional PCR from the mouse brain DNA using a specific primer set and Ex Taq (Takara) (Table 1). Real-time PCR was performed on each DNA sample using a LightCycler 480 Probe Master (Roche) and LightCycler (Roche) according to the manufacturer’s protocol. The copy numbers of MPyV DNA were normalized with reference to those of mouse β-actin DNA. In BALB/c mice, the amount of viral DNA in the brain peaked at 4 days p.i. and declined gradually at later time points (Fig. 1a). The MPyV DNA levels in the blood, kidney, and liver of BALB/c mice peaked at 6 days p.i. and were lower than those in the brain, while a marked and temporal elevation of viral DNA was seen in the spleen at 6 days p.i. (Fig. 1a). When KSN nude mice were inoculated with MPyV, the amount of viral DNA in the brain increased up to 4 days p.i. and remained unchanged during the observation period of 14 days (Fig. 1b). The viral DNA levels in the blood, kidney, and liver of KSN mice were similar to those of BALB/c mice (Fig. 1b). In contrast, the viral DNA in the spleen of KSN mice notably increased from 8 days p.i. and remained at a similar level up to 14 days p.i. (Fig. 1b).