HCV chimeric mouse study

Transgenic mice carrying the uPA gene driven by the major urinary protein promoter were crossed onto a SCID/Beige background (MUP-uPA-SCID/Beige) [31]. These transgenic mice are healthier than former Alb-uPA mice and provide an extended window from age 4 to 12 months for engraftment with human hepatocytes and infection with viral hepatitis viruses derived from serum of infected chimpanzees or from concentrated supernatant of HCV-replicating cell culture [31]. MUP-uPA-SCID/Beige mice (gift from A. Kumar) (4 months old) were transplanted with human hepatocytes (10⁷ cells/mouse) (we usually obtain ~300-500x10⁶ hepatocytes per donor, gift from D. Geller) as described previously [31]. In brief, fresh hepatocytes were transplanted immediately upon arrival within 12–16 hour after isolation. Viable cell counts were determined. One cm skin incision was made in the upper left quadrant of the mouse abdomen to visualize the spleen. Human hepatocytes were injected intra-splenically. Incision was then closed with Vetbond tissue adhesive (3M Animal Care Products, St. Paul, MN). To verify the degree of “humanization”, blood was weekly collected for human albumin quantification by ELISA (Bethyl Laboratories) according to the manufacturer’s protocol. Mice expressing >300 μg/mL of human albumin were randomized into groups of 10 mice. Note that the expression of the uPA transgene causes damage to the murine liver that is rapidly replaced by clusters of implanted human hepatocytes that can be visualized by human albumin immunostaining [31]. MUP-uPA-SCID/Beige mice that had been engrafted with human hepatocytes were infected intravenously (i.v.) with diluted plasma from HCV-infected chimpanzee (100 infectious doses (CID₅₀)/mL of genotype 2a HCV) (gift from R. Lanford) or concentrated viruses (genotype 1a, 2a, 3a and 4a) derived from cell culture [16]. CRV431 was dissolved in polyethylene glycol 300 molecular weight (PEG-300), NV556 was dissolved in 5% absolute ethanol, 5% Cremophor EL, and 90% sterile saline solution, and sofosbuvir was dissolved in DMSO and subsequently in 95% sterile saline solution. Drugs were administered once by oral gavage at 50 mg/kg at the indicated time points. Blood was collected retro-orbitally at the indicated time points.