2.3. HCV Chimeric Mouse Study

MUP-uPA-SCID/Beige mice (gift from A. Kumar), which have the uPA gene driven by the major urinary protein promoter [50], can be engrafted with human hepatocytes until the age of 12 months. MUP-uPA-SCID/Beige mice (4 months old) were engrafted with 10⁷ human hepatocytes per mouse. We normally acquired ~300–500 × 10⁶ hepatocytes per donor from D. Geller as we reported previously [39]. After viability confirmation, fresh human hepatocytes were engrafted straightaway upon arrival within 16 h post-isolation. A skin incision (1 cm) was made in the upper left abdomen quadrant to inspect the spleen for the intrasplenic injection of human hepatocytes. Vetbond tissue adhesive (3M Animal Care Products, St. Paul, MN, USA) was used to close the incision. Human albumin (hAlb) blood levels were quantified using ELISA (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s protocol to verify the degree of “humanization” of the animals. Mice expressing >300 μg/mL of hAlb were selected and randomized into groups (n = 10). Although the uPA transgene expression induces murine liver damage, engrafted human hepatocytes visualized via hAlb immunostaining rapidly restore the functionality of the humanized liver [50]. We confirmed that enhancing the number of engrafted human hepatocytes is critical to obtaining functional repopulated human hepatocytes by quantifying serum hAlb via ELISA a month after engraftment [50]. Humanized liver mice were then infected intravenously (i.v.) with serum from infected chimpanzees (100 infectious doses): HC-TN GT1a, HC-J6 GT2a, S52 GT3a, and ED43 GT4a (gift from Dr. Lanford). CRV431 was dissolved in PEG-300, while velpatasvir and sofosbuvir were dissolved in DMSO and subsequently in a 95% sterile saline solution. Drugs were administered once by oral gavage at 50 mg/kg, and blood was collected retro-orbitally at the indicated time points.