MAVS-dependent host species range and pathogenicity of human hepatitis A virus by Asuka Hirai-Yuki, Lucinda Hensley, David R. McGivern, Olga González-López, Anshuman Das, Hui Feng, Lu Sun, Justin E. Wilson, Fengyu Hu, Zongdi Feng, William Lovell, Ichiro Misumi, Jenny P.-Y. Ting, Stephanie Montgomery, John Cullen, Jason K. Whitmire, and Stanley M. Lemon Science Volume 353(6307):1541-1545 September 30, 2016 Published by AAAS

Fig. 1 HAV infection in DKO (Ifnar1−/−Ifngr1−/−), Ifnar1−/−, and Ifngr1−/− mice. HAV infection in DKO (Ifnar1−/−Ifngr1−/−), Ifnar1−/−, and Ifngr1−/− mice. (A) Representative course of infection in a DKO mouse inoculated intravenously with 107 genome equivalents (GE) of human HAV (chimpanzee fecal extract). (B) Hematoxylin and eosin–stained liver from representative infected (top) and control (bottom) DKO mice at 41 dpi showing inflammatory infiltrates; scale bar, 50 μm. Inset shows apoptotic hepatocytes; scale bar, 12.5 μm. (C) Summary of serial passage of HAV in DKO mice showing intrahepatic HAV RNA (left) and fecal HAV RNA (right), source and magnitude of HAV inocula (ptF, chimpanzee feces; mF, DKO mouse feces; mL, DKO mouse liver), and day of harvest. Data are means ± SEM or range; n = 2 or 3 animals (shown by open circles in each bar). *P < 0.05, ***P < 0.001 for p1 versus p5 [one-way analysis of variance (ANOVA)]. (D) Fecal HAV RNA (top) and serum ALT (bottom) in DKO, Ifnar1−/−, Ifngr1−/−, and wild-type (WT) BL6 mice challenged with fourth-passage DKO liver extract (2.6 × 108 GE). Data are means ± SEM; n = 4. *P < 0.05, ***P < 0.001 for Ifnar1−/− versus DKO (ANOVA). (E) Intrahepatic HAV RNA in WT, DKO, Ifnar1−/−, and Ifngr1−/− mice at 127 dpi (mean ± range, n = 2). (F) Intrahepatic HAV RNA in Ifnar1−/− mice infected with fourth-passage liver extract. Symbols represent individual mice. Dotted horizontal lines in panels indicate level of detection (RNA) or upper limit of normal (ALT). Asuka Hirai-Yuki et al. Science 2016;353:1541-1545 Published by AAAS

Fig. 2 HAV infection in Ifnar1−/− and Mavs−/− mice. HAV infection in Ifnar1−/− and Mavs−/− mice. (A) Fecal HAV RNA on day 7 and day 14 after intravenous challenge of different genetically deficient mice. Data are means ± SEM; n = 3 to 5. (B) Viral RNA in livers of Ifnar1−/− and Mavs−/− mice at 15 and 63 dpi. Data are means ± SEM; n = 2 to 5 as shown. *P < 0.05, ***P < 0.001 (two-sided t test). (C) Serum ALT at 7 and 14 dpi in genetically deficient mice, with expanded low ALT range at right. Data are means ± SEM; n = 5. **P < 0.01, ***P < 0.001 for combined day 7 and day 15 data (two-sided Mann-Whitney test); ns, not significant. (D) Immunohistochemical staining of cleaved caspase 3 in liver from representative (top) Ifnar1−/− versus (bottom) Mavs−/− mice at 15 dpi; scale bar, 100 μm. Inset shows an apoptotic hepatocyte; scale bar, 12.5 μm. (E) Tissue distribution of HAV RNA in infected Ifnar1−/− and Mavs−/− mice. Data are means ± SEM; n = 3 or 4. ***P < 0.001 (multiple t test with false discovery rate of 1%). (F) Fecal virus shedding in infected Ifnar1−/− or Mavs−/− mice over 56 days of infection. Data are means ± SEM; n = 3 to 5. Asuka Hirai-Yuki et al. Science 2016;353:1541-1545 Published by AAAS

Fig. 3 Cellular and cytokine response to HAV infection in DKO and Ifnar1−/− mice. Cellular and cytokine response to HAV infection in DKO and Ifnar1−/− mice. (A) Estimated intrahepatic leukocyte numbers in naïve and infected Ifnar1−/− mice at 7 dpi. Data are means ± SD; n = 5 (mean ALT = 372 IU/liter). **P < 0.01, ***P < 0.001 (two-way ANOVA with Tukey multiple comparison test). (B) Dual immunohistochemical staining of infected Ifnar1−/− liver for CD4 (magenta) and CD8 (brown) showing a mixed cellular infiltrate at 14 dpi. Scale bar, 10 μm. (C) Relative increase in liver cytokine levels in HAV-infected DKO mice (Luminex assay) with ALT >200 IU/liter. Data are means ± range; n = 2. (D) Serum IFN-β measured by ELISA at 7 dpi. Data are means ± SD; n = 4. (E) Relative increase in intrahepatic cytokine and chemokine mRNA abundance in Ifnar1−/− mice. Data are means ± SEM; n = 4 or 5. (F and G) Immunoblots of phospho-Ser396 and total IRF3 (F) and ISG15 (G) in livers from HAV-infected and naïve DKO mice; β-actin was included as a loading control. (H) Intrahepatic transcripts of IRF3-regulated ISGs, ISG15, IFIT1 (ISG56), CCL5 (RANTES), and ISG20 (not directly regulated by IRF3) in HAV-infected DKO (n = 4) and Mavs−/− (n = 3) mice and naïve animals at 18 to 28 dpi. *P < 0.05 (t test). Asuka Hirai-Yuki et al. Science 2016;353:1541-1545 Published by AAAS