Supporting Information Fujii et al. 10.1073/pnas.1217563110 A Human Cen chromosome 4q ART3 NUP54 SCARB2 FAM47E CCDC8 Tel B Bac clones RP11-54D17 RP11-628A4 Exon 1 2 34 5 6 7 8 9 10 11 12 5 3 PCR region a b c d a b c d (Size bp) non- RD non- RD non- RD non- RD 2,000 1,000 600 500 400 300 200 100 Fig. S1. Generation of transgenic () mice expressing human scavenger receptor class B, member 2 (hscarb2). (A) Schematic diagram of bacterial artificial chromosome (BAC) clones containing the hscarb2 gene used to produce the mice. The open and closed circles show the centromere (Cen) and telomere (Tel) of human chromosome 4, respectively. The white arrows indicate the genes located upstream and downstream of the SCARB2 gene (black arrow). The cloned region in each BAC construct is shown as a black line. (Scale bar, 100 kb.) (B) PCR genotyping of the hscarb2- mouse. The diagram shows the structure of the hscarb2 gene, which consists of 12 exons. The arrowheads indicate the primer sets used for genotype analysis. The results show PCR genotyping for hscarb2-10 animals. 1of8
A rabbit goat B Human non- Intestine Spleen Kidney Liver Lung Purkinje Thalamus Hippocampus Cerebral cortex Fig. S2. Immunohistochemical analysis of hscarb2 expression in human and mouse tissues. (A) The human cerebral cortex sample was stained with rabbit anti-human SCARB2 antibody (SIGMA: Left) or goat anti-human SCARB2 antibody (R&D: Right) and counterstained with hematoxylin. (B) Immunohistochemical analysis of hscarb2 expression in human and and non- mouse tissues. The cerebral cortex, hippocampus, thalamus, Purkinje layer, lung, liver, kidney, spleen, and intestine tissues were stained with rabbit anti-human SCARB2 antibody and counterstained with hematoxylin. (Scale bars, 50 and 100 μm in human and mouse tissue images, respectively.) 2of8
16 22 16 22 Cerebellar nucleus Intestine Spinal cord anterior horn Medulla oblongata Pons Cerebral cortex Spleen Kidney Liver Lung Fig. S3. Immunohistochemical analysis of hscarb2 expression in 16 and 22 mouse tissues. The cerebral cortex, pons, medulla oblongata, spinal cord anterior horn, cerebellar nucleus, lung, liver, kidney, spleen, and intestine tissues of 16 and 22 mice were stained with rabbit anti-human SCARB2 antibody and counterstained with hematoxylin. 3of8
A B 6-week-old 10-week-old 14-week-old 0 7 14 Days post-inoculation C 3-week-old D age (week-old) 16 16 22 22 10 18 18 49 49 0 7 14 Days post-inoculation no clinical sign ataxia 0 7 14 Days post-inoculation Fig. S4. Neurological signs observed in hscarb2- mice. (A) A 3-wk-old hscarb2-10 mouse inoculated intracerebrally (i.c.) with the enterovirus 71 (EV71) Isehara strain at 1 10 6 TCID 50 is shown. The arrowhead indicates the paralyzed left hindlimb. (B) hscarb2-10 mice at the indicated ages were infected i.c. with EV71 Isehara strain at 1 10 6 TCID 50. Mice were observed daily for up to 2 wk for clinical signs. (C and D) hscarb2-16, 22, and 49 mice of the indicated age were infected with the EV71 Isehara strain at 1 10 5 TCID 50 by the indicated inoculation route. Mice were observed daily for clinical signs for up to 2 wk. 4of8
A Brain 6 6 1 1 5 5 2 2 3 4 3 4 1.Cerebral cortex 2.Thalamus 3.Pons * 4.Medulla 5.Cerebellar nuclei * 6. Purkinje layer * Lumbar spinal cord B 16 22 49 C Oral mucosa Limbs non- non- Fig. S5. Sites of viral replication in hscarb2-10 mice. (A) Histopathological changes and viral antigens in the CNS of 3-wk-old mice inoculated i.v. with the EV71 Isehara strain (shown in Table 1 and Table S2). The brains and spinal cords are shown in low-magnification sagittal and coronal sections, respectively (Left, H&E staining; Right, immunohistochemistry). The numbers indicate the parts of the brain shown at higher magnification below (Upper, H&E staining; Lower, immunohistochemistry). The images of the pons, medulla, and cerebellar nuclei are the same as those shown in Fig. 2B. The squares correspond to the areas in the lumbar spinal cord that are shown at higher magnification (Fig. 2B). Open arrowheads, closed arrowheads, and asterisks indicate degenerated neurons, neuronophagia, and gliosis, respectively. (B) Histopathological changes and viral antigen expression in the spinal cord of hscarb2-16 (17 wk old), hscarb2-22 (18 wk old), and hscarb2-49 ( wk old) mice inoculated i.c. with the EV71 Isehara strain at 1 10 5 TCID 50. (C) Histopathology of the epithelium in neonatal hscarb2-10 or non- mice after s.c. inoculation with the Isehara strain at 1 10 4 TCID 50. A number of squamous epithelium cells in terminal regions of the oral mucosa (Left) and the limbs (Right) of hscarb2- mice were positive for the EV71 antigen, whereas those in non- mice did not exhibit EV71 antigen positivity (Lower). There was no inflammation in these sites (Upper). 5of8
Table S1. Position and sequences of primers for PCR genotyping Name of primer set Primer name Region Sequence Length of PCR product (bp) a hscarb2-int1-f Intron 1 5 -AGCTCTTCTTGCGTCCCTGT-3 594 hscarb2-int1-r 5 -GCTCTCCAGCCCAATCATCT-3 b hscarb2-ex2-f Exon 2 5 -AGGAGACTGCAGTTGAATGC-3 552 hscarb2-ex2-r 5 -AGTCTGCCAACTCAGGAGGA-3 c hscarb2-ex6-f Exon 6 5 -CTGTGGATAGACAGCTCCAG-3 651 hscarb2-ex6-r 5 -AGTTGATGCTTGACAGAGCA-3 d hscarb2-ex11-f Exon 11 5 -TAACAGGAGGACATTCCCAA-3 526 hscarb2-ex11-r 5 -GTTAACTCCCAGGGGATACA-3 6of8
Table S2. Viral antigen and inflammation in the CNS of mice Spinal cord Ganglion Animal Route Dose log10 TCID50 Clinical sign Killed (day p.i.) Cerebral cortex Hippocampus Cerebellum Brainstem Thalamus Hypothalamus Pons Midbrain Medulla Cervical cord Lumbar cord Trigeminal ganglion Dorsal root ganglion Skin Skeletal muscle Others i.c. 5 Day 7 ataxia 7 / * +/++ +/++ w/++ +/++ +/++ +/++ +/++ w/++ +/+ +/ +/++ / / / i.c. 5 Day 10 ataxia 10 /+ /+ /+ /+ /+ /+ /+ /+ w/+ +/++ +/ +/+ / / / i.c. 3 Day 14 14 w/+ +/+ /++ +/++ +/++ w/++ +/++ w/++ w/++ +/++ w/+ +/++ / / / i.c. 3 Day 9 ataxia 9 /+ /+ w/++ /w +/+ +/++ +/+ +/++ +/++ +/++ /+ /+ / / / i.v. 6 Day 6 6 ++/+ / ++/+ ++/+ ++/+ ++/+ ++/++ ++/++ ++/++ ++/++ +/+ ++/++ / / / i.v. 6 Day 5 5 +/+ / ++/+ ++/+ ++/+ ++/+ ++/+ ++/+ ++/+ ++/++ ++/+ ++/+ / / / Non- i.v. 4 Day 9 i.v. 4 Day 5 i.p. 6 Day 5 9 +/ / +/+ +/w +/w +/+ +/+ +/+ +/+ +/+ +/+ +/+ / / / 5 +/ / +/+ +/+ +/+ +/+ +/+ +/+ +/++ +/+ +/+ +/+ / / / 5 /+ / +/+ /+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ / / / i.p. 6 Day 7 ataxia 7 +/+ +/ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ / / / i.p. 4 Day 8 8 / / +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ / +/+ / / / i.p. 4 Day 9 9 +/ / +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ /+ +/+ / / / i.c. Mock / / / / / / / / / / / / / / / i.c. Mock / / / / / / / / / / / / / / / After killing the affected mice (i.c.: n = 11; i.v.: n = 13; i.p., n = 7), viral antigen and inflammation were observed. This table shows representative results. +, positive; w, weak positive;, negative; i.c., intracerebral; i.p., intraperitoneal; i.v., intravenous. *Viral antigen/inflammation. Heart, lung, kidney, spleen, pancreas, intestine and lymph node. 7of8
Table S3. Detection of viral antigen positive cells in the neonatal mice Animal Animal no. Dose log10 TCID50 Killed (day p.i.) Clinical sign Cerebral cortex Hippocampus Cerebellum Brainstem Thalamus Hypothalamus Pons Midbrain Medulla Spinal cord Skin Oral mucosa Limbs Trunk Skeletal muscle Heart Lung Liver Kidney Spleen Lymph node Gastrointestinal tract Non- 1-1 4 5 Day 5 1-2 4 5 Day 5 2-1 4 4 Day 4 2-2 4 4 Day 4 3-1 4 4 Day 4 3-2 4 4 Day 4 3-3 4 4 Day 4 4-1 4 3 Day 3 10-1 4 4 Day 4 10-2 4 4 Day 4 10-3 4 4 Day 4 10-4 4 4 Day 4 10-5 4 4 Day 4 + + + + +w +w + + +w +w +w + + +w +w +w +w + + + + +w +w +w + + + + + + + + + + + + + + + + + + + + + + + + + +w +w + +w + + + + + + + + + + + + + + + + +w + + + + + + + + + + +w + + + + + + +w + After killing the affected mice, viral antigens were observed. +, positive; +w, a few cells;, negative. 8of8