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1 Supporting Information Yen et al /pnas SI Materials and Methods Cells. Madin Darby canine kidney (MDCK) cells and human embryonic kidney 293T cells were obtained from the American Type Culture Collection and were maintained in minimum essential medium (MEM) with 5% FCS and Opti-MEM (Invitrogen) supplemented with 5% FCS, respectively. Differentiated normal human bronchial epithelial (dnhbe) cells were generated as described previously (1). Viruses. The swine influenza viruses sw4167 (H1N1), sw915 (H1N2), swns29 (H1N1), and sw201 (H1N1) were obtained through continuous 13-y surveillance at an abattoir in Hong Kong as previously described (2 4); swar2976 (H1N2) and the pandemic H1N1 virus CA04 were obtained from the virus repository of St. Jude Children s Research Hospital; the pandemic H1N1 virus HK415742, isolated from the first confirmed case in Hong Kong, was obtained from Queen Mary Hospital, Hong Kong. To generate recombinant viruses, the eight gene segments of sw915 and HK viruses were amplified by RT-PCR and cloned into plasmid vector phw2000 (5). Recombinant viruses were generated by cotransfecting human embryonic kidney 293T cells (TransIT-LT1; Mirus Bio) with eight plasmids and propagating the rescued viruses in MDCK cells twice at multiplicity of infection (MOI) = to prepare stocks. The full genome sequences of the recombinant viruses were verified. Human seasonal H3N2 RG-Wuhan virus was generated as described previously (6). Infectivity and Replication Kinetics. The 50% tissue culture infectious dose (TCID 50 ) and plaque-forming units (pfu) were determined by incubating serial dilutions of virus in MDCK cells at 37 C for 72 h. A hemagglutination assay was performed to determine the end point of infection and the TCID 50 was calculated by the Reed Muench method (7). Replication kinetics in MDCK cells was determined at an MOI of pfu per cell. After incubation, the MDCK cells were washed and overlaid with infection medium (MEM with 0.3% BSA), and supernatants were collected at specified time points and stored at 70 C for virus titration. Replication kinetics in differentiated NHBE cells was determined at an MOI of 0.01 pfu per cell. Cells were washed with PBS to remove mucin before infection and were infected via the apical side with 200 μl diluted virus in the absence of trypsin. After a 1-h incubation at 37 C, viral inoculum was removed and cells were washed and incubated for 24, 48, or 72 h. Cells were then preincubated with 200 μl fresh medium at 37 C for 30 min and samples were collected and stored at 70 C for virus titration. Minigenome Assay. Subconfluent monolayers of 293T cells ( cells in 35-mm dishes) were transfected (Mirus Bio) with 1 μg viral RNA-like reporter plasmid (firefly luciferase flanked by the noncoding region of segment 7 of influenza A virus driven by human PolI promoter), 0.1 μg Renilla luciferase reporter plasmid driven by CMV promoter for transfection control, and a mixture of PB2, PB1, PA, and NP plasmids of HK or sw915 virus at a quantity of 1, 1, 1, and 2 μg, respectively. At 24 h posttransfection, cell extracts were prepared in 500 μl lysis buffer and luciferase levels were assayed (Luciferase Assay System; Promega) using a Glomax microplate luminometer (Promega). Experiments were independently repeated three times in duplicates or triplicates. Ferret Organ Collection and Virus Titration. To determine the tropism and replication efficiency of the swine and pandemic H1N1 viruses in ferrets, we inoculated two ferrets with 10 5 TCID 50 of each virus and collected tissues at 3 d postinoculation (dpi). Tissues were weighed and homogenized in sterile PBS with antibiotics. Virus titers were measured in MDCK cells (lower detection limit, TCID 50 /g) and expressed as TCID 50 /g tissue. Tissues were also fixed in 10% neutral buffered formalin and embedded in paraffin for histology examination or immunohistochemistry. Histology Analysis and Immunohistochemistry. Formalin-fixed and paraffin-embedded tissue sections were stained with hematoxylin and eosin for histology or with pooled monoclonal antibodies against influenza A virus nucleoprotein (NP) for detection of influenza viral antigen. RT-PCR and Virus Sequencing. Viral RNAs were isolated from cellculture supernatant or directly from ferret nasal washes (RNeasy; Qiagen) and one-step RT-PCR was done with a universal primer set (8). The sequences were analyzed on Applied Biosystems 3700 DNA analyzers using BigDye Terminator (v. 3) chemistry and synthetic oligonucleotides. Hemagglutinin Glycan Array. The sialoside array containing 29 synthetic α2,3- or α2,6-linked sialyl glycans was prepared as described (9) at the Genomic Research Center, Academia Sinica. Sw915 and HK viruses were propagated in MDCK cells and inactivated with formaldehyde at a final concentration of 0.25%. Viruses ( and pfu/ml of sw915 and HK viruses, respectively, after ultrafiltration; Millipore) inactivated with oseltamivir carboxylate at a final concentration of 10 μm were overlaid on the slide and incubated for 1 h; the slides were then washed. Bound virus was detected by treatment with polyclonal rabbit anti-h1 antibodies followed by fluorescence-labeled secondary antibody. K d was determined by measuring the intensity of binding to serially diluted sialosides (10). The binding equilibrium data were fitted to the Langmuir isotherms (11), assuming a single ligand binding site, by using nonlinear regression (Prism; GraphPad). Statistical Analysis. Comparisons used one-way and two-way ANOVA followed by Tukey s posttest in GraphPad Prism software. 1. Chan MC, et al. (2010) Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract. Am J Pathol 176: Smith GJ, et al. (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459: Vijaykrishna D, et al. (2010) Reassortment of pandemic H1N1/2009 influenza A virus in swine. Science 328: Vijaykrishna D, et al. (2011) Long-term evolution and transmission dynamics of swine influenza A virus. Nature 473: Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 97: Yen HL, et al. (2005) Neuraminidase inhibitor-resistant influenza viruses may differ substantially in fitness and transmissibility. Antimicrob Agents Chemother 49: Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27: Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146: Liao HY, et al. (2010) Differential receptor binding affinities of influenza hemagglutinins on glycan arrays. J Am Chem Soc 132: Liang PH, Wang SK, Wong CH (2007) Quantitative analysis of carbohydrate-protein interactions using glycan microarrays: Determination of surface and solution dissociation constants. J Am Chem Soc 129: Haes AJ, Van Duyne RP (2002) A nanoscale optical biosensor: Sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles. J Am Chem Soc 124: of8

2 Fig. S1. Plaque morphology of the recombinant viruses in MDCK cells after a 72-h incubation at 37 C. Fig. S2. A panel of 29 α2,3- or α2,6-linked sialosides used for glycan array analysis. Table S1. Area under the curve as an estimate of the cumulative quantity of virus shed AUC (mean ± SD) Virus Donor Direct contact Droplet contact RG-Wuhan ± 0.7 (n =3) 23.5± 2.6 (n =3) 25.6± 2.9 (n =2) CA ± 0.9 (n =3) 25.2± 0.5 (n =3) 21.1± 7.7 (n =3) HK ± 3.7 (n =3) 25.0± 1.3 (n =3) 26.0± 1.9 (n =3) Sw ± 1.8 (n =3) 24.9± 4.7 (n =3) SwAR ± 0.7 (n =3) 21.1± 2.4 (n =3) SwNS ± 3.2 (n =3) 16.8± 7.9 (n =2) Sw ± 3.7 (n =3) 28.2± 1.5 (n =3) Sw915 (I and II) 11.1 ± 1.6* (n =3) 14.2± 2.6 (n =3) 10.0(n =1) RG-sw ± 2.9 (n =3) 20.7± 5.3 (n =3) 21.5(n =1) RG-sw915xHK NA 13.8 ± 1.6 (n =3) 17.3± 5.3 (n =3) 19.6± 1.6 (n =3) RG-sw915xHK PB2,PA,HA,NA 17.9 ± 1.0 (n =3) 24.5± 1.5 (n =3) 17.5± 9.2 (n =2) The area under the curve (AUC) represents total virus shedding (log 10 TCID 50 /ml) on days 2, 4, 6, 8, 10, 12, and 14 dpi as calculated from the nasal washes of each ferret and the mean ± SD. AUCs are shown. Number of ferrets per group is shown in parentheses. The detection limit was TCID 50 /ml. *The sw915-inoculated ferret shed significantly less virus than the swar2976-, sw4167-, or sw201-inoculated ferrets (P < 0.05, one-way ANOVA and Tukey s test). 2of8

3 Table S2. Rectal virus shedding and clinical signs Donor Direct contact Respiratory droplet contact Virus Rectal shedding ΔTemp ( C) Sneezing (no. of animals sneezed/total no. of animals) Weight loss (%) Rectal shedding ΔTemp ( C) Sneezing (no. of animals sneezed/total no. of animals) Rectal shedding ΔTemp ( C) Sneezing (no. of animals sneezed/total no. of animals) RG-Wuhan ND 0.9 ± 0.2 2/3 1.8 ± 2.1 (2/3) ND 0.8 ± 0.3 0/3 ND 0.5 ± 0.3 2/2 CA04 1/3 1.0 ± 0.1 2/3 5.4 ± 2.0 (3/3) 1/3 0.4 ± 0.3 1/3 0/3 1.2 ± 0.4 2/3 HK /3 0.8 ± 0.4 3/3 8.9 ± 0.8 (2/3) 1/3 0.9 ± 0.2 1/3 2/3 0.7 ± 0.1 2/2 SwHK4167 1/3 0.8 ± 0.1 1/3 5.0 ± 3.6 (2/3) 1/3 0.7 ± 0.3 1/3 0/3 NA NA SwAR2976 0/3 0.8 ± 0.7 1/3 2.3 ± 1.1 (2/3) 0/3 1.0 ± 0.3 2/3 0/3 NA NA SwNS29 1/3 0.8 ± 0.1 3/3 0.6 (1/3) 0/3 0.7 ± 0.4 0/3 0/3 NA NA Sw201 0/3 1.0 ± 0.5 2/3 2.8 ± 1.6 (3/3) 0/3 1.0 ± 0.0 2/3 0/3 NA NA Sw915 (I) 0/3 0.7 ± 0.3 2/3 4.3 (1/3) 0/3 0.7 ± 0.3 1/3 0/ /1 Sw915 (II) 0/3 0.6 ± 0.2 2/ (1/2) 0/3 0.6 ± 0.1 1/2 0/2 NA NA RG-sw915 1/3 0.7 ± 0.8 2/3 7.9 ± 4.8 (2/3) 0/3 0.7 ± 0.4 1/3 0/ /2 RG-sw915xHK NA 1/3 0.9 ± 0.2 2/3 3.4 (1/3) 0/3 0.5 ± 0.2 3/3 0/3 0.7 ± 0.5 1/3 RG-sw915xHK PB2,PA,HA,NA 0/3 1.5 ± 0.2 3/3 5.1 ± 4.2 (3/3) 1/3 1.3 ± 0.8 2/3 0/3 0.4 ± 0.5 1/2 ND, not done; NA, not available (no respiratory droplet transmission). 3of8

4 Table S3. Virus neutralization assay to determine pre- and postcontact antibody titers against homologous virus CA04 HK RG- Wuhan RGsw915xHK NA RGsw915xHK RGsw PB2,PA,HA,NA Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Direct contact Upper cage <20 1, >2, >2, >2, ,360 Middle cage , >2, >2, >2, ,200 Lower cage NA 1, >2, >2, >2, >2, ,920 Aerosol contact Upper cage >2, >2, >2, Middle cage , >2, >2, >2, ,560 Lower cage >2, sw4167 swar2976 swns29 sw201 sw915 (I) sw915 (II) Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Direct contact Upper cage >2, <20 >2, , Middle cage , >2,560 <20 >2, , Lower cage , >2,560 <20 2, ,920 NA NA Aerosol contact Upper cage , Middle cage Lower cage NA NA Sera were collected before and d after contact with the inoculated ferret. Sera were treated with receptor destroying enzyme (RDE) before the virus neutralization assay. Values are the mean from two to four replicates. 4of8

5 Table S4. Amino acid differences between the sw915 and HK viruses Gene Residue sw915 HK PB2 54 K R 104 P S 183 M L 184 T A 255 I V 292 I V 315 M I 344 M V 411 V I 453 P S 526 K R 559 T I 623 S A 645 M L 684 A S 740 D N PB1 113 I V 172 K E 175 D N 212 V L 298 L I 364 L I 386 R K 435 T I 480 R K 517 I V 587 A V 618 E D 678 N S 728 I V PA 44 I V 85 T I 186 G S 204 R K 213 R K 238 T P 254 S N 262 K R 275 P L 277 S H 304 I L 323 I V 336 L M 356 K R 404 S A 581 M L 626 K R 672 F L 684 R G 716 E K HA* 46 R K 70 L I 80 (Cb site) F S 91 N S 117 D E 145 (Ca2 site) N K 146 G S 149 R K 171 N D 219 (220-loop) T I 227 (220-loop) A E 260 I M 5of8

6 Table S4. Cont. Gene Residue sw915 HK K E 263 S A 273 (Ca1 site) S P 297 V I 300 V I 304 E K 313 R K 316 M L 347 I V 376 G E 390 T I 420 V I 445 R K 475 D N NP 53 E D 217 I V 313 F V 316 I M 373 A I 425 I V 444 I V 473 S N M2 11 I T 13 N S 14 G E 21 G D 43 V T 51 V I 54 L R NS1 6 V M 21 Q R 78 K R 91 A S 94 N T 114 S P 119 M L 124 I M 129 I V 145 V I 153 G E 171 D Y 180 I V 197 S N 198 L I 206 R C 207 N D 209 D N NS2 40 V I 52 T M 81 G E 83 V M 115 T A Residues located in the antigenic sites are set in italics and those in receptor-binding domains are underlined. *H3 numbering. 6of8

7 Table S5. Virus titers detected in ferret organs at 3 dpi Virus Olfactory bulb Nasal turbinate Trachea (pharynx) Trachea Right bronchi Left bronchi Right upper lung lobe Right lower lung lobe Left upper lung lobe Left lower lung lobe Descending colon RG-Wuhan < 5.4 ± ,<,<* <* <* 1.625,<,<* <* <* <* 3.0,<,<* < CA04 < 7.2 ± ± ± ± ± ± ± ± ± 0.5 < HK < 7.0 ± ± ± ± ± ± ± ± ± 0.6 < Sw ,<,< 6.9 ± ± ± ± ± ± ± 1.2* 4.6 ± ± ,< SwAR2976 < 5.4 ± ± ± ± ± ± ± 0.6* 6.3,< 6.3 ± 0.5 < SwNS29 < 5.8 ± ± 0.1* 3.5 ± ± ± 0.1 <, <* <, ± ± 1.3 < Sw201 < 4.5 ± ± ± ± ± ,< 4.5,< 6.0,< 5.8 ± 0.4 < Sw915 < 5.0 ± 0.4* 3.8 ± 0.4* 3.5 ± ± ± 0.7 <, <* 3.1 ± 0.4* 3.5 ± 1.2 <, 4.0 < RG-sw915 < 3.9 ± ± ± ± ± ± ± ± ± 0.9 < RG-swHK915xHK NA < 5.9 ± ± ± ± ± ± ± ± ± 0.7 < RG-swHK915xHK PB2,PA,HA,NA < 6.8 ± ± ± ± ± ± ± ± ± 1.1 < Ferrets were intranasally inoculated with 10 5 TCID50 of virus in 0.5 ml medium. Two or three ferrets per group were euthanized at 3 dpi for virus titration in organs. Values are mean ± SD log10tcid50/ml. <, below the detection limit of 1.5 log 10 TCID 50 /ml. *P < 0.05 compared with the CA04 and HK pandemic viruses (t test). 7of8

8 Table S6. Antigen detection in the ferret respiratory tract by immunohistochemistry Trachea Bronchi/lungs Virus Epithelium Submucosal glands Epithelium Submucosal glands Wuhan CA (bronchiole) + HK (bronchiole) +++ Sw (bronchiole and alveoli) + SwAR (bronchiole and alveoli) ++ SwNS29 + (bronchial and alveoli) + Sw201 + (bronchiole) +++ Sw915 + (bronchial) +++ RG-sw915 + (bronchiole and alveoli) ++ RG-sw915xHK NA (bronchiole and alveoli) ++ RG-sw915xHK PB2,PA,HA,NA + ++ (alveoli) +++ Table S7. Binding affinity of HK and sw915 for α2,6-linked sialyl glycans Affinity for sialosides (K surface-diss., mean ± SD μm) Virus Glycan 23 Glycan 27 Glycan 28 Glycan 29 HK ± ± ± ± 4.1 Sw ± ± 12.3* 38.3 ± ± 4.5 HA-binding affinity was assayed by determining the K d with serially diluted sialosides and fitting the data to the Langmuir isotherms (1) by nonlinear regression, assuming a single, independent ligand binding site. Mean ± SD of the K surface-diss. (μm) is shown for each sialoside. *P < 0.05, t test. 8of8