Relative activity (%) SC35M

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1 a 125 Bat (H17N) b 125 A/WSN (H1N1) Relative activity (%) Relative activity (%) Pos. Neg. PA PB1 Pos. Neg. NP PA PB1 PB2 0 Pos. Neg. NP PA PB1 PB2 SC35M Bat Supplementary Figure 1. Functional complementarities of bat and influenza A virus NP. a-b Relative polymerase activity of H17N (a) or WSN/33 (b) determined in HEK293T cells without (Pos.) or after substitution of the indicated single components of the influenza A virus strains A/ SC35M or bat H17N. Omission of the polymerase subunit PB1 (Neg.) served as a negative control. Shown are mean and SD of 3 independent experiments.

2 a CarperAEC.B b NPTr c A SC35M Bat C1 Bat C2 Bat C3 d EpoNi/ SC35M (37 C) SC35M (33 C) Bat C3 (37 C) Bat C3 (33 C) 9 CarperAEC.B SC35M (37 C) SC35M (33 C) Bat C3 (37 C) Bat C3 (33 C) EpoNi/ SC35M (37 C) SC35M (40 C) Bat C3 (37 C) Bat C3 (40 C) 9 CarperAEC.B SC35M (37 C) SC35M (40 C) Bat C3 (37 C) Bat C3 (40 C) Supplementary Figure 2. Viral growth of Bat C1, C2 and C3 in cell cultures. a-c, Bat CarperAEC.B-3 (a), newborn pig trachea (NPTr) (b) or human adenocarcinoma lung (A549) cells (c) were infected with the indicated viruses at an MOI of Shown are mean and SD of 3 independent experiments. d, Bat EpoNi/22.1 and CarperAEC.B-3 cells were infected with Bat C3 or SC35M at an MOI of 0.01 at either 33, 37 C or 40 C. Shown are mean and SD of 3 independent experiments.

3 a BALB/c (lungs) SC35M ( 3 PFU) Bat C1 ( 4 PFU) Bat C3 ( 4 PFU) Days p.i. b BALB/c (nasal turbinates) Bat C1 ( 4 PFU) Bat C3 ( 4 PFU) Days p.i. c 1 BALB/c Body weight (%) Bat C3 ( 6 PFU) Days p.i. d Bat Bat C1 Bat C2 NS C1 C2 C3 C1 C2 C3 B6 B6-STAT1 / Bat C3 NS Bat C1 * Bat C2 Bat C3 Supplementary Figure 3. Viral growth of Bat C1, C2 and C3 in mice. Viral titers in lung (a) and nasal turbinate (b) of BALB/c mice infected with Bat C1 or Bat C3 ( 4 PFU) for the indicated time. c, Weight of BALB/c mice (n=5/group) infected with the indicated viral dose. Shown are mean and SD. d, Viral lung titers (PFU per lung) in B6 or B6- STAT1-/- mice intranasally infected with 4 PFU of Bat C1, C2 and C3 72h post infection. Student s t-test was performed to determine the P value. *P<0.05; NS, not significant.

4 a HEK293T b LMH Relative activity (%) Relative activity (%) Bat+ Bat- PA S550R PB1 E656G Bat+ Bat- PA S550R PB1 E656G Supplementary Figure 4. Avian-adaptive mutations in PB1 and PA enhances viral polymerase activity. The bat polymerase activity was determined in avian LMH or human HEK293T cells in the presence of wt (Bat+), PA S550R or PB1 E656G. Omission of PB1 served as a negative control (Bat-). Shown are mean and SD of 3 independent experiments.

5 Supplementary Figure 5. Protein alignment of M2 host-specific consensus sequences. Protein sequences of M2 proteins of influenza isolates were grouped according to their host and aligned using MUSCLE. The four consensus sequences obtained from the alignments were compared. The consensus sequence is illustrated as sequence logo. Mean pairwise identity over all pairs in the column is given in green (0% identity), brown (at least 30% and under 0% identity) and red (below 30% identity). Transmembrane regions predicted by TMHMM are indicated in greyish bar. Arrow indicates position 31.

6 Supplementary Table 1. Reconstitution and detection of virus-like particles (VLP) using a GFP minigenome. Bat/SC35M HA-NA-3P*-NP-M1-M2-NEP VLP no** no no no no no** no** * 3P = PB2, PB1 and PA ** in the presence or absence of trypsin in the culture medium

7 Supplementary Table 2. Sequence variations observed in Bat C1, C2 and C3 virus stocks prepared in MDCK cells by deep sequencing. Segment Pos Change VarFreq* AA Change Bat C1 1_PB2 99 C>T 11% silent 3_PA 1672 T>G 99% S>R (550) 6_NA 621 G>A 98% V>I (167) Bat C2 2_PB A>G 98% E>G (656) 4_HA 1646 A>G 49% silent 8_NS 177 T>C 30% silent Bat C3 4_HA 34 T>C 0% 3`UTR 4_HA 1307 C>A 58% silent Nucleotides different to the published H17N sequence are highlighted in green. Nucleotide changes that cause amino acid changes are highlighted in orange. * VarFreq, variant frequency

8 Supplementary Table 3. Mutations identified in Bat C1, C2 and C3 after amplification in avian DF-1 cells. Segment Pos Change VarFreq* AA Change Bat C1 7_M2 803 A>G n.d.** N>S (31) 7_M2 919 A>G n.d. T>A (70) Bat C2 4_HA ab 1307 C>T 22 a;30 b% silent 7_M2 ab 802 A>C 22 a;19 b% N>H (31) 7_M1 b 341 G>A 14% silent 7_M1 b 347 T>G 14% silent 7_M1 b 365 A>G 16% silent 7_M1 b 369 G>T 16% A>S (116) 7_M1 a 441 A>G 11% T>A (140) 7_M2 a 803 A>G 15% N>S (31) Bat C3 3_PA 1835 C>T n.d. P>S (605) 4_HA 307 T>C n.d. I>T (59) 7_M1 441 A>G n.d. T>A (140) Bat C1 and C3 mutations were identified by sequencing RT-PCR amplification products. Bat C2 mutations were identified by deep sequencing from two independent virus stocks (a and b). Nucleotide changes are highlighted in pink. Amino acid changes are indicated in orange. * VarFreq, variant frequency; ** n.d., not determined

9 Supplementary Table 4. Sequence variations observed in Bat C1, C2 and C3 virus stocks prepared from eggs infected with MDCK- or DF-1-derived virus stocks by deep sequencing. MDCK-derived virus stock DF-1-derived virus stock Segment Pos Change VarFreq* AA Change Segment Pos Change VarFreq* AA Change Bat C1 4_HA ab 78 G>A 86 a;0 b% R>Q (316) Bat C1 1_PB2 ab 587 A>G 11 a;14 b% K>R (187) 4_HA b 967 A>G 34% D>G (279) 2_PB1 a 1686 G>A 16% D>N (554) 7_M2 a 803 A>G % N>S (31) 4_HA b 966 G>T 11% D>Y (279) 7_M2 a 986 T>C 39% V>A (92) 4_HA a 78 G>A 11% R>Q (316) 4_HA b 1379 A>G 13% silent (416) 4_HA b 1384 A>T 14% Q>L (418) 4_HA b 1386 A>T 13% I>F (419) 6_NA b 858 A>C 11% S>R (246) 6_NA b 861 A>T 12% S>C (247) Bat C2 3_PA ab 1671 G>A 27 b;74 a% S>N (550) Bat C2 4_HA ab 1307 C>T 61 a;66 b% silent (392) 4_HA ab 179 T>C 12 b;64 a% silent (16) 7_M2 ab 802 A>G 29 a;39 b% N> H (31) 4_HA ab 78 G>A 32 b;88 a% R>Q (316) 7_M2 ab 803 A>G 53 b;64 a% N>S (31) 4_HA b 1301 C>T 15% silent (390) 7_M2 ab 904 A>G 11 b;29 a% R>G (65) 4_HA b 1307 C>T 15% silent (392) 7_M2 ab 930 A>G 11 b;30 a% silent (73) 8_NS1 b 676 G>T 11% D>Y (217) 2_PB1 a 92 T>A 13% F>L (22) 8_NEP b 676 G>T 11% K>N (58) 7_M a 04 A>T 14% 5`UTR Bat C3 3_PA b 565 A>G 30% silent (181) Bat C3 5_NP a 639 T>G 13% M>R (200) 4_HA b 1153 G>A 43% R>Q (341) 7_M2 a 802 A>C 19% N>H (31) 6_NA a 824 A>G 40% silent (234) Nucleotide changes are highlighted in purple. Amino acid changes are highlighted in orange. Sequences were determined from two infected eggs (a and b) with highest titer. * VarFreq, variant frequency

10 Supplementary Table 5. Rescue attempts of Bat H17N and SC35M reassortant viruses by reverse genetics using authentic genome segments. Bat H17N SC35M Rescue 3P*, M, NP, HA, NA, NS no 3P, M, NP, HA, NA NS no 3P, M, NP, NS HA, NA no 3P, NP, NS HA, NA, M no 3P, NP HA, NA, M, NS no M, NS HA, NA, 3P, NP no NP HA, NA, 3P, M, NS no M HA, NA, 3P, NP, NS no NS HA, NA, 3P, NP, M no PB2 HA, NA, 2P**, NP, M, NS no HA, NA, 3P, NP, M, NS * 3P = PB2, PB1 and PA ** 2P = PB1 and PA

11 Supplementary Table 6. Reassortment events after co-infection of MDCKII cells. Strain Initial infection dose (MOI) Number of clones Number of parental strains Reassortment events Bat C3:SC35M 1: * 48 Bat C3 / 44 SC35M 0 Bat C3:WSN 1: Bat C3 / 2 WSN 0 Bat C3:WSN 1: Bat C3 / 27 WSN 0 Bat C3:WSN 1:1 0 3 Bat C3 / 97 WSN 0 SC35M:WSN 1:1 0 0 SC35M / 95 WSN 5 * after analysis, 6 clones contained a mixture of both parental viruses, but no segment reassortment.

12 Supplementary Table 7. A list of primers used to identify strain specific genome segments in reassortment experiments. WSN vs SC35M PB2-F WSN GCACAGGATGTAATCATGGAAGT PB2-R WSN CAGTTCTCTCTCCAACATGTATGC SC35M GCACAGGATGTAATCATGGAAGT SC35M ACCAATTCTCTTTCTAGCATGTATGC PB1-F WSN CTAATTAGGGCATTAACCCTGAACACAATGAC PB1-R WSN ACAAACCCCCTTATTTGCATCCC SC35M CTAATAAGAGCATTGACACTGAACACAATGAC SC35M ACAAATCCTCTGATTTGCATCCC PA-F WSN GGGGAGGAAATGGCCAC PA-R WSN CTGACGAAAGGAATCCCAGAG SC35M ACTGGAGAAGAAATGGCCAC SC35M GATTGACGAAAGGAATCCCAGAG HA-F WSN TCTATTTGGAGCCATTGCTGG HA-R WSN TGCTTTTTTGATCCGCTGCA SC35M GCTTTTGGGAGCAATTGCTGG SC35M GTGCTTTTGTAGTCAGCTGCA NP-F WSN CTTTGAAAGAGAGGGATACTCTCTAGT NP-975-R WSN CTGTTTTGAAGCAGTCTGAAAGGG SC35M GAGAGAGAGGGATACTCCCTAGT SC35M CTGTTTTGGAGCAGGCGGAAAG NA-F WSN GTTCCAGACATGGGTTTGAGATG NA-R WSN TCCGCTGTACCCTGACC SC35M cgttccagaagtggttttgagatg SC35M AAACTCCCTGAGTATCCTGACC M-F WSN GATAACATTCCATGGGGCCAA M-R WSN GCCACTTCAGTGGTCACAG SC35M GAAATTACATTCCATGGGGCCAA SC35M CCACTTCGGTGGTCACAG NS-F WSN GGACCAGGCGATCATGGATAA NS-R WSN TGTCCCCTCTTCGGTGAA WSN vs Bat C3 SC35M GGACCAGGCGATCATGGATAA SC35M CAATTGCTCCTTCTTCGGTGAA PB2-F WSN GCACAGGATGTAATCATGGAAGT PB2-R WSN CAGTTCTCTCTCCAACATGTATGC Bat GGCACAAGAAGTCATTATGGAAGT Bat TTCCCTTTCCAGCATGTATGC PB1-F WSN TGATGGGCATGTTCAATATGTTAAGTAC PB1-R WSN AGATTGAAGACCATCCCACCA Bat ATGATGGGAATGTTCAATATGCTTAGTAC Bat GGAGGCCATCCCACCA PA-F WSN CACAAATGGGAGAAGTACTGTGTTCTTGA PA-R WSN CTCATTTCCATCCCCCATTTCATTTT Bat CATAAGTGGGAGAAATACTGCGTACTAGA Bat CTCATTTCCATTCCCCATTTCATTTT HA-F WSN TCTATTTGGAGCCATTGCTGG HA-R WSN TGCTTTTTTGATCCGCTGCA (same as WSN/SC35M) Bat GCTTTTGGGAGCAATTGCTGG Bat GTGCTTTTGTAGTCAGCTGCA NP-F WSN GACATGAGAACCGAAATCATAAGGCTGATGGAAA NP-R WSN ACTCCTCTGCATTGTCTCCGAAGAAATAAGA Bat GACATGAGGACTGAAATAATCAGACTTATGGAAA Bat ACTCGTCTGCCTTGTCTCCAAAGAAATAAGA NA-F WSN GTTCCAGACATGGGTTTGAGATG NA-R WSN TCCGCTGTACCCTGACC (same as WSN/SC35M) SC35M cgttccagaagtggttttgagatg SC35M AAACTCCCTGAGTATCCTGACC M-F WSN GATAACATTCCATGGGGCCAA M-R WSN GCCACTTCAGTGGTCACAG Bat GATAACTTTCCATGGGGCCAA Bat GCCACTTCGGTTGTCACAG NS-F WSN GGACCAGGCGATCATGGATAA NS-R WSN TGTCCCCTCTTCGGTGAA SC35M vs Bat C3 Bat CATGGACCAAGCAGTAACCAATAA Bat ACTACTGTCCCTTCTTCTGTGAA PB2-F SC35M GCACAGGATGTAATCATGGAAGT PB2-R SC35M ACCAATTCTCTTTCTAGCATGTATGC Bat GGCACAAGAAGTCATTATGGAAGT Bat TTCCCTTTCCAGCATGTATGC PB1-F SC35M CTAATAAGAGCATTGACACTGAACACAATGAC PB1-R SC35M ACAAATCCTCTGATTTGCATCCC Bat CTAATAAGGGCACTGACCCTAAATACAATGAC Bat ACAAACCCTCTAATTTGCATCCC PA-F SC35M CACAAGTGGGAGAAGTATTGTGTCCTTGA PA-R SC35M CTCATTTCCATACCCCATTTCATTTT Bat CATAAGTGGGAGAAATACTGCGTACTAGA Bat CTCATTTCCATTCCCCATTTCATTTT 2HA-F SC35M AGCAGAAGCAGGGTCACTATTAC HA-common-R ATGGTGTCCAAGACATATTTTGTCTCC Bat AGCAAAAGCAGGGGATACAAAATG NP-F SC35M GAGAGAGAGGGATACTCCCTAGT NP-R13-41 SC35M TCCATACCAGTTGACTTTTATGTGC Bat ACTTTGAAAAAGAGGGATACTCTCTAGT Bat ACACTAGCTGACTTTTGTGTGC 2NA-F SC35M AGCAGAAGCAGGAGTTTTTAATAAAGTC NA-common-R CTTTAAGTGTAATGAGACATTCAGCACC Bat AGCAAAAGCAGGGTGATTGAG M-F SC35M GAAATTACATTCCATGGGGCCAA M-R SC35M CCACTTCGGTGGTCACAG Bat GATAACTTTCCATGGGGCCAA Bat GCCACTTCGGTTGTCACAG NS-F SC35M GGACCAGGCGATCATGGATAA NS-R SC35M CAATTGCTCCTTCTTCGGTGAA *Fluorescent probes PB2 Bat CATGGACCAAGCAGTAACCAATAA Bat ACTACTGTCCCTTCTTCTGTGAA [6FAM]-GAGCCAGGATACTAACATCGGAATCGCAAC-[BHQ1] M [6FAM]-AAGAAATAGCATTCAGTTATTCTGC-[BHQ1] NS [6FAM]-GTGATTTTTGACCGGCTGGAGACTC-[BHQ1] * In the indicated combinations we used a fluorescent probe with Light Cycler 480 genotyping master buffer (Roche) and manufacturer recommended conditions.

Supplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Supplementary Figure 1. SC35M polymerase activity in the presence of Bat or SC35M NP encoded from the phw2000 rescue plasmid. HEK293T