Molecular Characterization of Closely Related H6N2 Avian Influenza Viruses Isolated from Turkey, Egypt, and Uganda

Yavuz Mercan 1,2,†, Gladys Atim 3,†, Ahmed E. Kayed 4,†, M. Ekin Azbazdar 1,2, Ahmed Kandeil 4, Mohamed A. Ali 4, Adam Rubrum 5, Pamela McKenzie 5, Richard J. Webby 5, Bernard Erima 3, Fred Wabwire-Mangen 3,6, Qouilazoni A. Ukuli 3, Titus Tugume 3, Denis K. Byarugaba 3,7, Ghazi Kayali 8,9, Mariette F. Ducatez 10, and Zeynep A. Koçer 1,2,*

1 Emerging Viral Diseases Laboratory, Izmir Biomedicine and Genome Center, Izmir, 35340 Turkey; ya- [email protected] (Y.M.); [email protected] (M.E.A.) 2 Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Izmir, 35340 Turkey 3 Makerere University Walter Reed Project, P.O. Box 7062 Kampala, Uganda [email protected] (G.A.); [email protected] (B.E.); [email protected] (F.W.-M.); [email protected] (Q.A.U.); ttu- [email protected] (T.T.); [email protected] (D.K.B.) 4 Center of Scientific Excellence for Influenza Viruses, National Research Centre, Giza, 12311 Egypt; Ah- [email protected] (A.E.K.); [email protected] (A.K.); mohamedah- [email protected] (M.A.A.) 5 St Jude Children’s Research Hospital, Memphis, TN, 38105 USA; [email protected] (A.R.); Pam- [email protected] (P.M.); [email protected] (R.J.W.) 6 School of Public Health, Makerere University, P.O. Box 7062 Kampala, Uganda 7 College of Veterinary Medicine, Makerere University, P.O. Box 7062 Kampala, Uganda 8 Department of Epidemiology, Human Genetics, and Environmental Sciences, University of Texas, Houston, TX, 77030 USA; [email protected] (G.K.) 9 Human Link, Dubai, United Arab Emirates 10 IHAP, UMR1225, Université de Toulouse, INRAe, ENVT, 31076 Toulouse, France; [email protected] (M.F.D.) † These authors contributed equally to this work * Correspondence: [email protected] (Z.A.K.); Tel.: +90-232-299-4165

Citation: Lastname, F.; Lastname, F.; Lastname, F. Title. Viruses 2021, 13, x. https://doi.org/10.3390/xxxxx

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Academic Editor: Firstname Last- name Received: 27 February 2021 Accepted: 30 March 2021 Published: date

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Copyright: by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed un- der the terms and conditions of the Crea- tive Commons Attribution (CC BY) li- cense (http://creativecommons.org/li- censes/by/4.0/). Table S1. Pairwise nucleotide similarities of virus isolates. Gene GD/18 UG/731 EG/1566C EG/1556OP PB2 GD/18 1 UG/731 0.91506 1 EG/1566C 0.9853 0.91082 1 EG/1556OP 0.9978 0.91564 0.98484 1 PB1 GD/18 1 UG/731 0.961213 1 EG/1566C 0.97354 0.96076 1 EG/1556OP 0.999824 0.960759 0.99912 1 PA GD/18 1 UG/731 0.951806 1 EG/1566C 0.997671 0.950274 1 EG/1556OP 0.998138 0.950787 0.99535 1 HA GD/18 1 UG/731 0.966591 1 EG/1566C 0.982039 0.966632 1 EG/1556OP 0.981418 0.965985 0.999411 1 NP GD/18 1 UG/731 0.949747 1 EG/1566C 0.953384 0.975428 1 EG/1556OP 0.953384 0.975428 1 1 NA GD/18 1 UG/731 0.947689 1 EG/1566C 0.998772 0.948232 1 EG/1556OP 0.998772 0.948232 1 1 M GD/18 1 UG/731 0.975291 1 EG/1566C 0.9967 0.976016 1 EG/1556OP 0.9967 0.9760161 1 1 NS GD/18 1 UG/731 0.984703 1 EG/1566C 0.979922 0.971398 1 EG/1556OP 0.979922 0.971398 1 1

Table S2. Pairwise amino acid similarities of virus isolates. Protein GD/18 UG/731 EG/1566C EG/1556OP PB2 GD/18 1 UG/731 0.994785 1 EG/1566C 0.994785 0.989542 1 EG/1556OP 1 0.994785 0.994785 1 PB1 GD/18 1 UG/731 0.996029 1 EG/1566C 0.998678 0.994702 1 EG/1556OP 1 0.996029 0.998678 1 PB1-F2 GD/18 1 UG/731 0.966098 1 EG/1566C 0.988827 0.977527 1 EG/1556OP 0.988827 0.977527 1 1 PA GD/18 1 UG/731 0.992991 1 EG/1566C 0.998602 0.994398 1 EG/1556OP 0.998602 0.994398 1 1 PA-X GD/18 1 UG/731 0.984 1 EG/1566C 0.996024 0.979959 1 EG/1556OP 0.996024 0.979959 1 1 HA GD/18 1 UG/731 0.97314 1 EG/1566C 0.99113 0.98217 1 EG/1556OP 0.98934 0.98037 0.99823 1 NP GD/18 1 UG/731 0.989991 1 EG/1566C 0.987879 0.993958 1 EG/1556OP 0.987879 0.993958 1 1 NA GD/18 1 UG/731 0.95227 1 EG/1566C 0.99575 0.95707 1 EG/1556OP 0.99575 0.95707 0.99165 1 M1 GD/18 1 UG/731 1 1 EG/1566C 1 1 1 EG/1556OP 1 1 1 1 M2 GD/18 1 UG/731 0.989637 1 EG/1566C 1 0.989637 1 EG/1556OP 1 0.989637 1 1 NS1 GD/18 1 UG/731 0.968955 1 EG/1566C 0.995624 0.964598 1 EG/1556OP 0.995624 0.964598 1 1 NEP GD/18 1 UG/731 0.991701 1 EG/1566C 0.991701 0.983333 1 EG/1556OP 0.991701 0.983333 1 1

Table S3. Amino acid markers reported in the literature to be associated with host tropism, increased virulence, and es- cape from the host immune system.

Protein Amino Acid Phenotype Viruses with Reference(s)c Marker the marker PB2 I63 (with PB1 Pathogenic in mice All [27] T677) V89 (with, Enhanced polymerase activity, All [25] D309, K339, Increased virulence in mice G477, V495, T676) S590/R591 Increased polymerase activity, EG/1566C [26] Human host adaptation PB1 V3 (with N328 Increased polymerase activity, All [28, 31] and N375) Increased virulence in ferrets and mice P13 Increased polymerase activity, All [30] Adaptation to mice Y436 Increased polymerase activity All [29] and virulence in , fer- rets and mice V473 Increased polymerase activity All [32] and replication efficiency in mammalian cells T677 (with PB2 Pathogenic in mice All [27] I63) PB1-F2 S66 Increased virulence in mam- All [34, 35] mals L82 Increased pathogenicity in GD/18 [36] mice PA A37 Significantly increased viral All [38] growth and polymerase activ- ity in mammalian cells S277 (with Adaptation to mammalian All [37] Q278 and hosts P653) PA-X P28 Increased host shutdown in All [39] mammalian cells S65 Increased host shutdown in All [39] mammalian cells HAa A156 Increased virus binding to α2– All [40, 41] 6 sialic acid receptor, Trans- missibility in Guinea pigs T189 Increased virus binding to α2– UG/731, [42] 6 SA receptor EG/1566C, EG/1556OP NP V105 Adaptation of AIVs to GD/18, [43] chickens EG/1566C, EG/1556OP NAb T117 Reduced susceptibility to osel- All [44] tamivir and zanamivir M1 D30 Increased virulence in mice All [45] A215 Increased virulence in mice All [45]

NS1 S42 Increased virulence in mice, All [47] Antagonism of IFN induction F138 (with Increased replication in mam- All [46] ESEV in PDZ malian cells, Decreased inter- binding do- feron response main) A149 Increased virulence and de- All [48] creased interferon response in chickens No amino acid substitution was detected from the literature for M2 nor NEP (NS2) proteins. aAccording to H5 numbering bAccording to N2 numbering cReference numbers were given in accordance with the references in the main text

A/Whooper swans/Hokkaido/W14/2017(H5N2) A/Whooper swans/Hokkaido/W3/2017(H5N2) A/Whooper swans/Hokkaido/W73/2017(H5N2) A/Whooper swans/Hokkaido/W80/2017(H5N2) A/duck/Hokkaido/W151/2017(H3N2) A/Whooper swans/Hokkaido/W78/2017(H5N2) A/duck/Hokkaido/W154/2017(H3N2) 100 A/duck/Hokkaido/W144/2017(H3N2) A/duck/Hokkaido/W103/2017(H5N2) A/duck/Mongolia/154/2015(H1N2) 91 A/duck/Mongolia/520/2015(H1N1) A/teal/Egypt/MB-D-621C/2016(H7N9) 82 A/duck/Mongolia/245/2015(H10N3) 97 A/duck/Mongolia/326/2015(H10N3) A/duck/Hokkaido/56/2017(H12N2) A/teal/Egypt/MB-D-698OP/2016(H7N3) 99 A/duck/Mongolia/709/2015(H10N7) A/duck/Mongolia/626/2015(H10N7) 97 A//Egypt/MB-D-690OP/2016(H7N3) A/duck/Mongolia/499/2015(H10N7) 99 A/duck/Hokkaido/X9/2016(H8N4) 60 A/duck/Moscow/5662/2018(H1N2) A/teal/Egypt/MB-D-487OP/2016(H7N3) 100 A/duck/Moscow/5586/2018(H1N2) A/duck/Hubei/ZYSYG3/2015(H6N2) A/teal/Egypt/MB-D-698C/2016(H7N3) A/duck/Hubei/ZYSYG8/2015(H6N2) 99 96 A/duck/Hubei/ZYSYG7/2015(H6N2) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) A/Gallus gallus/Belgium/3497 0001/2019(H3N1) 73 A/teal/Egypt/MB-D-621C/2016(H7N9) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/teal/Egypt/MB-D-698OP/2016(H7N3) 94 99 A/northern shoveler/Egypt/MB-D-690OP/2016(H7N3) 60 A/teal/Egypt/MB-D-487OP/2016(H7N3) A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) A/teal/Egypt/MB-D-698C/2016(H7N3) 73 A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) A/wild /Uganda/MUWRP-731/2017(H6N2) 94 A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/duck/Mongolia/971/2018(H2N3) A/duck/Mongolia/971/2018(H2N3) A/duck/Mongolia/909/2018(H2N3) A/duck/Mongolia/909/2018(H2N3) 73 100 A/duck/Mongolia/975/2018(H2N3) A/duck/Mongolia/878/2018(H2N3) 73 100 A/duck/Mongolia/975/2018(H2N3) A/duck/Mongolia/927/2018(H2N3) 85 A/duck/Mongolia/871/2018(H2N3) A/duck/Mongolia/946/2018(H2N3) A/duck/Mongolia/878/2018(H2N3) 56 A/duck/Mongolia/899/2018(H2N3) A/duck/Mongolia/953/2018(H2N3) A/duck/Mongolia/927/2018(H2N3) A/duck/Mongolia/934/2018(H2N3) 85 89 A/duck/Mongolia/911/2018(H2N3) A/duck/Mongolia/871/2018(H2N3) A/ duck/Netherlands/26/2011(H11N9) A/mallard duck/Netherlands/26/2010(H4N6) A/mallard duck/Netherlands/6/2013(H3N8) A/duck/Mongolia/946/2018(H2N3) A/mallard duck/Netherlands/24/2014(H7N3) 56 85 A/mallard duck/Netherlands/10/2013(H4N6) A/duck/Mongolia/899/2018(H2N3) 62 A/mallard duck/Sweden/139703/2012(H15N5) A/mallard duck/Sweden/139752/2012(H15N5) 100 A/duck/Mongolia/953/2018(H2N3) A/mallard duck/Sweden/139579/2012(H15N5) A/mallard duck/Sweden/139647/2012(H15N5) A/white-fronted goose/Netherlands/3/2010(H5N2) A/duck/Mongolia/934/2018(H2N3) 100 A/greater white-fronted goose/Netherlands/2/2010(H5N2) A/mallard/Sweden/80261/2008(H4N9) 89 A/duck/Mongolia/911/2018(H2N3) 100 75 A/mallard/Nizhnegorsk/3-06-10/2010(H7N7) 74 A/mallard/Askania-Nova/39-15-02/2011(H7N7) 98 A/mallard/Novomyhalivka/10-23-12/2010(H7N7) A/mallard duck/Georgia/3/2012(H7N3) 95 A/teal/Egypt/MBD1566C/2018(H6N2) 100 A/teal/Egypt/MBD1556OP/2018(H6N2) 52 A/aquatic bird/Gediz Delta/1/2018(H6N2) 95 A/wild duck//KNU2018-91/2018(H5N3) A/duck/Mongolia/129/2015(H3N3) A//Georgia/1/2012(H2N3) 75 68 100 A/mallard/Nizhnegorsk/3-06-10/2010(H7N7) A/duck/Mongolia/837/2015(H1N1) 74 A/duck//11CL01/2014(H6N2) A/mallard/Askania-Nova/39-15-02/2011(H7N7) A/duck/Mongolia/20/2015(H3N8) 98 85 A/duck/Mongolia/167/2015(H3N8) A/duck/Mongolia/83/2015(H3N8) A/mallard/Novomyhalivka/10-23-12/2010(H7N7) 89 A/duck/Mongolia/208/2015(H3N8) 98 94 A/duck/Mongolia/211/2015(H3N8) A/mallard duck/Georgia/3/2012(H7N3) 50 A/duck/Bangladesh/26992/2015(H7N9) A/duck/Bangladesh/24692/2015(H7N1) 95 A/teal/Egypt/MBD1566C/2018(H6N2) 77 A/Cygnus olor/Belgium/2967/2017(H5N8) A/chicken/Czech Republic/206-17 2/2017(H5N8) 100 99 A/domestic duck/Georgia/1/2016(H4N6) A/teal/Egypt/MBD1556OP/2018(H6N2) 100 A/domestic duck/Georgia/12/2016(H4N6) A/domestic duck/Georgia/8/2016(H4N6) 52 A/aquatic bird/Gediz Delta/1/2018(H6N2) 98 A/domestic duck/Georgia/11/2016(H4N6) 95 A/domestic duck/Georgia/9/2016(H4N6) A/wild duck/South Korea/KNU2018-91/2018(H5N3) A/domestic duck/Georgia/7/2016(H4N6) A/domestic duck/Georgia/4/2016(H4N6) 62 A/duck/Mongolia/129/2015(H3N3) A/domestic duck/Georgia/10/2016(H4N6) A/domestic duck/Georgia/6/2016(H4N6) A/tufted duck/Georgia/1/2012(H2N3) 50 A/domestic duck/Georgia/2/2016(H4N6) A/domestic duck/Georgia/5/2016(H4N6) 68 A/mallard duck/Georgia/9/2016(H4N6) A/duck/Mongolia/837/2015(H1N1) A/domestic duck/Georgia/3/2016(H4N6) A/mallard duck/Georgia/8/2016(H4N6) A/duck/India/11CL01/2014(H6N2) A/mallard duck/Georgia/7/2016(H4N6) A/mallard duck/Georgia/6/2016(H4N6) A/mallard duck/Georgia/5/2016(H4N6) A/mallard duck/Georgia/4/2016(H4N6) A/mallard duck/Georgia/3/2016(H4N6) 100 A/duck /Zambia/03/2008(H6N2) A/duck/Zambia/02/2008(H6N2) 100 A/duck/Zambia/10/2009(H6N2) 88 A/duck/Zambia/08/2009(H6N2) A/Teal/Markeev/13-3-11/2016(H5N2) A/duck/Mongolia/140/2015(H10N2) 91 94 A/barnacle goose/Netherlands/2/2014(H3N6) 63 A/chicken/South Africa/AL25/2002(H6N2) 100 A/chicken/South Africa/UP1102/02(H6N2) 89 A/chicken/South Africa/AL19/02(H6N2) A/chicken/South Africa/W04/2002(H6N2) 100 A/chicken/South Africa/N2826/2016(H6N2) 100 A/chicken/South Africa/BKR2/2012(H6N2) 99 A/chicken/South Africa/BKR4/2012(H6N2) 98 A/chicken/South Africa/NWY/2012(H6N2) 97 A/chicken/South Africa/MAS/2013(H6N2) A/whiskered tern/Egypt/04/2004(H6N2) 100 A/duck/Egypt/053/2005(H6N2)

Figure S1. Phylogenetic relationships between GD/18, EG/1566C, EG/1556OP and UG/731 based on PB1 genes. All four vi- ruses were indicated with black circles. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. PB1 genes of GD/18, EG/1566C and EG/1556OP clustered with Korean and Mongo- lian sequences. PB1 gene of UG/731 mainly located in the same cluster with Egypt and Mongolia PB1 sequences. Reference viruses (most closely related strains for at least one gene segment) are color-coded: A/domestic duck/Georgia/1/2016 (H4N6) in red, A/Teal/Markeev/13-3-11/2016 (H5N2) in blue, and A/teal/Egypt/MB-D-487OP/2016 (H7N3) in green.

72 A/mallard duck/Netherlands/8/2014(H7N5) A/Mallard/Netherlands/8/2013(H3N8) 55 A/Teal/Markeev/13-3-11/2016(H5N2) 62 A/greater white-fronted goose/Netherlands/1/2012(H1N1) A/Mallard/Netherlands/3/2013(H3N6) A/Mallard/Netherlands/3/2013(H3N6) 99 A/mallard duck/Netherlands/2/2013(H3N6) A/mallard duck/Netherlands/24/2013(H4N6) 74 A/mallard duck/Netherlands/13/2013(H7N7) 97 96 A/mallard duck/Netherlands/11/2013(H7N7) 98 A/mallard duck/Netherlands/1/2014(H10N7) A/mallard duck/Netherlands/2/2014(H10N7) A/mallard/Republic of Georgia/3/2010(H7N3) 95 91 A//Netherlands/5/2007(H7N7) A/duck/Mongolia/17/2011(H4N3) A/northern shoveler/Georgia/1/2010(H2N3) 82 A/wild waterfowl/Dongting/C2383/2012(H1N2) 95 73 A/mallard duck/Georgia/3/2012(H7N3) A/shelduck/Yasnopolyanske/4-08-02/2011(H1N1) A/mallard/Czech Republic/13438-29K/2010(H11N9) A/tufted duck/Georgia/1/2012(H2N3) 98 A/duck/Mongolia/140/2015(H10N2) 83 A/mallard duck/Georgia/1/2014(H10N7) 99 A/mallard duck/Netherlands/22/2013(H2N3) A/duck/Moscow/4652/2011(H4N6) A/mallard/Republic of Georgia/3/2010(H7N3) A/northern shoveler/Georgia/1/2010(H2N3) 92 94 A/teal/Egypt/MBD1556OP/2018(H6N2) 95 A/shelduck/Yasnopolyanske/4-08-02/2011(H1N1) 99 98 A/duck/Mongolia/140/2015(H10N2) A/duck/Moscow/4652/2011(H4N6) A/teal/Egypt/MBD1566C/2018(H6N2) 92 94 A/teal/Egypt/MBD1556OP/2018(H6N2) 98 99 A/teal/Egypt/MBD1566C/2018(H6N2) A/aquatic bird/Gediz Delta/1/2018(H6N2) 98 A/aquatic bird/Gediz Delta/1/2018(H6N2) A/duck/Bangladesh/36395/2018(H4N6) A/duck/Bangladesh/36395/2018(H4N6) 99 A/duck/Bangladesh/36227/2018(H7N6) 86 A/mallard duck/Georgia/2/2016(H7N7) 99 A/duck/Bangladesh/36227/2018(H7N6) 82 A/Bewick swan/Netherlands/1/2014(H12N1) A/mallard/Netherlands/21/2016(H3N2) 86 A/mallard duck/Georgia/2/2016(H7N7) 59 99 A/mallard duck/Netherlands/59/2015(H6N5) 82 A/black-headed gull/Netherlands/21/2014(H11N1) A/Bewick swan/Netherlands/1/2014(H12N1) 88 A/mallard duck/Netherlands/56/2015(H3N2) 91 A/mallard/Netherlands/73/2017(H4N6) 78 A/mallard/Netherlands/21/2016(H3N2) 99 A/mallard/Netherlands/27/2017(H4N6) A/duck/Jiang Xi/2374/2005(H3N6) A/mallard duck/Netherlands/59/2015(H6N5) 99 A/mallard/Korea/GH170/2007(H7N7) 59 99 A/magpie/Korea/YJD174/2007(H7N7) 95 A/domestic green-winged teal/Hunan/3450/2006(H5N1) A/black-headed gull/Netherlands/21/2014(H11N1) A/duck/Mongolia/637/2010(H4N6) 88 70 99 A/duck/Mongolia/244/2010(H4N6) A/mallard duck/Netherlands/56/2015(H3N2) A/sharp-tailed sandpiper/Australia/6/2004(H11N9) 91 A/migratory duck/Jiang Xi/6568/2004(H4N6) 50 A/mallard/Netherlands/73/2017(H4N6) 96 A/duck/Suphanburi/AI157/2005(H6N1) 78 A/environment/Korea/CSM05/2004(H3N1) A/mallard/Netherlands/27/2017(H4N6) 64 A/duck/Jiang Xi/3345/2005(H5N2) 99 63 A/duck/Jiang Xi/1286/2005(H5N2) A/Baikal teal/Hongze/14/2005(H11N9) A/duck/Mongolia/131/2010(H3N8) 67 A/aquatic bird/Korea/CN5/2009(H6N5) 82 67 A/wild duck/Korea/CSM20-5/2009(H4N6) 88 A/duck/Shanxi/3180/2010(H10N7) 51 A/duck/Mongolia/675/2010(H1N1) A/duck/Mongolia/50/2010(H10N8) 99 A/duck/Mongolia/46/2010(H10N8) 88 A/duck/Mongolia/493/2010(H10N8) 98 95 A/duck/Mongolia/371/2010(H10N8) A/duck/Mongolia/3/2001(H10N3) A/avian//320/2001(H6N2) 56 A/barnacle goose/Netherlands/2/2014(H3N6) A/domestic duck/Georgia/1/2016(H4N6) 99 A/duck/Zambia/02/2008(H6N2) A/duck /Zambia/03/2008(H6N2) A/duck/Zambia/08/2009(H6N2) 78 99 A/duck/Zambia/10/2009(H6N2) A/mallard duck/Netherlands/63/2006(H5N2) A//Altai/1213/2007(H5N2) A/duck/Mongolia/340/2011(H4N8) A/mallard duck/Netherlands/10/2013(H4N6) 56 A/mallard duck/Netherlands/5/2013(H1N1) 98 A/mallard duck/Netherlands/20/2012(H1N1) 95 A/mallard duck/Netherlands/7/2012(H1N1) 99 A/mallard duck/Netherlands/37/2011(H1N1) A/mallard duck/Netherlands/10/2012(H6N1) 81 A///0921/2011(H6N8) 94 A/greylag goose/Iceland/0911/2011(H6N8) A/duck/Mongolia/122/2015(H3N3) 99 A/duck/Bangladesh/26980/2015(H7N9) A/duck/Bangladesh/26992/2015(H7N9) 79 () 88 56 A/Anser fabalis/China/Anhui/L144/2014(H6N1) 99 95 A//Anhui/S107/2014(H1N1) A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/duck/Mongolia/124/2015(H3N2) A/duck/Hokkaido/X9/2016(H8N4) 65 A/mallard/Republic of Georgia/13/2011(H6N2) A/duck/Bangladesh/33676/2017(H4N6) A/duck/Mongolia/173/2015(H3N8) 64 77 99 A/duck/Mongolia/179/2015(H3N8) A/duck/Mongolia/575/2018(H3N1) 99 A/muscovy duck/Vietnam/LBM201/2012(H3N2) A/muscovy duck/Vietnam/LBM189/2012(H3N2) A/mallard duck/Georgia/10/2016(H7N7) 99 A/wild goose/Dongting/C1037/2011(H12N8) 79 A/duck/Hokkaido/491003/2014(H4N2) A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/duck/Bangladesh/26918/2015(H3N6) 65 A/duck/Bangladesh/33676/2017(H4N6) 64 A/duck/Mongolia/575/2018(H3N1) A/duck/Mongolia/777/2015(H3N8) A/mallard duck/Georgia/10/2016(H7N7) A/duck/Bangladesh/26918/2015(H3N6) A/duck/Mongolia/777/2015(H3N8) A/duck/Mongolia/211/2015(H3N8) A/duck/Mongolia/211/2015(H3N8) A/duck/Mongolia/83/2015(H3N8) A/duck/Mongolia/83/2015(H3N8) A/duck/Mongolia/211/2015(H3N8) 84 87 A/duck/Mongolia/167/2015(H3N8) A/duck/Mongolia/211/2015(H3N8) 99 A/duck/Mongolia/208/2015(H3N8) 84 A/duck/Mongolia/30/2015(H3N8) 53 A/duck/Mongolia/518/2015(H10N3) A/duck/Mongolia/167/2015(H3N8) A/teal/Egypt/MB-D-487OP/2016(H7N3) 87 99 A/teal/Egypt/MB-D-621C/2016(H7N9) A/duck/Mongolia/208/2015(H3N8) A/teal/Egypt/MB-D-148OP/2015(H10N7) 99 58 A/duck/Bangladesh/24035/2014(H10N1) A/duck/Mongolia/30/2015(H3N8) 93 A/duck/Bangladesh/25891/2015(H4N6) A/black-tailed godwit/Bangladesh/24734/2015(H7N5) 53 A/duck/Bangladesh/24704/2015(H15N9) A/duck/Mongolia/518/2015(H10N3) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) A/teal/Egypt/MB-D-487OP/2016(H7N3) 99 A/teal/Egypt/MB-D-698C/2016(H7N3) A/teal/Egypt/MB-D-698OP/2016(H7N3) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) 99 A/teal/Egypt/MB-D-621C/2016(H7N9) 80 A/chicken/South Africa/AL19/02(H6N2) 97 A/chicken/South Africa/AL25/2002(H6N2) 63 A/chicken/South Africa/UP1102/02(H6N2) A/chicken/South Africa/W04/2002(H6N2) 99 A/chicken/South Africa/N2826/2016(H6N2) A/chicken/South Africa/MAS/2013(H6N2) 99 A/chicken/South Africa/BKR2/2012(H6N2) 98 99 A/chicken/South Africa/BKR4/2012(H6N2) A/whiskered tern/Egypt/04/2004(H6N2) 99 A/duck/Egypt/053/2005(H6N2)

Figure S2. Phylogenetic relationships between GD/18, EG/1566C, EG/1556OP and UG/731 based on PA genes. All four vi- ruses were indicated with black circles. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. PA genes of GD/18, EG/1566C and EG/1556OP clustered together with Bangladesh and Netherlands sequences. PA of UG/731 clustered with Mongolia, Egypt, and Bangladesh sequences. Reference viruses (most closely related strains for at least one gene segment) are color-coded: A/domestic duck/Georgia/1/2016 (H4N6) in red, A/Teal/Markeev/13-3-11/2016 (H5N2) in blue, and A/teal/Egypt/MB-D-487OP/2016 (H7N3) in green.

A/duck/Bangladesh/34191/2017(H3N1) A/duck/Bangladesh/34192/2017(H3N1) A/duck/Bangladesh/34193/2017(H3N1) 100 A/duck/Bangladesh/34186/2017(H3N1) A/duck/Bangladesh/34194/2017(H3N1) A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/teal/Egypt/MB-D-487OP/2016(H7N3) 79 A/teal/Egypt/MB-D-621C/2016(H7N9) 65 A/teal/Egypt/MB-D-621C/2016(H7N9) A/pintail/Egypt/MB-D-384C/2015(H3N6) 92 A/Tufted duck/Denmark/11740-LWPL/2016(H5N8) A/tufted duck/Denmark/11740/2016(H5N8) A/duck/Bangladesh/34191/2017(H3N1) 99 A/peacock/Belgium/1017/2017(H5N8) 100 A/chicken/Belgium/807/2017(H5N8) A/duck/Bangladesh/34192/2017(H3N1) 72 A/Brahma chicken/Belgium/6153/2017(H5N8) A/Guinea fowl/Belgium/6102/2017(H5N8) 72 98 A/duck/Bangladesh/34193/2017(H3N1) A/teal/Egypt/MBD1566C/2018(H6N2) 100 100 A/teal/Egypt/MBD1556OP/2018(H6N2) A/duck/Bangladesh/34186/2017(H3N1) 89 A/duck/Egypt/N13736E/2017(H5N8) A/teal/Egypt/MB-D-487OP/2016(H7N3) 99 A/duck/Bangladesh/34194/2017(H3N1) 99 A/chicken/Egypt/Q13804A/2017(H5N8) A/teal/Egypt/MB-D-698OP/2016(H7N3) A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/teal/Egypt/MB-D-698C/2016(H7N3) 81 100 A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) A/teal/Egypt/MB-D-487OP/2016(H7N3) A/teal/Egypt/MB-D-698OP/2016(H7N3) A/mallard duck/Georgia/8/2016(H4N6) 79 A/teal/Egypt/MB-D-621C/2016(H7N9) A/domestic duck/Georgia/6/2016(H4N6) 100 A/mallard duck/Georgia/7/2016(H4N6) 65 A/teal/Egypt/MB-D-621C/2016(H7N9) A/mallard duck/Georgia/6/2016(H4N6) A/mallard duck/Georgia/5/2016(H4N6) A/pintail/Egypt/MB-D-384C/2015(H3N6) A/mallard duck/Georgia/4/2016(H4N6) A/mallard duck/Georgia/3/2016(H4N6) 92 A/Tufted duck/Denmark/11740-LWPL/2016(H5N8) A/domestic duck/Georgia/9/2016(H4N6) A/domestic duck/Georgia/8/2016(H4N6) A/tufted duck/Denmark/11740/2016(H5N8) A/domestic duck/Georgia/7/2016(H4N6) A/domestic duck/Georgia/5/2016(H4N6) 99 A/peacock/Belgium/1017/2017(H5N8) A/domestic duck/Georgia/4/2016(H4N6) 100 A/domestic duck/Georgia/3/2016(H4N6) A/chicken/Belgium/807/2017(H5N8) A/domestic duck/Georgia/2/2016(H4N6) A/domestic duck/Georgia/12/2016(H4N6) 72 A/Brahma chicken/Belgium/6153/2017(H5N8) A/domestic duck/Georgia/11/2016(H4N6) A/domestic duck/Georgia/10/2016(H4N6) 98 A/Guinea fowl/Belgium/6102/2017(H5N8) 98 A/domestic duck/Georgia/1/2016(H4N6) 72 A/domestic duck/Georgia/1/2016(H4N6) A/teal/Egypt/MBD1566C/2018(H6N2) A/mallard duck/Netherlands/37/2011(H1N1) A/mallard duck/Netherlands/5/2013(H1N1) 100 A/teal/Egypt/MBD1556OP/2018(H6N2) A/mallard duck/Netherlands/23/2013(H7N3) A/mallard duck/Netherlands/20/2012(H1N1) A/duck/Egypt/N13736E/2017(H5N8) A/mallard duck/Netherlands/31/2013(H10N7) 89 68 A/mallard duck/Netherlands/32/2013(H10N7) A/teal/Egypt/MB-D-487OP/2016(H7N3) A/mallard duck/Netherlands/15/2011(H6N8) 99 59 A/mallard duck/Netherlands/20/2011(H6N8) A/chicken/Egypt/Q13804A/2017(H5N8) 96 A/greater white-fronted goose/Netherlands/6/2011(H6N8) 99 A/white-fronted goose/Netherlands/5/2011(H6N8) A/teal/Egypt/MB-D-698OP/2016(H7N3) A/mallard duck/Netherlands/12/2012(H6N1) A/greater white-fronted goose/Netherlands/11/2009(H6N1) A/Mallard/Netherlands/8/2013(H3N8) A/teal/Egypt/MB-D-698C/2016(H7N3) A/mallard duck/Netherlands/24/2009(H6N1) 100 100 A/mallard duck/Netherlands/11/2013(H7N7) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/mallard duck/Netherlands/13/2013(H7N7) A/mallard duck/Netherlands/18/2012(H4N2) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) A/Mallard/Netherlands/37/2015(H3N8) 71 A/barnacle goose/Netherlands/2/2014(H3N6) A/teal/Egypt/MB-D-698OP/2016(H7N3) 70 A/barnacle goose/Netherlands/2/2014(H3N6) A/mallard duck/Netherlands/23/2012(H11N9) A/mallard duck/Netherlands/16/2012(H3N2) 63 70 A/Mallard/Netherlands/21/2013(H3N2) 99 A/duck /Zambia/03/2008(H6N2) 100 A/duck/Zambia/02/2008(H6N2) A/duck/Zambia/08/2009(H6N2) 98 A/duck/Zambia/10/2009(H6N2) 98 A/chicken/South Africa/AL19/02(H6N2) A/chicken/South Africa/W04/2002(H6N2) 84 A/chicken/South Africa/UP1102/02(H6N2) 99 A/chicken/South Africa/AL25/2002(H6N2) 99 A/chicken/South Africa/BKR4/2012(H6N2) 83 A/chicken/South Africa/BKR2/2012(H6N2) 100 A/chicken/South Africa/MAS/2013(H6N2) 99 A/chicken/South Africa/N2826/2016(H6N2) 80 A/duck/Bangladesh/36227/2018(H7N6) 99 A/duck/Mongolia/211/2015(H3N8) A/duck/Mongolia/140/2015(H10N2) 99 A/Mallard/Netherlands/3/2013(H3N6) 96 A/greater white-fronted goose/Netherlands/1/2006(H6N2) A/mallard duck/Netherlands/23/2006(H1N1) A/mallard duck/Netherlands/2/2013(H3N6) 79 A/mallard duck/Netherlands/86/2008(H7N1) A/mallard/Netherlands/29/2006(H7N2) 77 99 A/greater white-fronted goose/Netherlands/6/2008(H5N2) A/aquatic bird/Gediz Delta/1/2018(H6N2) 61 A/greater white-fronted goose/Netherlands/5/2008(H5N2) A/Eurasian wigeon/Netherlands/2/2009(H12N5) A/teal/Egypt/MB-D-148OP/2015(H10N7) A/turnstone/Netherlands/3/2008(H3N8) 92 90 A/ruddy turnstone/Netherlands/6/2008(H3N8) 97 A/turnstone/Netherlands/4/2008(H3N8) A/mallard/Sweden/100881/2009(H4N6) A/duck/Moscow/4643/2011(H4N6) 64 A/mallard/Sweden/101754/2009(mixed) A/mallard/Sweden/101081/2009(H4N6) A/mallard duck/Netherlands/24/2005(H11N3) 90 99 A/Mallard/Netherlands/3/2013(H3N6) A/mallard duck/Netherlands/2/2013(H3N6) A/Teal/Markeev/13-3-11/2016(H5N2) 77 A/aquatic bird/Gediz Delta/1/2018(H6N2) A/teal/Egypt/MB-D-148OP/2015(H10N7) A/mallard/Sweden/100881/2009(H4N6) A/mallard duck/Netherlands/18/2014(H1N1) 64 91 90 A/mallard/Sweden/101081/2009(H4N6) A/Teal/Markeev/13-3-11/2016(H5N2) A/tufted duck/Georgia/1/2012(H2N3) A/mallard duck/Netherlands/18/2014(H1N1) 91 A/tufted duck/Georgia/1/2012(H2N3) A/mallard duck/Netherlands/13/2007(H6N5) A/mallard duck/Netherlands/13/2007(H6N5) 55 55 89 A/mallard/Netherlands/11/2007(H6N5) A/barnacle goose/Netherlands/1/2011(H6N8) 89 A/mallard/Netherlands/11/2007(H6N5) 99 A/mallard duck/Netherlands/5/2012(H10N7) A/mallard duck/Netherlands/29/2009(H11N9) A/mallard duck/Netherlands/26/2010(H4N6) A/barnacle goose/Netherlands/1/2011(H6N8) A/ crecca/Spain/1460/2008(H7N9) A/mallard/Sweden/79436/2008(H11N9) A/mallard duck/Netherlands/5/2012(H10N7) A/barnacle goose/Netherlands/1/2010(H6N8) 99 A/pink-footed goose/Netherlands/1/2006(H9N2) A/mallard duck/Netherlands/19/2005(H9N2) A/mallard duck/Netherlands/29/2009(H11N9) 83 A/Eurasian wigeon/Netherlands/4/2005(H9N2) A/mallard duck/Netherlands/1/2005(H9N2) A/mallard/Sweden/7146/2004(H9N2) A/mallard duck/Netherlands/26/2010(H4N6) 61 A/mallard duck/Netherlands/36/2008(H11N9) 100 A/bewicks swan/Netherlands/5/2008(H5N2) A/Anas crecca/Spain/1460/2008(H7N9) 55 A/mallard duck/Netherlands/62/2008(H3N6) A/bewicks swan/Netherlands/3/2006(H1N1) A/mallard/Sweden/79436/2008(H11N9) 99 A/Bewick Swan/Netherlands/2/2006(H1N1) A/mallard/Sweden/5824/2005(H10N7) A/mallard/Sweden/52462/2006(H4N6) A/barnacle goose/Netherlands/1/2010(H6N8) 83 A/Bewick swan/Netherlands/5/2007(H3N6) A/mallard duck/Netherlands/38/2008(H5N3) 51 A/mallard/Sweden/51933/2006(H10N9) A/mallard/Sweden/52057/2006(mixed) A/mallard/Sweden/52903/2006(H10N9) A/mallard duck/Netherlands/24/2011(H2N3) A/mallard duck/Netherlands/18/2010(H6N8) 99 A/ duck/Netherlands/1/2011(H6N2) A/Eurasian teal/Netherlands/1/2011(H3N8) A/duck/Egypt/053/2005(H6N2) A/whiskered tern/Egypt/04/2004(H6N2) 100

Figure S3. Phylogenetic relationships between GD/18, EG/1566C, EG/1556OP and UG/731 based on NP genes. All four vi- ruses were indicated with black circles. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. NP of UG/731, EG/1566C and EG/1556OP clustered with Bangladesh, Egypt, and some European sequences. NP of GD/18 clustered with European sequences. Reference viruses (most closely related strains for at least one gene segment) are color-coded: A/domestic duck/Georgia/1/2016 (H4N6) in red, A/Teal/Markeev/13-3- 11/2016 (H5N2) in blue, and A/teal/Egypt/MB-D-487OP/2016 (H7N3) in green.

A/domestic duck/Georgia/5/2016(H4N6) A/domestic duck/Georgia/1/2016(H4N6) A/domestic duck/Georgia/6/2016(H4N6) A/domestic duck/Georgia/7/2016(H4N6) A/domestic duck/Georgia/8/2016(H4N6) A/domestic duck/Georgia/9/2016(H4N6) 99 A/mallard duck/Georgia/3/2016(H4N6) A/mallard duck/Georgia/9/2016(H4N6) A/mallard duck/Georgia/8/2016(H4N6) A/mallard duck/Georgia/7/2016(H4N6) A/mallard duck/Georgia/6/2016(H4N6) A/mallard duck/Georgia/5/2016(H4N6) A/mallard duck/Georgia/4/2016(H4N6) 97 A/duck/Mongolia/709/2015(H10N7) A/duck/Mongolia/499/2015(H10N7) A/Anser fabalis/China/664/2014(H11N8) g() 98 A/wild bird/Wuhan/CDHN09/2015(H6N2) A/wild bird/Wuhan/CDHN15/2015(H6N2) 97 A/duck/Mongolia/709/2015(H10N7) A/teal/Egypt/MB-D-621C/2016(H7N9) 98 A/teal/Egypt/MB-D-487OP/2016(H7N3) A/duck/Mongolia/499/2015(H10N7) A/duck/Mongolia/173/2015(H3N8) A/mallard duck/Georgia/1/2014(H10N7) A/Anser fabalis/China/664/2014(H11N8) A/pintail/Egypt/MB-D-384C/2015(H3N6) A/mallard duck/Georgia/2/2016(H7N7) 98 A/wild bird/Wuhan/CDHN09/2015(H6N2) 98 A/duck/Mongolia/127/2015(H4N6) A/duck/Mongolia/101/2015(H4N6) A/wild bird/Wuhan/CDHN15/2015(H6N2) A/mallard duck/Georgia/10/2016(H7N7) 57 A/aquatic bird/Gediz Delta/1/2018(H6N2) A/duck/Bangladesh/33676/2017(H4N6) A/teal/Egypt/MB-D-621C/2016(H7N9) 98 A/teal/Egypt/MBD1566C/2018(H6N2) A/teal/Egypt/MBD1556OP/2018(H6N2) 98 A/teal/Egypt/MB-D-487OP/2016(H7N3) A/Duck/Mongolia/652/2017(H7N3) 51 A/Duck/Mongolia/751/2017(H7N3) A/duck/Mongolia/173/2015(H3N8) 99 A/Duck/Mongolia/786/2017(H7N3) A/Duck/Mongolia/782/2017(H7N3) A/mallard duck/Georgia/1/2014(H10N7) A/duck/Mongolia/782/2017(H7N3) A/Anser fabalis/China/Anhui/S148/2014(H6N2) 78 A/pintail/Egypt/MB-D-384C/2015(H3N6) A/duck/Cambodia/C72W14M1/2018(mixed) 57 100 A/duck/Cambodia/C70W14M/2018(H7N5) A/mallard duck/Georgia/2/2016(H7N7) 68 A/mallard duck/Netherlands/2/2015(H7N7) A/mallard duck/Netherlands/3/2015(H7N7) 98 A/duck/Mongolia/127/2015(H4N6) A/duck/Cambodia/C50W8M1/2018(H7N4) 77 A/mallard duck/Netherlands/8/2014(H7N5) A/duck/Mongolia/101/2015(H4N6) A/mallard duck/Netherlands/41/2015(H5N1) A/mallard duck/Netherlands/59/2015(H6N5) A/mallard duck/Georgia/10/2016(H7N7) 97 A/Teal/Markeev/13-3-11/2016(H5N2) A/mallard duck/Netherlands/60/2015(H6N5) 57 A/teal/Egypt/MB-D-698OP/2016(H7N3) A/aquatic bird/Gediz Delta/1/2018(H6N2) A/teal/Egypt/MB-D-698C/2016(H7N3) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) A/duck/Bangladesh/33676/2017(H4N6) A/northern shoveler/Egypt/MB-D-690OP/2016(H7N3) 85 98 A/teal/Egypt/MBD1566C/2018(H6N2) 91 A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) A/teal/Egypt/MB-D-148OP/2015(H10N7) A/teal/Egypt/MBD1556OP/2018(H6N2) A/duck/Bangladesh/36227/2018(H7N6) 96 A/mallard/Netherlands/73/2017(H4N6) A/Duck/Mongolia/652/2017(H7N3) 97 A/mallard/Netherlands/89/2017(H4N6) 51 A/duck/Moscow/5662/2018(H1N2) A/Duck/Mongolia/751/2017(H7N3) 98 A/duck/Mongolia/326/2015(H10N3) 99 72 A/duck/Mongolia/245/2015(H10N3) A/Duck/Mongolia/786/2017(H7N3) A/duck/Mongolia/62/2013(H3N1) 85 A/wild goose/dongting /121/2018(H6N2) A/Duck/Mongolia/782/2017(H7N3) A/wild bird/Wuhan/CDHN01/2015(H11N9) 57 A/wild bird/Wuhan/CDHN22/2015(H11N9) 86 A/duck/Mongolia/782/2017(H7N3) A/wild bird/Wuhan/CDHN173/2015(H11N9) A/duck/Wuhan/WHYF05/2014(H9N2) A/mallard duck/Netherlands/9/2014(H6N2) A/mallard/Netherlands/25/2013(mixed) 67 A/Eurasian wigeon/Netherlands/2/2008(H7N4) 50 A/mallard duck/Netherlands/77/2008(H7N1) A/mallard duck/Netherlands/1/2007(H10N7) A/greater white-fronted goose/Netherlands/1/2010(H6N1) A/avian/Israel/824/2005(H10N2) A/Mallard/Netherlands/31/2014(H4N3) 64 A/mallard/Netherlands/32/2014(mixed) A/duck/Zambia/08/2009(H6N2) 100 A/duck/Zambia/10/2009(H6N2) A/mallard duck/Netherlands/17/2009(H6N8) A/quail/Lebanon/272/2010(H9N2) 93 A/mallard duck/Netherlands/2/2014(H10N7) A/mallard duck/Netherlands/1/2014(H10N7) A/mallard duck/Netherlands/18/2009(H6N2) A/duck/Shanghai/602/2009(H10N8) A/mallard/Netherlands/22/2015(mixed) 99 A/mallard duck/Netherlands/18/2015(H7N7) 85 A/barnacle goose/Netherlands/1/2011(H6N8) A/barnacle goose/Netherlands/1/2010(H6N8) A/mallard duck/Netherlands/32/2008(H6N1) A/greater white-fronted goose/Netherlands/8/2009(H6N2) g() A/mallard duck/Netherlands/51/2008(H3N2) A/mallard duck/Netherlands/25/2009(H1N1) 53 A/mallard duck/Netherlands/26/2009(H1N1) 86 A/wild bird/Uganda/MUWRP-731/2017(H6N2) 95 A/mallard duck/Netherlands/28/2009(H1N1) 89 A/Bewick Swan/Netherlands/4/2007(H4N6) A/mallard duck/Netherlands/17/2011(H3N8) A/bewicks swan/Netherlands/3/2007(H4N6) A/mallard/Netherlands/17/2012(mixed) A/mallard duck/Netherlands/11/2006(H8N4) A/duck /Zambia/03/2008(H6N2) 85 A/mallard duck/Netherlands/14/2006(H8N4) A/Eurasian wigeon/Netherlands/6/2005(H6N2) 100 A/duck/Zambia/02/2008(H6N2) 86 A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/mallard duck/Netherlands/17/2011(H3N8) A/mallard duck/Netherlands/3/2004(H4N6) A/duck /Zambia/03/2008(H6N2) 100 A/duck/Zambia/02/2008(H6N2) A/mallard duck/Netherlands/3/2004(H4N6) 52 A/Eurasian wigeon/Netherlands/6/2007(H1N1) 52 A/Eurasian wigeon/Netherlands/6/2007(H1N1) A/mallard duck/Netherlands/9/2001(H4N6) A/mallard duck/Netherlands/9/2001(H4N6) A/mallard duck/Netherlands/10/1999(mixed) 100 A/mallard/Netherlands/10-Nmkt/1999(mixed) A/mallard duck/Netherlands/10/1999(mixed) A/mallard duck/Netherlands/8/1999(H1N1) 100 A/duck/Hokkaido/15/2015(H3N8) A/mallard/Netherlands/10-Nmkt/1999(mixed) 97 A/mallard/Korea/GH170/2007(H7N7) 99 A/avian/Israel/320/2001(H6N2) A/avian/Israel/289/2001(H6N2) A/mallard duck/Netherlands/8/1999(H1N1) 100 A/mallard duck/Netherlands/5/2009(H5N2) A/greater white-fronted goose/Netherlands/4/2009(H5N2) A/avian/Canada/IM-1474/1994(H7N1) A/mallard duck/Netherlands/6/2006(H4N6) A/mallard/Netherlands/12/2000(H7N3) 77 A/mallard/Netherlands/5/2001(H3N6) 94 A/duck/Mongolia/211/2015(H3N8) A/duck/Mongolia/140/2015(H10N2) 100 A/chicken/South Africa/BKR4/2012(H6N2) A/chicken/South Africa/BKR2/2012(H6N2) 87 A/chicken/South Africa/UP1102/02(H6N2) 99 A/chicken/South Africa/AL25/2002(H6N2) A/chicken/South Africa/AL19/02(H6N2) A/chicken/South Africa/MAS/2013(H6N2) 100 100 A/chicken/South Africa/N2826/2016(H6N2) A/barnacle goose/Netherlands/2/2014(H3N6) A/whiskered tern/Egypt/04/2004(H6N2) 100 A/duck/Egypt/053/2005(H6N2)

Figure S4. Phylogenetic relationships between GD/18, EG/1566C, EG/1556OP and UG/731 based on M genes. All four vi- ruses were indicated with black circles. Phylogenetic trees were constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. M gene sequences of GD/18, EG/1566C and EG/1556OP clustered together with Bangladesh, Mongolia, and Georgia sequences. M gene of UG/731 clustered with Netherlands sequences. Reference viruses (most closely related strains for at least one gene segment) are color-coded: A/domestic duck/Georgia/1/2016 (H4N6) in red, A/Teal/Markeev/13-3-11/2016 (H5N2) in blue, A/teal/Egypt/MB-D-487OP/2016 (H7N3) in green, and A/duck/Mos- cow/5662/2018 (H1N2) in purple.

87 A/duck/Cambodia/14T-24-1-D11/2018(N4) A/mallard duck/Netherlands/6/2009(H7N7) 81 A/mallard duck/Netherlands/3/2009(H7N7) A/mallard duck/Netherlands/4/2009(H7N7) A/mallard duck/Netherlands/2/2009(H7N7) A/mallard duck/Netherlands/7/2009(H7N7) A/mallard duck/Netherlands/23/2012(H11N9) A/mallard/Netherlands/22/2015(mixed) 91 A/Black-headed Gull/Netherlands/22/2014(mixed) 96 A/mallard/Netherlands/22/2015(mixed) A/teal/Egypt/MB-D-487OP/2016(H7N3) 97 A/teal/Egypt/MB-D-621C/2016(H7N9) A/duck/Bangladesh/821/2009(H10N7) A/domestic green-winged teal/Hunan/2036/2007(H3N6) 54 g() A/mallard duck/Georgia/11/2011(H1N1) 94 A/mallard duck/Netherlands/19/2009(H5N3) 70 A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/mallard duck/Netherlands/20/2009(H5N3) A/mallard/Netherlands/25/2013(mixed) A/duck/Bangladesh/36227/2018(H7N6) A/mallard duck/Netherlands/3/2008(H6N2) A/mallard/Moscow/3661/2008(H4N6) A/aquatic bird/Gediz Delta/1/2018(H6N2) 54 A/canvasback/Mongolia/2-69/2007(H10N6) 70 A/wild bird/Uganda/MUWRP-731/2017(H6N2) A/mallard duck/Netherlands/4/2011(H12N5) A/duck/Bangladesh/36227/2018(H7N6) A/aquatic bird/Gediz Delta/1/2018(H6N2) A/mallard duck/Netherlands/23/2010(H10N7) A/mallard duck/Netherlands/4/2011(H12N5) 84 A/mallard duck/Netherlands/23/2010(H10N7) A/mallard duck/Netherlands/21/2010(H10N7) 84 A/mallard duck/Netherlands/21/2010(H10N7) 57 57 A/mallard duck/Netherlands/22/2010(H10N7) A/mallard duck/Netherlands/22/2010(H10N7) A/mallard duck/Netherlands/50/2010(H10N7) 80 A/mallard/Korea/GH170/2007(H7N7) A/mallard duck/Netherlands/50/2010(H10N7) A/wild duck/Korea/SH5-26/2008(H4N6) A/mallard/Netherlands/32/2014(mixed) 80 A/mallard/Korea/GH170/2007(H7N7) A/mallard duck/Netherlands/49/2010(H4N6) A/mallard duck/Netherlands/78/2006(H6N8) A/wild duck/Korea/SH5-26/2008(H4N6) 87 91 A/Eurasian wigeon/Netherlands/1/2006(H6N2) A/mallard duck/Netherlands/34/2006(H3N8) A/mallard/Netherlands/32/2014(mixed) 52 A/bewicks swan/Netherlands/4/2006(H9N2) A/mallard duck/Netherlands/86/2008(H7N1) A/mallard duck/Netherlands/49/2010(H4N6) A/mallard duck/Netherlands/32/2008(H6N1) A/Eurasian teal/Netherlands/1/2008(H6N1) A/mallard duck/Netherlands/78/2006(H6N8) 75 A/mallard duck/Netherlands/8/2008(H6N1) 87 A/Eurasian wigeon/Netherlands/1/2008(H6N1) 91 A/Eurasian wigeon/Netherlands/1/2006(H6N2) A/Eurasian teal/Netherlands/3/2008(H6N1) A/mallard duck/Netherlands/31/2008(H6N1) A/aquatic bird/Korea/CN5/2009(H6N5) A/wild bird/Mongolia/2066/2011(H5N3) 57 A/common shelduck/Mongolia/2076/2011(H3N8) A//Alaska/16-041335-5/2016(H5N2) A/mallard duck/Netherlands/13/2005(H6N2) 93 A/wild bird/Wuhan/CDHN01/2015(H11N9) 100 A/teal/Egypt/MBD1566C/2018(H6N2) 64 A/wild bird/Wuhan/CDHN22/2015(H11N9) A/wild bird/Wuhan/CDHN173/2015(H11N9) A/teal/Egypt/MBD1556OP/2018(H6N2) A/wild bird/Wuhan/CDHN09/2015(H6N2) A/greater white-fronted goose/Netherlands/4/2011(H1N1) A/domestic duck/Georgia/12/2016(H4N6) A/mallard duck/Netherlands/50/2010(H10N7) 51 A/domestic duck/Georgia/1/2016(H4N6) 100 A/mallard duck/Netherlands/2/2011(H10N7) 99 A/duck/Zambia/10/2009(H6N2) A/domestic duck/Georgia/11/2016(H4N6) A/duck/Zambia/08/2009(H6N2) A/domestic duck/Georgia/2/2016(H4N6) A/mallard duck/Netherlands/13/2005(H6N2) 100 A/teal/Egypt/MBD1566C/2018(H6N2) A/domestic duck/Georgia/3/2016(H4N6) A/teal/Egypt/MBD1556OP/2018(H6N2) A/mallard duck/Georgia/3/2016(H4N6) A/domestic duck/Georgia/12/2016(H4N6) A/domestic duck/Georgia/1/2016(H4N6) A/mallard duck/Georgia/4/2016(H4N6) A/domestic duck/Georgia/11/2016(H4N6) 83 A/mallard duck/Georgia/5/2016(H4N6) A/domestic duck/Georgia/2/2016(H4N6) 86 A/domestic duck/Georgia/3/2016(H4N6) A/mallard duck/Georgia/7/2016(H4N6) 96 A/mallard duck/Georgia/3/2016(H4N6) A/mallard duck/Georgia/8/2016(H4N6) A/mallard duck/Georgia/4/2016(H4N6) 56 83 A/mallard duck/Georgia/5/2016(H4N6) 86 A/teal/Egypt/MB-D-125OP/2015(H7N3) 53 A/mallard duck/Georgia/7/2016(H4N6) 96 A/mallard duck/Georgia/8/2016(H4N6) A/Duck/Mongolia/652/2017(H7N3) 56 A/teal/Egypt/MB-D-125OP/2015(H7N3) 63 A/Bewick swan/Netherlands/1/2014(H12N1) A/Duck/Mongolia/652/2017(H7N3) 56 63 A/Bewick swan/Netherlands/1/2014(H12N1) A/Mallard/Netherlands/39/2015(H4N6) 56 A/Mallard/Netherlands/39/2015(H4N6) 70 A/mallard duck/Netherlands/6/2013(H3N8) 70 A/mallard duck/Netherlands/6/2013(H3N8) A/mallard duck/Netherlands/23/2014(H1N1) A/mallard duck/Netherlands/23/2014(H1N1) 96 A/mallard duck/Netherlands/26/2014(H1N1) 96 A/mallard duck/Netherlands/26/2014(H1N1) 62 A/Mallard/Netherlands/8/2013(H3N8) A/tufted duck/Georgia/1/2012(H2N3) 62 A/Mallard/Netherlands/8/2013(H3N8) A/mallard duck/Netherlands/13/2012(H3N8) A/tufted duck/Georgia/1/2012(H2N3) 59 A/mallard duck/Netherlands/22/2013(H2N3) A/mallard duck/Netherlands/18/2010(H6N8) A/mallard duck/Netherlands/13/2012(H3N8) A/duck/Cambodia/12T-24-1-D17/2018(H7N4) A/mallard duck/Netherlands/7/2015(H8N4) A/mallard duck/Netherlands/22/2013(H2N3) 57 A/northern shoveler/Egypt/MB-D-690OP/2016(H7N3) A/mallard duck/Netherlands/18/2010(H6N8) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) 82 A/duck/Cambodia/12T-24-1-D17/2018(H7N4) A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) A/teal/Egypt/MB-D-698C/2016(H7N3) A/mallard duck/Netherlands/7/2015(H8N4) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) 57 A/teal/Egypt/MB-D-698OP/2016(H7N3) A/northern shoveler/Egypt/MB-D-690OP/2016(H7N3) A/duck /Zambia/03/2008(H6N2) A/northern shoveler/Egypt/MB-D-690C/2016(H7N3) 100 A/duck/Zambia/02/2008(H6N2) 82 DD323186.1 Inspection Method for AIV Infection A/northern shoveler/Egypt/MB-D-695C/2016(H7N3) 55 A/duck/Mongolia/211/2015(H3N8) A/teal/Egypt/MB-D-698C/2016(H7N3) A/barnacle goose/Netherlands/2/2014(H3N6) A/duck/Egypt/053/2005(H6N2) A/northern shoveler/Egypt/MB-D-686OP/2016(H7N3) 98 A/whiskered tern/Egypt/04/2004(H6N2) A/teal/Egypt/MB-D-698OP/2016(H7N3) 99 A/chicken/South Africa/BKR2/2012(H6N2) A/chicken/South Africa/BKR4/2012(H6N2) 97 A/chicken/South Africa/MAS/2013(H6N2) 99 A/chicken/South Africa/N2826/2016(H6N2) 99 A/chicken/South Africa/W04/2002(H6N2) A/chicken/South Africa/AL19/02(H6N2) 94 A/chicken/South Africa/AL25/2002(H6N2) 78 A/chicken/South Africa/UP1102/02(H6N2) A/Teal/Markeev/13-3-11/2016(H5N2) 100 A/duck/Mongolia/140/2015(H10N2)

Figure S5. Phylogenetic relationship between GD/18, EG/1566C, EG/1556OP and UG/731 based on NS gene. All four viruses were indicated with black circles. Phylogenetic tree was constructed using the Maximum Likelihood method based on the Hasegawa-Kishino-Yano model. NS gene of EG/1566C and EG/1556OP clustered together Georgia, Netherlands, and Egypt sequences. NS of UG/731 and GD/18 clustered with Bangladesh, Korea, and Netherland sequences. Reference viruses (most closely related strains for at least one gene segment) are color-coded: A/domestic duck/Georgia/1/2016 (H4N6) in red, A/Teal/Markeev/13-3-11/2016 (H5N2) in blue, and A/teal/Egypt/MB-D-487OP/2016 (H7N3) in green.