1. A/chicken/Egypt/RIMD17-3/2009
A/duck/Egypt/D3Lu5/2007
A/duck/Egypt/D1Br2/2007
A/duck/Egypt/D1Tr220/2007
A/duck/Egypt/D3Li420/2007
A/duck/Egypt/D1Li10/2007
A/chicken/Egypt/RIMD27-4/2009
A/chicken/Egypt/2253-1/2006
A/chicken/Egypt/RIMD2-1/2008
A/chicken/Egypt/3/2006
A/turkey/Egypt/2253-2/2006
A/Egypt/3401-NAMRU3/2008
A/Egypt/14725-NAMRU3/2006
A/turkey/Egypt/7/2007
A/chicken/Egypt/1889N3-SM26/2007
A/chicken/Egypt/RIMD8-7/2008
A/chicken/Egypt/RIMD9-2/2008
A/chicken/Egypt/RIMD22-3/2009
A/chicken/Egypt/1/2006
A/duck/Egypt/2253-3/2006
A/chicken/Egypt/C1Li27/2007
A/chicken/Egypt/C1Tr1/2007
A/chicken/Egypt/12379N3-CLEVB/2006
A/chicken/Egypt/1129N3-HK9/2007
A/Egypt/1604-NAMRU3/2007
A/chicken/Egypt/0891/2008
A/chicken/Egypt/RIMD10-2/2008
A/chicken/Egypt/RIMD1-2/2008
A/chicken/Egypt/0813-NLQP/2008
A/goose/Egypt/13009N3-SM2/2006
A/duck/Egypt/0871/2008
A/chicken/Egypt/RIMD5-3/2008
A/duck/Egypt/D2Br213/2007
A/duck/Egypt/D2Li14/2007
A/duck/Egypt/D2br10/2007
A/chicken/Egypt/1/2008
A/chicken/Egypt/1/2009
A/chicken/Egypt/0869h3-NLQP/2008
A/Egypt/3300-NAMRU3/2008
A/chicken/Egypt/RIMD4-5/2008
A/chicken/Egypt/9402-CLEVB213/2007
A/chicken/Qalubia/CAI5/2008
A/chicken/Egypt/0836/2008
A/chicken/Egypt/1709-5/2008
A/chicken/Qalubia/CAI19/2008
A/chicken/Qalubia/CAI14/2008
A/chicken/Qalubia/CAI21/2008
A/chicken/Giza/CAI7/2008
A/chicken/Giza/CAI37/2009
A/duck/Qalubia/CAI11/2010
A/chicken/Egypt/N05811-CLEVB521/2009
A/chicken/Egypt/RIMD11-4/2008
A/chicken/Egypt/083/2008
A/chicken/Qalubia/CAI18/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI12/2008
A/chicken/Egypt/RIMD28-3/2009
A/chicken/Egypt/RIMD6-1/2006
A/chicken/Egypt/RIMD21-1/2009
A/chicken/Egypt/RIMD25-1/2009
A/chicken/Egypt/RIMD29-1/2009
A/chicken/Egypt/RIMD13-1/2008
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Egypt/RIMD26-1/2009
A/Pigeon/Egypt/RIMD18/2009
A/Egypt/N04979/2009
A/chicken/Egypt/RIMD12-2/2008
A/duck/Egypt/07322S-NLQP/2007
A/Egypt/N03439/2009
A/Egypt/N11981/2009
A/duck/Egypt/09184SM-NLQP/2009
A/chicken/Egypt/pt49/2014
A/Egypt/N02554/2010
A/Egypt/N03071/2010
A/duck/Egypt/1113SD/2011
A/duck/Egypt/Q4596C/2012
A/chicken/Egypt/14VIR S/2013
A/duck/Egypt/14VIR S/2013
A/chicken/Egypt/BSU-BS-F27/2012
A/Egypt/MOH-NRC-7271/2014
A/chicken/Egypt/A10351A/2014 99 75 73 90 66 79 87 91 98 97 72 61 65 92 63 94 93
0.002
Supplemental Fig. 1 Phylogenetic tree reconstructed from nucleotide sequences of the NA genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

2. A/chicken/Sharkia/CAI38/2009
A/chicken/Menofia/CAI36/2010
A/duck/Qalubia/CAI11/2010
A/duck/El Fayoum/CAI9/2010
A/chicken/Egypt/1063/2010
A/chicken/Egypt/Q1112E/2010
A/chicken/Egypt/Q1118D/2010
A/chicken/Sharkia/CAI16/2009
A/chicken/Egypt/096L-NLQP/2009
A/chicken/Qalubia/CAI21/2008
A/chicken/Qalubia/CAI14/2008
A/chicken/Qalubia/CAI19/2008
A/chicken/Giza/CAI8/2008
A/chicken/Sharkia/CAI41/2009
A/chicken/Sharkia/CAI27/2009
A/chicken/Giza/CAI15/2008
A/chicken/Qalubia/CAI5/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI12/2008
A/Pigeon/Egypt/RIMD18/2009
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Egypt/083/2008
A/chicken/Egypt/0836/2008
A/chicken/Egypt/9402-CLEVB213/2007
A/duck/Egypt/D1Tr8/2007
A/duck/Egypt/D1Li216/2007
A/duck/Egypt/D1Br22/2007
A/chicken/Egypt/06553-NLQP/2006
A/chicken/Egypt/07118-NLQP/2007
A/duck/Egypt/D2Li218/2007
A/duck/Egypt/D2br10/2007
A/duck/Egypt/D2Br212/2007
A/duck/Egypt/0871/2008
A/chicken/Egypt/0813-NLQP/2008
A/duck/Egypt/0923-NLQP/2009
A/chicken/Egypt/0891/2008
A/chicken/Egypt/08124S-NLQP/2008
A/duck/Egypt/2253-3/2006
A/chicken/Egypt/2253-1/2006
A/turkey/Egypt/2253-2/2006
A/duck/Egypt/D3Lu27/2007
A/duck/Egypt/D3Li212/2007
A/chicken/Egypt/C1Lu10/2007
A/chicken/Egypt/C1Li22/2007
A/chicken/Egypt/CL6/2007
A/chicken/Egypt/C3Br212/2007
A/chicken/Egypt/C1Tr4/2007
A/chicken/Egypt/102d/2010
A/Egypt/MOH-NRC-7271/2014
A/chicken/Egypt/A10351A/2014
A/chicken/Egypt/F9514E/2014
A/chicken/Egypt/F9514A/2014
A/duck/Egypt/S6419C/2012
A/chicken/Egypt/M2773A/2011
A/chicken/Egypt/S3280D/2011
A/chicken/Egypt/S3280B/2011
A/chicken/Egypt/S3280E/2011
A/duck/Egypt/Q4596A/2012
A/goose/Egypt/M2788E/2011
A/goose/Egypt/M2788A/2011
A/duck/Egypt/M3075B/2011
A/duck/Egypt/M2583C/2010
A/duck/Egypt/M2583A/2010
A/duck/Egypt/M2583B/2010
A/duck/Egypt/M2583D/2010
100 98 90 57 49 56 89 99 51 53 63 93 96 88 68 83 78
0.005
Supplemental Fig. 2 Phylogenetic tree reconstructed from nucleotide sequences of the PB2 genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

3. A/chicken/Qalubia/CAI19/2008
A/chicken/Giza/CAI24/2008
A/chicken/Giza/CAI8/2008
A/chicken/Giza/CAI7/2008
A/chicken/Sharkia/CAI27/2009
A/chicken/Qalubia/CAI5/2008
A/chicken/Sharkia/CAI16/2009
A/duck/El Fayoum/CAI1/2010
A/chicken/Menofia/CAI29/2009
A/chicken/Egypt/1/2009
A/chicken/Sharkia/CAI38/2009
A/chicken/Egypt/1063/2010
A/chicken/Egypt/1052s/2010
A/chicken/Egypt/Q1995D/2010
A/chicken/Egypt/Q2247A/2010
A/chicken/Egypt/096L-NLQP/2009
A/chicken/Egypt/083/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI18/2008
A/chicken/Qalubia/CAI12/2008
A/Pigeon/Egypt/RIMD18/2009
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Egypt/0836/2008
A/chicken/Egypt/1/2008
A/chicken/Giza/CAI6/2008
A/chicken/Egypt/9402-CLEVB213/2007
A/duck/Egypt/D1Li33/2007
A/duck/Egypt/D3Lu220/2007
A/duck/Egypt/D1Tr712/2007
A/duck/Egypt/D1Br423/2007
A/duck/Egypt/D3Li748/2007
A/duck/Egypt/D2Li449/2007
A/duck/Egypt/D2Br111/2007
A/chicken/Egypt/06553-NLQP/2006
A/turkey/Egypt/2253-2/2006
A/chicken/Egypt/07118-NLQP/2007
A/chicken/Egypt/2253-1/2006
A/chicken/Egypt/0813-NLQP/2008
A/duck/Egypt/0923-NLQP/2009
A/chicken/Egypt/0891/2008
A/duck/Egypt/2253 3/2006
A/chicken/Egypt/C1Lu2/2007
A/chicken/Egypt/C1Tr39/2007
A/chicken/Egypt/C1Li31/2007
A/chicken/Egypt/C3Br327/2007
A/duck/Egypt/0871/2008
A/chicken/Egypt/08124S-NLQP/2008
A/chicken/Egypt/102d/2010
A/duck/Egypt/M2583C/2010
A/turkey/Egypt/S6405A/2012
A/duck/Egypt/S6419C/2012
A/goose/Egypt/M2788E/2011
A/goose/Egypt/M2794A/2011
A/duck/Egypt/M2583D/2010
A/duck/Egypt/M3075B/2011
A/chicken/Egypt/S3280E/2011
A/chicken/Egypt/Q5283C/2012
A/chicken/Egypt/A10351A/2014
A/chicken/Egypt/F9514A/2014
A/chicken/Egypt/F9514E/2014
100 99 65 81 98 79 75 80 91 92 61 71 78 96 93 60 87
0.002
Supplemental Fig. 3 Phylogenetic tree reconstructed from nucleotide sequences of the PB1 genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

4. A/chicken/Qalubia/CAI21/2008
A/chicken/Giza/CAI7/2008
A/chicken/Qalubia/CAI19/2008
A/chicken/Sharkia/CAI27/2009
A/chicken/Qalubia/CAI5/2008
A/chicken/Sharkia/CAI41/2009
A/chicken/Menofia/CAI42/2009
A/chicken/Giza/CAI6/2008
A/chicken/Egypt/1/2008
A/chicken/Menofia/CAI39/2010
A/duck/El Fayoum/CAI9/2010
A/duck/El Fayoum/CAI1/2010
A/chicken/Sharkia/CAI38/2009
A/chicken/Egypt/Q1185/2010
A/chicken/Egypt/1/2009
A/chicken/Egypt/Q1184B/2010
A/chicken/Egypt/1063/2010
A/chicken/Egypt/9402-CLEVB213/2007
A/chicken/Egypt/083/2008
A/chicken/Egypt/0836/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI12/2008
A/Pigeon/Egypt/RIMD18/2009
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Egypt/096L-NLQP/2009
A/Egypt/902786/2006
A/chicken/Egypt/07118-NLQP/2007
A/duck/Egypt/0923-NLQP/2009
A/chicken/Egypt/0813-NLQP/2008
A/chicken/Egypt/0891/2008
A/duck/Egypt/2253-3/2006
A/chicken/Egypt/3/2006
A/duck/Egypt/D3Lu13/2007
A/duck/Egypt/D1Tr2/2007
A/duck/Egypt/D1Br17/2007
A/chicken/Egypt/2253-1/2006
A/Egypt/902782/2006
A/chicken/Qalubia/1/2006
A/turkey/Egypt/2253-2/2006
A/chicken/Egypt/06553-NLQP/2006
A/duck/Egypt/D2Li5/2007
A/duck/Egypt/D3Li13/2007
A/duck/Egypt/D1Li10/2007
A/duck/Egypt/D2Br236/2007
A/chicken/Egypt/C1Lu3/2007
A/chicken/Egypt/C1Li9/2007
A/chicken/Egypt/C1Tr211/2007
A/chicken/Egypt/C3Br4/2007
A/duck/Egypt/0871/2008
A/chicken/Egypt/08124S-NLQP/2008
A/chicken/Egypt/102d/2010
A/chicken/Egypt/14VIR S/2013
A/duck/Egypt/14VIR S/2013
A/Egypt/MOH-NRC-7271/2014
A/chicken/Egypt/A10351A/2014
A/Egypt/MOH-NRC-7305/2014
A/duck/Egypt/14VIR AD/2013
A/chicken/Egypt/F9514E/2014
A/chicken/Egypt/F9514A/2014
A/chicken/Egypt/14VIR AL/2013
A/duck/Egypt/M2583A/2010
A/chicken/Egypt/M2773A/2011
A/goose/Egypt/M2794A/2011
A/goose/Egypt/M2788A/2011
A/goose/Egypt/M2794E/2011
A/goose/Egypt/M2788E/2011 99 74 95
100 64 81 92 86 98 90 97 82 61 63 73 58 60 65
0.002
Supplemental Fig. 4 Phylogenetic tree reconstructed from nucleotide sequences of the PA genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

5. A/chicken/Egypt/1/2009
A/chicken/Egypt/Q2247A/2010
A/chicken/Egypt/09519S NLQP/2009
A/chicken/Egypt/1063/2010
A/chicken/Sharkia/CAI3/2009
A/chicken/Sharkia/CAI38/2009
A/duck/El Fayoum/CAI9/2010
A/duck/Qalubia/CAI11/2010
A/duck/El Fayoum/CAI1/2010
A/chicken/Egypt/10127s/2010
A/chicken/Egypt/115AD/2011
A/chicken/Menofia/CAI39/2010
A/chicken/Menofia/CAI35/2010
A/chicken/Egypt/1/2008
A/chicken/Qalubia/CAI14/2008
A/chicken/Qalubia/CAI21/2008
A/chicken/Qalubia/CAI19/2008
A/chicken/Menofia/CAI28/2009
A/chicken/Giza/CAI15/2008
A/chicken/Sharkia/CAI41/2009
A/chicken/Qalubia/CAI5/2008
A/chicken/Egypt/9402-CLEVB213/2007
A/chicken/Giza/CAI17/2009
A/chicken/Egypt/0836/2008
A/chicken/Egypt/1031/2010
A/chicken/Egypt/096L-NLQP/2009
A/chicken/Egypt/083/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI12/2008
A/chicken/Qalubia/CAI18/2008
A/Pigeon/Egypt/RIMD18/2009
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Egypt/0891/2008
A/duck/Egypt/0923-NLQP/2009
A/duck/Egypt/2253-3/2006
A/duck/Egypt/D3Lu13/2007
A/duck/Egypt/D2Br3/2007
A/duck/Egypt/D1Br3/2007
A/duck/Egypt/D3Li4/2007
A/duck/Egypt/D1Tr214/2007
A/chicken/Egypt/2253-1/2006
A/duck/Egypt/D2br10/2007
A/chicken/Egypt/0960-NLQP/2009
A/turkey/Egypt/2253-2/2006
A/duck/Egypt/0871/2008
A/chicken/Egypt/C3Br20/2007
A/duck/Egypt/D2Li10/2007
A/chicken/Egypt/C1Li2/2007
A/duck/Egypt/D1Li2/2007
A/chicken/Egypt/C1Lu1/2007
A/chicken/Egypt/C1Tr12/2007
A/chicken/Egypt/CL6/2007
A/chicken/Egypt/101604v/2010
A/chicken/Egypt/119S/2011
A/chicken/Egypt/0894-NLQP/2008
A/chicken/Egypt/pt49/2014
A/chicken/Egypt/1034/2010
A/chicken/Egypt/14VIR S/2013
A/chicken/Egypt/11316SS/2011
A/chicken/Egypt/B9039A/2013
A/chicken/Egypt/B9040A/2013
A/chicken/Egypt/F9514A/2014
A/Egypt/MOH-NRC-7305/2014
A/chicken/Egypt/A10351A/2014
A/Egypt/MOH-NRC-7271/2014
A/duck/Egypt/14VIR S/2013
A/Egypt/N02137/2012
A/duck/Egypt/14VIR AD/2013
A/chicken/Egypt/14VIR AL/2013
A/chicken/Egypt/S2938D/2011
A/chicken/Egypt/11669S/2011
A/Egypt/N00951/2012 97 98 80 52 73 46 68 56 47 95 99 57 71 88 96 83 89 92 58 66 72 61 43
0.002
Supplemental Fig. 5 Phylogenetic tree reconstructed from nucleotide sequences of the M genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

6. A/chicken/Qalubia/CAI14/2008
A/chicken/Giza/CAI8/2008
A/chicken/Sharkia/CAI41/2009
A/chicken/Qalubia/CAI19/2008
A/chicken/Sharkia/CAI27/2009
A/chicken/Qalubia/CAI5/2008
A/chicken/Giza/CAI15/2008
A/duck/Qalubia/CAI11/2010
A/duck/El Fayoum/CAI1/2010
A/duck/El Fayoum/CAI9/2010
A/chicken/Sharkia/CAI3/2009
A/chicken/Menofia/CAI36/2010
A/chicken/Sharkia/CAI38/2009
A/chicken/Egypt/Q2247B/2010
A/chicken/Egypt/1063/2010
A/chicken/Giza/CAI22/2008
A/chicken/Sharkia/CAI16/2009
A/Pigeon/Egypt/RIMD18/2009
A/Pigeon/Egypt/RIMD20/2009
A/chicken/Qalubia/CAI18/2008
A/chicken/Qalubia/CAI20/2008
A/chicken/Qalubia/CAI12/2008
A/chicken/Egypt/0836/2008
A/chicken/Egypt/083/2008
A/chicken/Egypt/9402-CLEVB213/2007
A/chicken/Giza/CAI6/2008
A/chicken/Giza/CAI17/2009
A/chicken/Egypt/096L-NLQP/2009
A/chicken/Egypt/08124S-NLQP/2008
A/chicken/Egypt/C3Br14/2007
A/chicken/Egypt/CL6/2007
A/chicken/Egypt/C1Lu14/2007
A/chicken/Egypt/C1Li13/2007
A/chicken/Egypt/C1Tr10/2007
A/duck/Egypt/D2Br24/2007
A/turkey/Egypt/2253-2/2006
A/chicken/Qalubia/1/2006
A/chicken/Egypt/07118-NLQP/2007
A/chicken/Egypt/2253-1/2006
A/duck/Egypt/D1Tr4/2007
A/chicken/Egypt/0891/2008
A/chicken/Egypt/0813-NLQP/2008
A/duck/Egypt/0871/2008
A/turkey/Egypt/7/2007
A/duck/Egypt/D3Li2/2007
A/duck/Egypt/D1Li8/2007
A/domestic poultry/Egypt/906089/2006
A/duck/Egypt/2253-3/2006
A/chicken/Egypt/06553-NLQP/2006
A/duck/Egypt/D3Lu1/2007
A/duck/Egypt/D1Br6/2007
A/duck/Egypt/D2Li417/2007
A/duck/Egypt/D2br10/2007
A/chicken/Egypt/14VIR AL/2013
A/chicken/Egypt/S2938D/2011
A/chicken/Egypt/S3280E/2011
A/chicken/Egypt/S3280B/2011
A/chicken/Egypt/Q5283C/2012
A/chicken/Egypt/A10351A/2014
A/chicken/Egypt/F9514A/2014
A/chicken/Egypt/F9514E/2014
A/Egypt/MOH-NRC-7271/2014
A/Egypt/MOH-NRC-7305/2014
A/duck/Egypt/14VIR S/2013
A/chicken/Egypt/IT-2/2013
A/chicken/Egypt/IT-1/2013 98 63 64 94 96 77 71
100 99 95 90 92 74 62 73 80 57 82 55 46
0.005
Supplemental Fig. 6 Phylogenetic tree reconstructed from nucleotide sequences of the NS genes of EG/P18, EG/P20 and the H5N1 Egyptian reference strains. The pigeon-derived viruses isolated in this study are bold underlined and marked with black circles. The tree was reconstructed using the neighbour-joining method, and phylogenetic relationships were estimated based on a bootstrap value of 1,000.

7. V3D
K363R
PB1
N-ext
RdRp
b-hairpin
RdRp
C-ext 1 35
352
398
669
757
A369V
V602I PA
PA-Nter
Endonuclease
PA-linker
PA-C
PA-arch
PA-C 1
195
256
367
396
716
R38K NP
RNA-binding
NP-NP self interaction
NP-NP self interaction 1
181
189
358
371
465
D120N
RNA-binding
Linker
region
NS1
Effector
C-terminal 1 73 88
202
237
F55C
NEP
Protease-sensitive
Protease-resistant 1 54
121
Supplemental Fig. 7 Overview of the unique and rarely reported mutations identified in this study. The unique and rarely detected mutations identified in this study are shown with an arrowhead. The functional domains of viral internal proteins are shown in gray and white.