Virus Genes DOI 10.1007/s11262-014-1037-0
Molecular evolution of H9N2 avian influenza viruses in Israel Irit Davidson • Alice Fusaro • Alireza Heidari Isabella Monne • Giovanni Cattoli
•
Received: 24 October 2013 / Accepted: 12 January 2014 Ó Springer Science+Business Media New York 2014
Abstract While the previous phylogenetic analyses of AIV H9N2 in Israel had mainly focused on phylogenetics and on describing different virus introductions into the country, for the first time, the H9N2-HA gene evolutionary history has been examined taking into account its origin, evolution and phylodynamics. The present study reveals the Israeli H9N2 molecular evolution rate, the virus molecular clock and skyline plot. The molecular skyline plot showed two major increments in population diversity sizes, the first which had occurred in 2003, the second between the end of 2007 and the first half of 2008. Between 2004 and 2007 the population size had proved to be constant. The two peaks correspond to the appearance of the 3rd and 4th major genetic groups, as well as to the introduction of two H9N2 vaccines. The mean evolution rate was 6.123 E-3 substitutions/site/year, typical of avian influenza viruses. The time interval from the most recent common ancestor was 12.3 years, corresponding to the year 2000, when H9N2 was first isolated in Israel. Keywords Avian influenza virus subgroup H9N2 Molecular evolution HA gene Phylodynamics Molecular clock
I. Davidson (&) Division of Avian Diseases, Kimron Veterinary Institute, P.O.Box 12, 50250 Bet Dagan, Israel e-mail: [email protected] A. Fusaro A. Heidari I. Monne G. Cattoli Research and Development Department, Istituto Zooprofilattico Sperimentale delle Venezie, OIE/FAO and National Reference Laboratory for Avian/Animal Influenza and Newcastle Disease, OIE Collaborating Centre for Diseases at the Human-Animal Interface, Legnaro, Padova, Italy
Introduction Avian influenza viruses (AIV) of the H9N2 subtype are widespread worldwide, being endemic in poultry populations in Asia and the Middle East [1, 2]. Outbreaks of AIV H9N2 in poultry have occurred in the western hemisphere since 1994, including Germany, Italy, Ireland, South Africa and the USA, as reviewed by Capua and Alexander [3]. Fusaro et. al. [4] and Davidson et. al. [5] have, respectively, described the Middle East AIV H9N2 and the Israeli AIV H9N2 isolates, focusing on distinct virus populations. Fusaro et. al. [4] described Middle Eastern and Central Asia isolates, including only limited Israeli H9N2 viruses, while Davidson et. al. [5] focused on Israeli AIV H9N2 isolates including only limited Asian isolates. The present study fulfils this gap by including the most comprehensive AIV H9N2 collection since their emergence back in 1997 in the Middle East and Asia and constitutes the missing ring between the two previous studies by reflecting the evolutionary history, origin and phylodynamics of the Israeli AIV H9N2. In Israel, AIV H9N2 have been isolated and molecularly characterised since the year 2000 [5–7]. All AIV H9N2 Israeli isolates belong to the G1 lineage, possess a cleavage site characteristic of low pathogenic AIV and are classified into five phylogenetic groups [5]. As no intermediate phylogenetic forms have ever been isolated, it was assumed that the source of the new virus introductions in Israel had been external, although no proofs had been provided until now. The first and second AIV H9N2 introductions prevailed from May 2000 to April 2003, the third introduction circulated from April 2003 to December 2006, while after 2006, the fourth genetic group, subdivided into two subgroups, A and B, prevailed in Israel. In 2012, the fifth group emerged, although viruses belonging to the fourth group were still circulating. The isolates A/ty/Israel/965/02
123
Virus Genes Table 1 The HA gene sequences employed in the present study, including their name, accession, country and year of isolation: the HA sequences from Israel, Central Asian and Middle East countries used to construct the phylogenetic tree in Fig. 1 Country
Year of isolation
No. of sequences
Israel
2002–2012
123
Jordan
2004–2007
11
Lebanon
2004–2010
3
Egypt
2011–2012
11
Middle East
1999
1
Libia
2006
1
Emirates
2002–2011
2
Qatar
2008
1
Saudi Arabia
2002–2011
22
Dubai
2000–2008
10
Iraq
2005–2008
2
Iran
1998–2010
63
Pakistan
1999–2010
18
Afganistan Kurdistan
2008–2009 2009
7 2
Bangladesh
2007
3
India
2006–2008
3
Hong Kong
1997–2010
18
Japan
1998
Total
1 303
and A/ck/Israel/215/07, belonging to the second and fourth genetic groups, respectively, were selected as prototype vaccine strains. The use of the first vaccine (A/ty/Israel/
965/02) commenced in 2003 and lasted until the end of 2008, when the second vaccine (strain A/ck/Israel/215/07) was introduced. In the previous study, the genetic grouping of the Israeli AIV H9N2 viruses was associated to separate introductions and phylogenetically deducted by analysing the genomic homology extent between viruses [5]. In the present study, the application of different and more advanced computational algorithms made it possible to perform a deeper investigation on the Israeli AIV H9N2 molecular viral evolution. Furthermore, the genetic relations of the various introductions of AIV H9N2 in Israel with the AIV H9N2 isolated in the surrounding countries have been investigated for the first time. The new analysis reveals the time and location of the most recent ancestor, highlights the nucleotide substitution rates and depicts the population dynamics and the extent of its molecular change since the AIV H9N2 first detection.
Materials and methods Viruses included in the study Only AIV H9N2 HA genes publicly available in the Gene Bank were included. The complete Israeli HA sequences included 97 viruses (Table 2). As well, the sequence dataset (n = 303; Table 1) included those from surrounding countries [4] and the newly depicted GenBank database viruses after 2010.
Table 2 The HA gene sequences employed in the present study, including their name, accession, country and year of isolation: Israeli complete HA sequences with specific specification of the collection date Year of isolation
No. of HA sequences
Accession no.
2000
2
EF492221, AY738451
2001 2002
4 9
AY738454, DQ104466-DQ104468 EF462494, AY738452, EF462495, EF492237, DQ104459-DQ104461, DQ104463, EF462496
2003
22
DQ108908-10, DQ108921, DQ104484, DQ104480-2, DQ104474, DQ108906, DQ108913-14, DQ108911, AY738455-6, EF462497, EF46250-3, EF462498, EF492240
2004
11
EF462504-7, DQ108919-20, AY738453, DQ108916-7, EF501983, EF492243
2005
11
EF492222, EF492225-6, EF492228, EF492232, EF492234-5, EF492236, EF492238, EF492230, EF462493
2006
4
FJ464729-30, FJ464714, EF492224,
2007
9
GQ120557, FJ464724-5, FJ464715-6, GQ120553, FJ464720, FJ464721-2
2008 2009
5 5
GQ120558, JQ347954, JQ347957, JQ347959, GQ120554 JQ347952-3, JQ347945, JQ254940, JQ347950,
2010
5
JQ254942, JQ254945, JQ254948, JQ254952, JQ254954
2011
4
JQ973657, JQ973658, JQ973659, JQ973660
2012
6
JQ973651-2, JQ973653-6
TOTAL
97
123
Virus Genes 89 A/chicken/Egypt/11vir4453-276/2010 A/chicken/Egypt/11vir4453-280/2011 A/chicken/Egypt/11vir4453-275/2011 A/chicken/Egypt/114915V/2011 A/chicken/Egypt/11vir4453-274/2011 A/chicken/Egypt/A2-D/2011 A/chicken/Egypt/11vir4453-273/2011 74,7% 91 A/chicken/Egypt/11vir4453-272/2011 98 A/chicken/Egypt/115617V/2011 A/chicken/El_Fayoum/CAI25/2011 A/chicken/Egypt/S4454B/2011 93,9% A/chicken/Egypt/S4454E/2011 97% A/chicken/Egypt/S4456B/2011 73,3% A/chicken/Egypt/12186F-9/2012 88,8% A/chicken/Egypt/114940v/2011 0% A/chicken/Egypt/115636V/2011 95,1% 77,4% A/quail/Egypt/113413v/2011 A/chicken/Egypt/11vir4453-132/VRLCU/2011
83 88 95
99
92,2% 75,8%
Egypt 2010-2012
B
turkey/Israel/311/2009_Kfar_Ezion chicken/Israel/663/2009_Mishmar_Hanegev 99,7% 93,1% chicken/Israel/794/2009_Bizaron turkey/Israel/117/2009_Sde_Yoav 87,1% turkey/Israel/1257/2008_Rosh_Zurim turkey/Israel/1608/2006_Nahalal
76,9%
chicken/Israel/50/2012_Klahim 96,4% chicken/Israel/58/2012_Talme_Bilu 85,4% chicken/Israel/3/2012_Kalia 99,3% chicken/Israel/1067/2010_Haifa 76% turkey/Israel/746/2009_Ein_Yron 93,3%chicken/Israel/724/2009_Mezer 0% 100% chicken/Israel/1302/2010_Shuval chicken/Israel/742/2009_Sde_Eliahu
0%
100%
0% 0% chicken/Israel/1525/2006_Gshor 0% chicken/Israel/1638/2006_Givat_Hen chicken/Israel/386/2007_Dovev 99,2% chicken/Israel/182/2008_Gilat 99,9% chicken/Israel/54/2008_Gilat 78% 0% chicken/Israel/1548/2006_Metzer chicken/Israel/215/2007_Midrach_Oz 0% chicken/Israel/728/2007_Afek 0% chicken/Israel/402/2007_Goren 83,3% chicken/Israel/449/2007_Goren chicken/Israel/760/2008_Goren 92,1% chicken/Israel/884/2008_Avdon 0% 0% chicken/Israel/330/2008_Gilat turkey/Israel/900/2007_Zarchia 0% chicken/Israel/869/2007_Ein_Gev 84,5% chicken/Israel/524/2008_Azrikam 0% chicken/Israel/702/2008_Kabri avian/Israel/314/2008_Bat_Yam 0% 0% chicken/Israel/310/2008_Gilat 92,8% chicken/Israel/292/2008_Sde_David 0% 75,4% turkey/Israel/689/2008_Ein_Hashlosha chicken/Israel/694/2008_Even_Menahem chicken/Israel/1293/2010_Zipori 77,5% 0% chicken/Israel/1167/2010_Mehola 90,2% chicken/Israel/1184/2010_Elipelet 99,9% chicken/Israel/1164/2011_Pardes_Hanna 95,7% A/quail/Lebanon/272/2010 100% A/quail/Lebanon/273/2010 89% A/chicken/Israel/184/2009 87,1% 95,3% chicken/Israel/184/2009_Gilat chicken/Israel/1256/2008_Afula 78,8% A/chicken/Jordan/VIR436-2/2010 86,9% 90,5%chicken/Israel/712/2009_Nahalal turkey/Israel/775/2009_Mei_Ami 94% 99,8% 86% A/chicken/Jordan/VIR436-1/2010 chicken/Israel/883/2008_Elipelet chicken/Israel/1195/2008_Ramat_Zvi 92% chicken/Israel/32/2009_Tel_Kazir 74,5% chicken/Israel/43/2009_Ziveon 66% chicken/Israel/1205/2008_Eilon 95,3%chicken/Israel/1018/2008_Goren turkey/Israel/1223/2008_Ein_Zurim
Israel Introduction IV
Lebanon 2010 Jordan 2010
94%
74,5%
83,8% 99,9% 94,4%
97,4% A/ck/Qatar/4576v08-4/2008 A/ck/Dubai/3771v09-2/2008 88,8% A/ck/Dubai/3771v09-3/2008 A/avian/Libya/RV35D/2006
chicken/Israel/51/2012_Nahalal 0% chicken/Israel/1115/2011_Avital 83% chicken/Israel/1089/2011_Bezet 0% 78,8%chicken/Israel/46/2012_Nahalal chicken/Israel/11/2012_Um_El_Phahem 100% chicken/Israel/1163/2011_Avital A/chicken/Saudi_Arabia/H29TR/2011 A/chicken/United_Arab_Emirates/F1P7/2011 A/chicken/Saudi_Arabia/D-36363/2010 A/chicken/Saudi_Arabia/C-36362/2010
Israel Introduction V
93,8%
100%
99,4% 0%
96,7%
83,9%
88,4%
Saudi Arabia UAE 2005-2011 Libia Qatar
A/ck/SaudiArabia/3489v08-51as366/2008 47,3% A/ck/SaudiArabia/3489v08-48as997/2007 100% A/ck/SaudiArabia/3489v08-50as118/2008 A/chicken/Saudi_Arabia/E-36364/2006 99,4% A/chicken/Saudi_Arabia/E-364/2006 73,5% A/chicken/Saudi_Arabia/F-36365/2006
97,6%
74,8% 100% A/chicken/Saudi_Arabia/F-6365/2006 A/chicken/Saudi_Arabia/2BL09/2006 84,7% A/ck/SaudiArabia/3489v08-38as92/05 82,5% A/chicken/Saudi_Arabia/582/2005 A/avian/Saudi/Arabia/910136/2006 98,6% 0% A/ck/SaudiArabia/3489v08-47as215/2007 91,1% A/avian/Saudi/Arabia/910134/2006 78% A/ck/SaudiArabia/3489v08-33as638/2005 99,5% A/falcon/SaudiArabia/3489v08-40as977/2006 A/ck/SaudiArabia/3489v08-44as253/2006 100% A/avian/Saudi/Arabia/910135/2006
99,9%A/chicken/Iran/10VIR854-3/2009 A/chicken/Iran/EBGV-88/2010 96,3% A/ck/Afghanistan/329v09-7AFG-Heart6/2009 99,4% A/ck/Afghanistan/329v09-8AFG-Heart7/2009 99,9% A/chicken/Sihala/NARC-12103/2008 0% A/chicken/Pakistan/UDL-03/2007 97,5% A/chicken/Pakistan/UDL-01/2008 A/chicken/Pakistan/UDL-04/2006 90,3% A/chicken/Pakistan/UDL-01/2006 A/chicken/Sawabi/NARC-2434/2006 92,8% 100% A/chicken/Pakistan/NARC-2434/2006 A/chicken/Pakistan/UDL-02/2006 100% A/chicken/Pakistan/UDL-04/2007 80,9% A/chicken/Pakistan/UDL-02/2005 100% 100%60,8% A/chicken/Pakistan/UDL-03/2005 A/chicken/Pakistan/UDL-01/2007 A/chicken/Pakistan/UDL-01/2005 A/chicken/Pakistan/47/2003 A/chicken/Iran/THLBM867/2007 95,3% A/chicken/Iran/THLBM868/2007 96,8% A/chicken/Iran/THLBM861/2007 95,9% A/Chicken/Iran/TH386/2007 A/chicken/Iran/THLBM863/2007 83,5% 90,7% A/chicken/Iran/THLBM866/2007 18,8% A/chicken/Iran/THLBM865/2007 75,5% 76,7% A/Chicken/Iran/TH286/2007 90,9% A/Chicken/Iran/TH186/2007 0% 99,9% A/chicken/Iran/10VIR854-5/2008 98,6% A/Chicken/Iran/TH85/2007 A/chicken/Iran/TH486/masoumi/2007 96,9% A/chicken/Iran/RZ28/2008 84,5% 0% A/chicken/Iran/RZ42/2008 A/chicken/Iran/RZ37/2008 100% 72,2% A/chicken/Iran/RZ36/2008 62,6% A/chicken/Iran/RZ53/2008 89,5% A/chicken/Iran/133/2004 A/chicken/Iran/B102/2005 88,7% A/chicken/Iran/68/2006 A/chicken/Iran/SH2/2007 76,8% A/chicken/Iran/20/2006 76,4% 76,9% A/chicken/Iran/B11A/2005 87,2%A/chicken/Iran/B99/2005 100% 74,4% A/chicken/Iran/L248/2003 75,4% A/chicken/Iran/B326/2005 A/chicken/Iran/B76/2004 A/ck/Kurdistan/4693v09-88/2009 66,9% 99,5% A/ck/Kurdistan/4693v09-9/2009 99,6% A/chicken/Iran/10VIR854-4/2009 20,2% 99,9% A/chicken/Iraq/EKI_14/2008 0% A/chicken/Iran/B308B/2004 A/chicken/Iran/320/2003 A/chicken/G-Karachi/2003 A/ck/Afghanistan/329v09-3AFG-Khost2/2008 0% A/ck/Afghanistan/329v09-5AFG-Khost4/2008 0% A/ck/Afghanistan/329v09-6AFG-Khost9/2008 0% A/ck/Afghanistan/329v09-2AFG-Khost1/2008 99,7% A/ck/Afghanistan/329v09-4AFG-Khost3/2008 100% A/chicken/Pakistan/UDL-02/2008 100% A/chicken/Pakistan/UDL-03/2008 A/chicken/Bangladesh/VP01/2007 100% A/chicken/Bangladesh/627/2007 50,9% A/chicken/Uchal/8286/2006 67,7% 99,2% A/chicken/India/3/2003 A/chicken/Bangladesh/FDIL/112/2007 81,8%
97,5%
98,5%
99,4%
96,2%
72,5% 97,9% 79,2% 31,3%
Iran Afghanistan Pakistan
A/chicken/Iran/THLBM862/2007
Iran 2003-2008 Iraq
G1 India Bangladesh Afghanistan Pakistan
A/chicken/Iran/SS2/2008 A/chicken/Iran/THLBM864/2007 54,4%
96,5% 94,3%
100% A/Chicken/Pakistan/5/1999 99,2% A/Chicken/Pakistan/2/1999 A/Chicken/Pakistan/4/1999
90,3%
A/chicken/Dubai/383/2002 78,6% A/chicken/Dubai/339/2001 99,8% A/chicken/Dubai/338/2001 A/chicken/Emirates/R66/2002 A/chicken/Dubai/463/2003
0%
94,5%
99,7%
0% A/HongKong/1074/1997 89,2% 0% A/HongKong/1074/1999 95,3% A/HongKong/1073/1999 A/guineafowl/HongKong/WF10/1999 A/Quail/HongKong/G1/1997
99,7% 99,9%
A/Chicken/HongKong/G9/97 42,8% A/Chicken/HongKong/G23/97
C
Pakistan 1999
A Israel Introduction III
58,5% 82,5% 0% 99,2% 0% chicken/Maale_HaHamisha/90658/2000_Maale_Hahamisha chicken/Israel/90658/2000_Maale_Hahamisha A/chicken/Israel/1990658/2000 A/chicken/Middle_East/ED-1/1999 A/chicken/Saudi/Arabia/EPD-22-01/2002 A/chicken/Iran/10VIR854-1/2002 76,5% 0% A/garganey/Iran/K8/2003 A/chicken/Iran/B314/2003 85,1% A/chicken/Iran/119/2005 69,7% A/chicken/Iran/284/2002 100% A/chicken/Iran/B322/2004 0% A/Chicken/Iran/TH78/1999 74,8% A/Chicken/Iran/TH79/2000 49,7% A/chicken/Iran/705/1999 77,9% A/chicken/Iran/B263/2003 100% 76,2% A/chicken/Iran/152/2004 100% A/chicken/Iran/B308A/2004 99,3% 74% 38%A/chicken/Iran/772/1999 A/chicken/Iran/233/2001 46,8% A/chicken/Iran/565/2000 86,3% A/Chicken/Iran/11T/1999 A/chicken/Iran/698/1998 83,3% A/chicken/Iran/SH1/2007 0% A/chicken/Iran/450/2001 96,6% A/chicken/Iran/86/2005 100% 90,2% A/chicken/Iran/L252/2003 A/Chicken/Iran/TH77/1998 49,5% 92,1% A/chicken/Iran/661/1998 86,8% A/chicken/Iran/SS1/1998 82,4% A/chicken/Saudi/Arabia/CP7/1998 A/parakeet/Narita/1992A/1998 96,5%
2003-2008 A/chicken/Pakistan/NCVI-09/2010
UAE 2000-2002
A/Iraq/579v09-8/2005 79,3% A/avian/Jordan/7-Y1/2004 77,4% 0% turkey/Nahalel/1547/2004_Nahalal 0% chicken/Tel_Adashim/1469/2003_Tel_Adashim chicken/Alonim/1552/2003_Alonim 0% 83,5% chicken/Tel_Adashim/1506/03_Tel_Adashim turkey/Beit_HaLevi/1566/04_Bet_Halevi 0% A/ck/Jordan/1436v04-1451/2004 84,3% 90,5% chicken/Neve_Ilan/1504/03_Neve_Ilan chicken/Pardes_Hana_Carcur/1475/2003_Pardes_Hanna 0% chicken/Israel/1475/2003_Pardes_Hanna chicken/Israel/1966/2004_Kalanit 0% chicken/Beit_Aran/29/2005_Beit_Aran 0% 0% chicken/Israel/29/2005_Kalanit 95,8% chicken/Klanit/1966/2004_Kalanit 0% turkey/Israel/747/2005_Neot_Golan 100% 97,2% turkey/Israel/884/2005_Ramot 0% A/chicken/Lebanon/1080/2004 chicken/Gan_Shomron/1465/03_Gan_Shomron 83,7% chicken/Israel/909/2005_Hadera 0% chicken/Israel/853/2005_Maanit 97,8% chicken/Israel/793/2005_Maanit 73,8% A/ck/Jordan/579v09-4/2007 99,7% 100% chicken/Israel/114/2007_Gevim 0% chicken/Israel/375/2007_Shazor 0% chicken/Israel/1953/2004_Alonim 0% chicken/Alonim/1953/2004_Alonim 93,9% 0% turkey/Israel/425/2005_Mishmar_Hasharon 91,2% chicken/Amioz/1527/03_Amioz chicken/Iarah/1376/03_Iarah chicken/Tel_Adashim/1321/2003_Tel_Adashim avian/Israel/584/2005_Sahnin 99,7% chicken/Israel/178/2006_Dalton 0% chicken/Israel/282/2005_Goren 43,7% A/chicken/Israel/421201/2004 0% 98% turkey/Tzur_Moshe/1565/2004_Tzur_Moshe chicken/Israel/554/2005_Elipelet 99,4% turkey/Bazat/89/2005_Bezet 0% 0% A/ck/Jordan/579v09-1/2007 93,7% turkey/Beit_HaLevi/1562/04_Beit_Halevi 100% turkey/Gyvat_Haim_Ehud/1544/03_Gyvat_Haim 81,1% chicken/Tzrofa/1568/2004_Tzrofa 0% A/ck/Jordan/1436v04-997/2004 0% A/ck/Jordan/1436v04-643/2004 0% 91,6% A/duck/Jordan/1436v04-890/2004 A/ck/Jordan/579v09-2/2007 0% 90,4% A/ck/Jordan/579v09-3/2007 86,1% turkey/Ein_Tzurim/1738/2004_Ein_Tzurim chicken/Ysodot/1362/2003_Yesodot 0% 0% chicken/Beit_Alfa/1282/2003_Beit_Alpha 0% turkey/Beit_Herut/1267/2003_Bet_Herut 0% chicken/Uzziyyahu/1651/2004_Uziyahu 0% chicken/Netohah/1373/03__Neohah 0% chicken/Talmei_Elazar/1304/03_Talmei_Elazar chicken/Telmond/1308/03_Tel_Mond turkey/Qevuzat_Yavne/1242/2003_Yavne 0% turkey/Avigdor/1209/2003_Avigdor 0% turkey/Israel/1209/2003_Avigdor 99,4% turkey/Brosh/1276/2003_Brosh 74,3% turkey/Israel/1013/2002_Naharia 0% 85,8% turkey/Hod_Ezyon/699/2002_Hod_Ezyon 0% turkey/Givat_Haim/965/2002_Givat_Haim turkey/Mishmar_Hasharon/619/2002_Mishmar_Hasharon 39,9% turkey/Yedidia/625/02_Yedidia 91,6% ostrich/Eshkol/1436/03_Eshkol 0% turkey/Kfar_Vitkin/616/2002_Kfar_Vitkin 84,6% turkey/Kfar_Vitkin/615/2002_Kfar_Vitkin 91,2% 0% chicken/Kfar_Monash/636/2002_Kfar_Monash turkey/Givat_Haim/810/01_Givat_Haim 0%0% chicken/Tel_Adashim/812/2001_Tel_Adashim 99,9% chicken/Tel_Adashim/811/2001_Tel_Adashim chicken/Tel_Adashim/786/2001_Tel_Adashim turkey/Neve_Ilan/90710/2000_Neve_Ilan
86,1%
99,7%
91,8%
A/quail/Dubai/301/2000 88,6% 90,3%A/quail/Dubai/302/2000 A/quail/Dubai/303/2000
2005-2010
Israel Introduction I+II A/quail/SaudiArabia/3489v08-46as104/2006
Iran 1998-2005 Saudi Arabia
A/HongKong/33982/2009 100% A/HongKong/35820/2009
D
Hong Kong 1997-2009 A/mallard/Iran/T366/2007 0% A/mallard/Iran/C370/2007 0% A/mallard/Iran/T370/2007 100% A/mallard/Iran/C364/2007
A/Duck/HongKong/Y280/97
0.04
Fig. 1 Phylogenetic tree created by the ML algorithm by PhyML of GenBank AIV H9N2 HA complete sequences—Israel until 2012 and Eastern countries, including isolates included Blast similar and
isolates of Fusaro et al. [4]. Colours and classification A–D were by Fusaro et al. [4]. Blue—Israeli H9N2 isolates, Black—global H9N2 isolates (Color figure online)
Phylogenetic analysis
Substitution rates and mean time to the most recent common ancestor (tMRCA)
The maximum likelihood (ML) tree (Fig. 1) was estimated by the PhyML software using the general time-reversible (GTR) model of nucleotide substitution with gamma-distributed rate variation among sites, and a heuristic SPR branch swapping search.
Rates of nucleotide substitution per site per year and the tMRCA of the sampled data were estimated as before [4], using the BEAST program version 1.7 [8], which employs a Bayesian Markov chain Monte Carlo (MCMC) approach.
123
Virus Genes
A
A/chicken/Israel/330/2008#2008.163_Gilat 14,88% A/turkey/Israel/900/2007#2007.874_Zarchia 7,91% A/chicken/Israel/869/2007#2007.847_EinGev 90,62% A/chicken/Israel/760/2008#2008.34_Goren 99,92% A/chicken/Israel/884/2008#2008.453_Avdon 24,38% A/chicken/Israel/524/2008#2008.192_Azrikam 10,53% A/chicken/Israel/292/2008#2008.068_SdeDavid 27,42% A/avian/Israel/314/2008#2008.086_BatYam 98,92% 90,92%A/chicken/Israel/310/2008#2008.074_Gilat A/turkey/Israel/689/2008#2008.292_EinHashlosha 97,82%
A/chicken/Israel/1167/2010#2010.893_Mehola 33,01% 96,7% A/chicken/Israel/1293/2010#2010.958_Zipori A/chicken/Israel/1184/2010#2010.901_Elipelet 100% A/chicken/Israel/1164/2011#2011.96_PardesHanna A/chicken/Israel/694/2008#2008.3_EvenMenahem A/chicken/Israel/702/2008#2008.308_Kabri A/turkey/Israel/775/2009#2009.927_MeiAmi 100% A/chicken/Israel/712/2009#2009.877_Nahalal 93,06% A/chicken/Israel/1256/2008#2008.96_Afula 100% 99,99% 98,39% A/chicken/Israel/184/2009#2009.125_Gilat A/chicken/Israel/883/2008#2008.453_Elipelet A/chicken/Israel/32/2009#2009.025_TelKazir 100% A/chicken/Israel/1195/2008#2008.901_RamatZvi 79% 99,96% A/chicken/Israel/43/2009#2009.033_Ziveon 93,96% A/chicken/Israel/1205/2008#2008.91_Eilon 100% A/chicken/Israel/1018/2008#2008.774_Goren A/turkey/Israel/1223/2008#2008.927_EinZurim A/chicken/Israel/1302/2010#2010.958_Shuval 12,47% A/chicken/Israel/742/2009#2009.896_SdeEliahu 9,01% A/chicken/Israel/724/2009#2009.89_Mezer 100% A/turkey/Israel/746/2009#2009.904_EinYron 100% A/chicken/Israel/58/2012#2012.03_TalmeBilu 100%A/chicken/Israel/50/2012#2012.027_Klahim 99,97% A/chicken/Israel/3/2012#2012.003_Kalia 100% A/chicken/Israel/1067/2010#2010.821_Haifa 83,72% A/chicken/Israel/794/2009#2009.941_Bizaron 100% A/chicken/Israel/663/2009#2009.838_MishmarHanegev 88,77% A/turkey/Israel/311/2009#2009.127_KfarEzion 31,47% 100% A/turkey/Israel/1257/2008#2008.944_RoshZurim 99,67% A/turkey/Israel/117/2009#2009.079_SdeYoav 6,72%A/chicken/Israel/386/2007#2007.186_Dovev A/chicken/Israel/449/2007#2007.262_Goren 29,15% A/chicken/Israel/728/2007#2007.573_Afek 2,49% 60,09% A/chicken/Israel/402/2007#2007.205_Goren A/chicken/Israel/1525/2006#2006.947_Gshor 9,12% 7,58% A/chicken/Israel/215/2007#2007.086_MidrachOz A/chicken/Israel/182/2008#2008.027_Gilat 100% A/chicken/Israel/54/2008#2008.003_Gilat 100%49,02% A/chicken/Israel/1548/2006#2006.955_Metzer 3,82% A/turkey/Israel/1608/2006#2006.977_Nahalal A/chicken/Israel/1638/2006#2006.99_GivatHen A/chicken/Israel/1089/2011#2011.941_Bezet 32,8% A/chicken/Israel/51/2012#2012.027_Nahalal 96,59% A/chicken/Israel/1115/2011#2011.949_Avital 100% A/chicken/Israel/46/2012#2012.016_Nahalal 18,68% A/chicken/Israel/11/2012#2012.008_UmElPhahem 38,04% A/chicken/Israel/1163/2011#2011.971_Avital 28,75% 99,76%22,83%
99,86%
100%
100%
A/chicken/Alonim/1552/2003#2004.004_Alonim 40,18% A/chicken/Tel_Adashim/1506/03#2003.93_TelAdashim 24,33% A/turkey/Beit_HaLevi/1566/04#2004.038_BetHalevi 95,11% 97,26% A/turkey/Nahalel/1547/2004#2004.033_Nahalal 65,28%A/chicken/Tel_Adashim/1469/2003#2003.918_TelAdashim A/chicken/Israel/1475/2003#2003.941_PardesHanna 40,54% A/chicken/Pardes_Hana_Carcur/1475/2003#2003.941_PardesHanna A/chicken/Israel/793/2005#2005.91_Maanit 5,37% 32,97% A/chicken/Israel/909/2005#2005.996_Hadera 100% A/chicken/Israel/853/2005#2005.936_Maanit 82,34% A/chicken/Israel/114/2007#2007.03_Gevim 100% A/chicken/Israel/375/2007#2007.184_Shazor 1,43% A/turkey/Israel/425/2005#2005.596_MishmarHasharon 29,29% 99,6% 100%A/chicken/Alonim/1953/2004#2004.879_Alonim A/chicken/Israel/1953/2004#2004.879_Alonim 99,96% 6,57% A/chicken/Amioz/1527/03#2003.985_Amioz A/chicken/Gan_Shomron/1465/03#2003.922_GanShomron 15,78% A/chicken/Israel/1966/2004#2004.936_Kalanit 19,82% A/chicken/Beit_Aran/29/2005#2005.005_BeitAran 100% A/chicken/Klanit/1966/2004#2004.936_Kalanit 17,9% A/chicken/Israel/29/2005#2005.011_Kalanit 100% 2,29% A/turkey/Israel/884/2005#2005.985_Ramot 11,45% 100% A/turkey/Israel/747/2005#2005.912_NeotGolan 1,52% A/chicken/Uzziyyahu/1651/2004#2004.175_Uziyahu A/chicken/Talmei_Elazar/1304/03#2003.348_TalmeiElazar A/chicken/Iarah/1376/03#2003.54_Iarah 13,28% A/chicken/Telmond/1308/03#2003.367_TelMond 100%A/avian/Israel/584/2005#2005.807_Sahnin 48,85% A/chicken/Israel/178/2006#2006.085_Dalton 72,84% A/turkey/Bazat/89/2005#2005.063_Bezet 1,13% 100% 94,58% A/chicken/Israel/554/2005#2005.804_Elipelet 99% A/chicken/Israel/282/2005#2005.378_Goren A/turkey/Tzur_Moshe/1565/2004#2004.036_TzurMoshe 79,86% A/turkey/Gyvat_Haim_Ehud/1544/03#2003.996_GyvatHaim 47,19% A/turkey/Beit_HaLevi/1562/04#2004.996_BeitHalevi 59,09% 3,2% A/chicken/Tzrofa/1568/2004#2004.025_Tzrofa 32,55% 40,02% A/chicken/Ysodot/1362/2003#2003.529_Yesodot 28,84% A/turkey/Ein_Tzurim/1738/2004#2004.337_EinTzurim 29,22% A/chicken/Neve_Ilan/1504/03#2003.979_NeveIlan 100% 18,98% A/chicken/Beit_Alfa/1282/2003#2003.286_BeitAlpha A/chicken/Tel_Adashim/1321/2003#2003.405_TelAdashim A/chicken/Netohah/1373/03_#2003.289_Neohah 7,19% A/turkey/Beit_Herut/1267/2003#2003.267_BetHerut A/chicken/Israel/90658/2000#2000.416_MaaleHahamisha A/chicken/Maale_HaHamisha/90658/2000#2000.397_MaaleHahamisha A/turkey/Brosh/1276/2003#2003.256_Brosh 47,36% A/turkey/Israel/1209/2003#2005.014_Avigdor 23,16% A/turkey/Avigdor/1209/2003#2003.014_Avigdor 100% A/turkey/Qevuzat_Yavne/1242/2003#2003.127_Yavne 87,37% 30,27% 18,04% A/turkey/Hod_Ezyon/699/2002#2002.459_HodEzyon A/turkey/Israel/1013/2002#2002.303_Naharia 100% 97,23% A/turkey/Naharia/1013/2002#2002.303_Naharia A/turkey/Givat_Haim/965/2002#2002.214_GivatHaim 96,58% A/turkey/Yedidia/625/02#2002.111_Yedidia 99,88% A/ostrich/Eshkol/1436/03#2003.834_Eshkol 39,81% A/turkey/Mishmar_Hasharon/619/2002#2002.082_MishmarHasharon 99,36% A/turkey/Kfar_Vitkin/616/2002#2002.082_KfarVitkin 98,62% A/turkey/Kfar_Vitkin/615/2002#2002.082_KfarVitkin 20,18% A/chicken/Kfar_Monash/636/2002#2002.13_KfarMonash 19,23% 35,47% A/chicken/Tel_Adashim/812/2001#2001.971_TelAdashim 23,56% A/turkey/Givat_Haim/810/01#2001.988_GivatHaim 100% A/chicken/Tel_Adashim/811/2001#2001.938_TelAdashim A/chicken/Tel_Adashim/786/2001#2001.938_TelAdashim A/turkey/Neve_Ilan/90710/2000#2000.416_NeveIlan
2.0
2000,0
2005,0
2010,0
2015,0
1,E2
B
1,E1
1,E0
0 2002,5
2005
2007,5
Time
123
2010
Virus Genes b Fig. 2 a Phylogenetic tree of Israeli AIV H9N2 HA gene segments
(Table 1). b Bayesian Skyride plot of the HA gene showing changes in genetic diversity in H5N1 Israeli viruses (2000–2012). A measure of genetic diversity (Net) is given on the y-axis with 95 % HPD values shown in blue. Vertical blue shaded lines highlight peaks of drastic increase in genetic diversity (Color figure online)
The dataset was analysed with the codon-based SRD06 nucleotide substitution model and the Skyline coalescent prior. To reflect the population complexity and dynamics of the AIV H9N2 viruses in Israel, the Bayesian and skyline coalescent tree were used (Fig. 2). Two molecular clocks models—a strict (constant) and an uncorrelated lognormal relaxed clock—were compared by estimating the marginal likelihood implemented in the Tracer program [8]. In addition, an independent estimate of the nucleotide substitution rate was obtained using a rootto-tip regression method available in the Path-O-Gen program (kindly provided by Andrew Rambaut, University of Edinburgh) http://tree.bio.ed.ac.uk/software/pathogen), in which genetic distances are estimated from a maximum likelihood (ML) tree and are plotted against the day of sampling. Analysis of selection pressures Gene- and site-specific selection pressures for the AIV HA protein of the Israeli AIV H9N2 viruses were measured as the ratio of non-synonymous (dN) to synonymous (dS) nucleotide substitutions per site. In all the cases, dN/dS ratios were estimated using the single-likelihood ancestor counting (SLAC) and, fixed-effects likelihood (FEL). The codon-based maximum likelihood (IFEL) and FUBAR [9]. (http://mbe.oxfordjournals.org/content/early/2013/02/18/mol bev.mst030.short) methods were available at the DataMonkey online version of the HY-Phy package http://www. datamonkey.org [10].
Results As AIV H9N2 continuously evolve from a genetic point of view and considering that molecular characteristics are mainly described on a country basis, timely and continental surveillance are essential. A recent molecular portrayal of the AIV H9N2 viruses which had emerged in Israel until 2012 revealed the introduction of thea fifth subgroup. However, only a limited number of AIV H9N2 isolated until 2011 from Eastern countries were analysed [5]. Fusaro et. al. [4] focused on the phylogeography and molecular evolution of AIV H9N2 in the Middle East and in Central Asia countries, but included only a limited number of Israeli H9N2 isolates until 2006. In addition, Monne et al.
[11] further identified and characterised the AIV H9N2 cocirculating with the AIV H5N1 in the Egyptian poultry farms. Figure 1 illustrates the completion of the gap in the global perception regarding the possible precursor viruses of the AIV H9N2 isolated during the last 12 years in Israel. For the first time, the spatiotemporal phylogenetic analysis has revealed the most recent molecular evolution of H9N2 AIVs isolated in Israel through a comparison with a large collection of AIV H9N2 from surrounding countries (Table 1). Basically, the inclusion of all Israeli AIV H9N2 viruses into 5 phylogenetic clades [5] is also confirmed in Fig. 1, but innovatively, the inclusion of AIV H9N2 from other countries has enabled a more comprehensive evaluation regarding their origin. Moreover, Fig. 1 shows the classification into 4 divisions, A–D by their high bootstrap values and long branches between the clades. It is evident that Israeli groups I, II and III differ from groups IV and V, as they belong to classes A and B, respectively [4]. According to the phylogenetic data illustrated in Fig. 1, the Hong Kong AIV H9N2 isolates of 1997 evolved into the viruses detected in Iran in 1998 and were the closest to the virus subclade of the Ist Introduction of the Israeli H9N2 between May 2000 and December 2001. The IInd group of viruses was isolated until April 2003, and the IIIrd group until December 2006. AIV H9N2 belonging to groups I–III cladded together with AIV H9N2 isolated from Jordan and Lebanon. In a separate clade, the AIV H9N2 isolated from the more distant Central Asian countries, such as Pakistan, Kurdistan, Iran and Saudi Arabia, (1999–2009) cladded with the Vth group of Israeli H9 viruses, identified starting from November 2011. The Egyptian H9N2 AIV appeared between 2010 and 2012 [11] and shared a common clade with the Israeli H9N2 group IV viruses, which had been isolated between December 2006 and early 2013. The present spatiotemporal phylogenetic analysis redefines the presence of successive, separate H9N2 virus introductions and provides evidence that each virus group introduced in Israel shared distinct ancestors with viruses which had been circulating in different geographical areas, thus suggesting potential epidemiological links. For example, viruses of groups I, II and III originated in the Far East, sharing common ancestors with Iranian isolates and had co-populated Israel, Jordan and Lebanon. Differently, group IV viruses are linked to the H9N2 strains recently described in Egypt, also circulating in the region, as demonstrated by similar viruses in Jordan and Lebanon. Israeli group V viruses are linked to viruses circulating in Saudi Arabia and in other Persian Gulf countries, confirming the previous findings [4]. Furthermore, we intended to analyse the Israeli H9N2 AIV molecular evolution, evolutionary rates and population dynamics, as represented by the HA gene segment. The Bayesian coalescent approach was applied using 65
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Virus Genes
complete HA gene sequences and their collection date (Table 1; Fig. 2). Figure 2a shows the Maximum Clade Credibility (MCC) tree of the Israeli AIV H9N2 isolates. The five genetic groups of viruses are distinctly separated on different nodes, consistent with the previous phylogenetic tree obtained by the maximum likelihood (ML) algorithm [5]. However, a more robust dissection was obtained now by the Bayesian algorithm, as compared with the previous tree obtained by the ML algorithm [12]. In addition, while genetic groups I and II had previously been associated to two separate introductions [5], the molecular epidemiological evidence from the present investigation indicates that groups I and II likely represent one single introduction that re-emerged subsequent to their first appearance in the country. Based on the phylogenetic tree (Fig. 2a), the virus population dynamics Skyline was calculated (Fig. 2b). The similar range of variability throughout the 12 year Skyline molecular evolution is indicative of its robustness. The analysis of virus population genetic diversity revealed two distinct remarkable increases, the first between the years 2003–2004 and the second at the beginning of 2008. Between 2004 and 2007 no increase in population size was detected. The two peaks in the Skyline genetic variability of the Israeli isolates reflect the sharp increase of the population genetic diversity, and are determined by both the number of isolates tested and by their genetic diversity. Interestingly, the first peak corresponds to the appearance of AIV H9N2 belonging to group III, while the second to the detection of several H9N2 outbreaks caused by group IV viruses. These two peaks corresponded in time also to the introduction of the two vaccines; the first consisting of isolate A/ty/Israel/965/02, which had been used starting from 2003, one year before the genetic variability of the viruses peaked. The second vaccine, containing the virus A/ck/Israel/215/07, was adopted at the end of 2008, the same year when the second dramatic increase in the virus population diversity occurred. The H9N2 vaccine introductions might be considered as important factors to keep in mind [13], but cannot be considered alone as responsible for the increased evolutionary rate of the AIV H9N2 in Israel. Besides, the selection pressures of the Israeli AIV H9N2 were estimated by assessing the overall number of non-synonymous to synonymous substitutions (dN/dS) The same two sites in the HA gene were under a positive selection pressure by 4 computational models with significant statistical values (http://www.datamonkey.org), FUBAR (posterior probability 0.99), FEL, IFEL and SLAC (p value for all 0.1–0.0006). The two amino acids which were found to be prone to changes were 198 and 234, corresponding to positions 190 and 226 by the AIV H3 nomenclature. These
123
two positions have been previously shown to be the place for amino acid substitutions in H9N2 viruses circulating in poultry [8] and responsible for potential antigenic and adaptive changes. Additionally, substitutions E190 and L226 together with H183 and A189 at the receptor binding domain of the HA molecule are essential for the horizontal spread in mammals of reassortant viruses carrying the surface proteins of avian AIV H9N2 in a human H3N2 backbone. Furthermore, position 189, located on the tip of the globular head of HA1 is considered a critical antigenic site and has been implicated in the H9 escape mutants [14]. Similar evolutionary rate for the Israeli HA-H9N2 genes was found by two methods, Beast and Path-O-Gen, thus a double confirmation was obtained. In fact, the mean rate of the nucleotide substitution of all the Israeli isolates collected between 2000 and 2012 obtained by the Beast method was 6.123 E-3, while by the Path-O-Gen method it was of 6.58 E-3 substitutions/site/year (correlation coefficient: 0.98, R squared–0.96). Interestingly, similar values were also obtained for the Egyptian and the Indonesian H5N1 viruses [13]. The time interval from the most recent common ancestor (tMRCA) was 12.3 years, as calculated by both molecular clock models, corresponding to the year 2000, when H9N2 virus was first isolated in Israel.
Discussion The present evaluation of the phylogenetic evolution of the AIV H9N2 HA segment has indeed provided novel information to better understand the molecular epidemiology of the H9N2 virus subtype in Israel and in the region. On the basis of the results of the tMRCA presented herein, the first detection of H9N2 in Israel occurred soon after its introduction in the country. However, the early detection in year 2000 did not prevent the virus to circulate, evolve and reemerge in subsequent years (2001–2003). As for other infectious diseases, the H9N2 infection control, mainly based on vaccination alone, limited the disease and the associated economic losses but probably l impacted only slightly in limiting the virus circulation and preventing further re-introductions. In the present study, the comparison of Israeli H9N2 viruses with the strains circulating at a continental level has allowed to better infer the epidemiology of H9N2 in Israel and in the region. It is interesting to note that both in the case of the recent H9N2 epidemic (caused by group IV) and the H5N1 incursions the strains isolated in Israel are about the same as the viruses circulating in Egypt and in other bordering countries, confirming the transboundary spread of the infections and the need to increase the efficacy of the control measures at a transnational level.
Virus Genes Acknowledgements We wish to thank Drs. A. Panshin, S. Perk, A. Lublin, E. Rosenbluth, Y. Paranoushi, L. Simanov, Mrs. E. Lapin, Mrs. I. Shkoda, Mrs. A. Altouri and Ms. I. Reibshtein, involved in the AIV diagnosis and sequencing at the Division of Avian Diseases at KVI, Israel, for the facilitation of the Gene Bank Published sequences. This study was supported by Grant US-4379-11 from the USAIsrael Agricultural Research and Development Fund (BARD).
References 1. D.J. Alexander, Avian Dis 51(1 Suppl), 161–166 (2007) 2. I. Capua, D.J. Alexander, Influenza Other Respir. Viruses 1, 11–18 (2007) 3. I. Capua, D.J. Alexander, in Avian Influenza and Newcastle Disease, ed. by I. Capua, D.J. Alexander (Springer, Italia, 2009), p. 9 4. A. Fusaro, I. Monne, A. Salviato, V. Valastro, A. Schivo, N.M. Amarin, C. Gonzalez, M.M. Ismail, A.R. Al-Ankari, M.H. AlBlowi, O.A. Khan, A.S. Ali Maken, A.S. Hedayati, J. Garcia, J. Ziay, A. Shoushtari, K.N. Al Qahtani, I. Capua, E.C. Holmes, G. Cattoli, J. Virol 85, 8413–8421 (2011) 5. I. Davidson, I. Shkoda, N. Golander, S. Perk, K. Lapin, Y. Khinich, A. Panshin, Virus Genes 46, 255–263 (2013)
6. N. Golander, A. Panshin, C. Banet-Noach, S. Nagar, S. Pokamunski, M. Pirak, Y. Tendler, I. Davidson, M.C. Garcia, S. Perk, Virus Genes 37, 289–297 (2008) 7. S. Perk, N. Golender, C. Banet-Noach, E. Shihmanter, S. Pokamunsky, M. Pirak, Y. Tendler, M. Lipkind, A. Panshin, Comp. Immunol. Microbiol. Infect. Dis. 32, 221–238 (2009) 8. A.J. Drummond, A. Rambout, BEAST: bayesian evolutionary analysis by sampling trees. BMC Evol. Biol 7, 214 (2007) 9. FUBAR http://mbe.oxfordjournals.org/content/early/2013/02/18/ molbev.mst030.short 10. S.L. Kosakovsky-Pond, D.W.S. Frost, Mol. Biol. Evol 22, 1208–1222 (2005) 11. I. Monne, H.A. Hussein, A. Fusaro, V. Valastro, M.M. Hamoud, R.A. Khalefa, S.N. Dardir, M.I. Radwan, I. Capua, G. Cattoli, Influenza Other Respir. Viruses (2012). doi:10.1111/j.1750-2659. 2012.00399.x 12. B. Wrobel, J. Appl Genet 49, 49–67 (2008) 13. G. Cattoli, A. Fusaro, I. Monne, F. Coven, T. Joannis, H.S. Abd El-Hamid, A.A. Hussein, C. Cornelious, N.M. Amarin, M. Mancin, E.C. Holmes, I. Capua, Vaccine 39, 9368–9375 (2011) 14. E.M. Sorrell, H. Wan, Y. Araya, H. Song, D.R. Perez, Proc. Natl. Acad. Sc. USA. 106, 7565–7570 (2009)
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Molecular evolution of H9N2 avian influenza viruses in Israel Irit Davidson • Alice Fusaro • Alireza Heidari Isabella Monne • Giovanni Cattoli
•
Received: 24 October 2013 / Accepted: 12 January 2014 Ó Springer Science+Business Media New York 2014
Abstract While the previous phylogenetic analyses of AIV H9N2 in Israel had mainly focused on phylogenetics and on describing different virus introductions into the country, for the first time, the H9N2-HA gene evolutionary history has been examined taking into account its origin, evolution and phylodynamics. The present study reveals the Israeli H9N2 molecular evolution rate, the virus molecular clock and skyline plot. The molecular skyline plot showed two major increments in population diversity sizes, the first which had occurred in 2003, the second between the end of 2007 and the first half of 2008. Between 2004 and 2007 the population size had proved to be constant. The two peaks correspond to the appearance of the 3rd and 4th major genetic groups, as well as to the introduction of two H9N2 vaccines. The mean evolution rate was 6.123 E-3 substitutions/site/year, typical of avian influenza viruses. The time interval from the most recent common ancestor was 12.3 years, corresponding to the year 2000, when H9N2 was first isolated in Israel. Keywords Avian influenza virus subgroup H9N2 Molecular evolution HA gene Phylodynamics Molecular clock
I. Davidson (&) Division of Avian Diseases, Kimron Veterinary Institute, P.O.Box 12, 50250 Bet Dagan, Israel e-mail: [email protected] A. Fusaro A. Heidari I. Monne G. Cattoli Research and Development Department, Istituto Zooprofilattico Sperimentale delle Venezie, OIE/FAO and National Reference Laboratory for Avian/Animal Influenza and Newcastle Disease, OIE Collaborating Centre for Diseases at the Human-Animal Interface, Legnaro, Padova, Italy
Introduction Avian influenza viruses (AIV) of the H9N2 subtype are widespread worldwide, being endemic in poultry populations in Asia and the Middle East [1, 2]. Outbreaks of AIV H9N2 in poultry have occurred in the western hemisphere since 1994, including Germany, Italy, Ireland, South Africa and the USA, as reviewed by Capua and Alexander [3]. Fusaro et. al. [4] and Davidson et. al. [5] have, respectively, described the Middle East AIV H9N2 and the Israeli AIV H9N2 isolates, focusing on distinct virus populations. Fusaro et. al. [4] described Middle Eastern and Central Asia isolates, including only limited Israeli H9N2 viruses, while Davidson et. al. [5] focused on Israeli AIV H9N2 isolates including only limited Asian isolates. The present study fulfils this gap by including the most comprehensive AIV H9N2 collection since their emergence back in 1997 in the Middle East and Asia and constitutes the missing ring between the two previous studies by reflecting the evolutionary history, origin and phylodynamics of the Israeli AIV H9N2. In Israel, AIV H9N2 have been isolated and molecularly characterised since the year 2000 [5–7]. All AIV H9N2 Israeli isolates belong to the G1 lineage, possess a cleavage site characteristic of low pathogenic AIV and are classified into five phylogenetic groups [5]. As no intermediate phylogenetic forms have ever been isolated, it was assumed that the source of the new virus introductions in Israel had been external, although no proofs had been provided until now. The first and second AIV H9N2 introductions prevailed from May 2000 to April 2003, the third introduction circulated from April 2003 to December 2006, while after 2006, the fourth genetic group, subdivided into two subgroups, A and B, prevailed in Israel. In 2012, the fifth group emerged, although viruses belonging to the fourth group were still circulating. The isolates A/ty/Israel/965/02
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Virus Genes Table 1 The HA gene sequences employed in the present study, including their name, accession, country and year of isolation: the HA sequences from Israel, Central Asian and Middle East countries used to construct the phylogenetic tree in Fig. 1 Country
Year of isolation
No. of sequences
Israel
2002–2012
123
Jordan
2004–2007
11
Lebanon
2004–2010
3
Egypt
2011–2012
11
Middle East
1999
1
Libia
2006
1
Emirates
2002–2011
2
Qatar
2008
1
Saudi Arabia
2002–2011
22
Dubai
2000–2008
10
Iraq
2005–2008
2
Iran
1998–2010
63
Pakistan
1999–2010
18
Afganistan Kurdistan
2008–2009 2009
7 2
Bangladesh
2007
3
India
2006–2008
3
Hong Kong
1997–2010
18
Japan
1998
Total
1 303
and A/ck/Israel/215/07, belonging to the second and fourth genetic groups, respectively, were selected as prototype vaccine strains. The use of the first vaccine (A/ty/Israel/
965/02) commenced in 2003 and lasted until the end of 2008, when the second vaccine (strain A/ck/Israel/215/07) was introduced. In the previous study, the genetic grouping of the Israeli AIV H9N2 viruses was associated to separate introductions and phylogenetically deducted by analysing the genomic homology extent between viruses [5]. In the present study, the application of different and more advanced computational algorithms made it possible to perform a deeper investigation on the Israeli AIV H9N2 molecular viral evolution. Furthermore, the genetic relations of the various introductions of AIV H9N2 in Israel with the AIV H9N2 isolated in the surrounding countries have been investigated for the first time. The new analysis reveals the time and location of the most recent ancestor, highlights the nucleotide substitution rates and depicts the population dynamics and the extent of its molecular change since the AIV H9N2 first detection.
Materials and methods Viruses included in the study Only AIV H9N2 HA genes publicly available in the Gene Bank were included. The complete Israeli HA sequences included 97 viruses (Table 2). As well, the sequence dataset (n = 303; Table 1) included those from surrounding countries [4] and the newly depicted GenBank database viruses after 2010.
Table 2 The HA gene sequences employed in the present study, including their name, accession, country and year of isolation: Israeli complete HA sequences with specific specification of the collection date Year of isolation
No. of HA sequences
Accession no.
2000
2
EF492221, AY738451
2001 2002
4 9
AY738454, DQ104466-DQ104468 EF462494, AY738452, EF462495, EF492237, DQ104459-DQ104461, DQ104463, EF462496
2003
22
DQ108908-10, DQ108921, DQ104484, DQ104480-2, DQ104474, DQ108906, DQ108913-14, DQ108911, AY738455-6, EF462497, EF46250-3, EF462498, EF492240
2004
11
EF462504-7, DQ108919-20, AY738453, DQ108916-7, EF501983, EF492243
2005
11
EF492222, EF492225-6, EF492228, EF492232, EF492234-5, EF492236, EF492238, EF492230, EF462493
2006
4
FJ464729-30, FJ464714, EF492224,
2007
9
GQ120557, FJ464724-5, FJ464715-6, GQ120553, FJ464720, FJ464721-2
2008 2009
5 5
GQ120558, JQ347954, JQ347957, JQ347959, GQ120554 JQ347952-3, JQ347945, JQ254940, JQ347950,
2010
5
JQ254942, JQ254945, JQ254948, JQ254952, JQ254954
2011
4
JQ973657, JQ973658, JQ973659, JQ973660
2012
6
JQ973651-2, JQ973653-6
TOTAL
97
123
Virus Genes 89 A/chicken/Egypt/11vir4453-276/2010 A/chicken/Egypt/11vir4453-280/2011 A/chicken/Egypt/11vir4453-275/2011 A/chicken/Egypt/114915V/2011 A/chicken/Egypt/11vir4453-274/2011 A/chicken/Egypt/A2-D/2011 A/chicken/Egypt/11vir4453-273/2011 74,7% 91 A/chicken/Egypt/11vir4453-272/2011 98 A/chicken/Egypt/115617V/2011 A/chicken/El_Fayoum/CAI25/2011 A/chicken/Egypt/S4454B/2011 93,9% A/chicken/Egypt/S4454E/2011 97% A/chicken/Egypt/S4456B/2011 73,3% A/chicken/Egypt/12186F-9/2012 88,8% A/chicken/Egypt/114940v/2011 0% A/chicken/Egypt/115636V/2011 95,1% 77,4% A/quail/Egypt/113413v/2011 A/chicken/Egypt/11vir4453-132/VRLCU/2011
83 88 95
99
92,2% 75,8%
Egypt 2010-2012
B
turkey/Israel/311/2009_Kfar_Ezion chicken/Israel/663/2009_Mishmar_Hanegev 99,7% 93,1% chicken/Israel/794/2009_Bizaron turkey/Israel/117/2009_Sde_Yoav 87,1% turkey/Israel/1257/2008_Rosh_Zurim turkey/Israel/1608/2006_Nahalal
76,9%
chicken/Israel/50/2012_Klahim 96,4% chicken/Israel/58/2012_Talme_Bilu 85,4% chicken/Israel/3/2012_Kalia 99,3% chicken/Israel/1067/2010_Haifa 76% turkey/Israel/746/2009_Ein_Yron 93,3%chicken/Israel/724/2009_Mezer 0% 100% chicken/Israel/1302/2010_Shuval chicken/Israel/742/2009_Sde_Eliahu
0%
100%
0% 0% chicken/Israel/1525/2006_Gshor 0% chicken/Israel/1638/2006_Givat_Hen chicken/Israel/386/2007_Dovev 99,2% chicken/Israel/182/2008_Gilat 99,9% chicken/Israel/54/2008_Gilat 78% 0% chicken/Israel/1548/2006_Metzer chicken/Israel/215/2007_Midrach_Oz 0% chicken/Israel/728/2007_Afek 0% chicken/Israel/402/2007_Goren 83,3% chicken/Israel/449/2007_Goren chicken/Israel/760/2008_Goren 92,1% chicken/Israel/884/2008_Avdon 0% 0% chicken/Israel/330/2008_Gilat turkey/Israel/900/2007_Zarchia 0% chicken/Israel/869/2007_Ein_Gev 84,5% chicken/Israel/524/2008_Azrikam 0% chicken/Israel/702/2008_Kabri avian/Israel/314/2008_Bat_Yam 0% 0% chicken/Israel/310/2008_Gilat 92,8% chicken/Israel/292/2008_Sde_David 0% 75,4% turkey/Israel/689/2008_Ein_Hashlosha chicken/Israel/694/2008_Even_Menahem chicken/Israel/1293/2010_Zipori 77,5% 0% chicken/Israel/1167/2010_Mehola 90,2% chicken/Israel/1184/2010_Elipelet 99,9% chicken/Israel/1164/2011_Pardes_Hanna 95,7% A/quail/Lebanon/272/2010 100% A/quail/Lebanon/273/2010 89% A/chicken/Israel/184/2009 87,1% 95,3% chicken/Israel/184/2009_Gilat chicken/Israel/1256/2008_Afula 78,8% A/chicken/Jordan/VIR436-2/2010 86,9% 90,5%chicken/Israel/712/2009_Nahalal turkey/Israel/775/2009_Mei_Ami 94% 99,8% 86% A/chicken/Jordan/VIR436-1/2010 chicken/Israel/883/2008_Elipelet chicken/Israel/1195/2008_Ramat_Zvi 92% chicken/Israel/32/2009_Tel_Kazir 74,5% chicken/Israel/43/2009_Ziveon 66% chicken/Israel/1205/2008_Eilon 95,3%chicken/Israel/1018/2008_Goren turkey/Israel/1223/2008_Ein_Zurim
Israel Introduction IV
Lebanon 2010 Jordan 2010
94%
74,5%
83,8% 99,9% 94,4%
97,4% A/ck/Qatar/4576v08-4/2008 A/ck/Dubai/3771v09-2/2008 88,8% A/ck/Dubai/3771v09-3/2008 A/avian/Libya/RV35D/2006
chicken/Israel/51/2012_Nahalal 0% chicken/Israel/1115/2011_Avital 83% chicken/Israel/1089/2011_Bezet 0% 78,8%chicken/Israel/46/2012_Nahalal chicken/Israel/11/2012_Um_El_Phahem 100% chicken/Israel/1163/2011_Avital A/chicken/Saudi_Arabia/H29TR/2011 A/chicken/United_Arab_Emirates/F1P7/2011 A/chicken/Saudi_Arabia/D-36363/2010 A/chicken/Saudi_Arabia/C-36362/2010
Israel Introduction V
93,8%
100%
99,4% 0%
96,7%
83,9%
88,4%
Saudi Arabia UAE 2005-2011 Libia Qatar
A/ck/SaudiArabia/3489v08-51as366/2008 47,3% A/ck/SaudiArabia/3489v08-48as997/2007 100% A/ck/SaudiArabia/3489v08-50as118/2008 A/chicken/Saudi_Arabia/E-36364/2006 99,4% A/chicken/Saudi_Arabia/E-364/2006 73,5% A/chicken/Saudi_Arabia/F-36365/2006
97,6%
74,8% 100% A/chicken/Saudi_Arabia/F-6365/2006 A/chicken/Saudi_Arabia/2BL09/2006 84,7% A/ck/SaudiArabia/3489v08-38as92/05 82,5% A/chicken/Saudi_Arabia/582/2005 A/avian/Saudi/Arabia/910136/2006 98,6% 0% A/ck/SaudiArabia/3489v08-47as215/2007 91,1% A/avian/Saudi/Arabia/910134/2006 78% A/ck/SaudiArabia/3489v08-33as638/2005 99,5% A/falcon/SaudiArabia/3489v08-40as977/2006 A/ck/SaudiArabia/3489v08-44as253/2006 100% A/avian/Saudi/Arabia/910135/2006
99,9%A/chicken/Iran/10VIR854-3/2009 A/chicken/Iran/EBGV-88/2010 96,3% A/ck/Afghanistan/329v09-7AFG-Heart6/2009 99,4% A/ck/Afghanistan/329v09-8AFG-Heart7/2009 99,9% A/chicken/Sihala/NARC-12103/2008 0% A/chicken/Pakistan/UDL-03/2007 97,5% A/chicken/Pakistan/UDL-01/2008 A/chicken/Pakistan/UDL-04/2006 90,3% A/chicken/Pakistan/UDL-01/2006 A/chicken/Sawabi/NARC-2434/2006 92,8% 100% A/chicken/Pakistan/NARC-2434/2006 A/chicken/Pakistan/UDL-02/2006 100% A/chicken/Pakistan/UDL-04/2007 80,9% A/chicken/Pakistan/UDL-02/2005 100% 100%60,8% A/chicken/Pakistan/UDL-03/2005 A/chicken/Pakistan/UDL-01/2007 A/chicken/Pakistan/UDL-01/2005 A/chicken/Pakistan/47/2003 A/chicken/Iran/THLBM867/2007 95,3% A/chicken/Iran/THLBM868/2007 96,8% A/chicken/Iran/THLBM861/2007 95,9% A/Chicken/Iran/TH386/2007 A/chicken/Iran/THLBM863/2007 83,5% 90,7% A/chicken/Iran/THLBM866/2007 18,8% A/chicken/Iran/THLBM865/2007 75,5% 76,7% A/Chicken/Iran/TH286/2007 90,9% A/Chicken/Iran/TH186/2007 0% 99,9% A/chicken/Iran/10VIR854-5/2008 98,6% A/Chicken/Iran/TH85/2007 A/chicken/Iran/TH486/masoumi/2007 96,9% A/chicken/Iran/RZ28/2008 84,5% 0% A/chicken/Iran/RZ42/2008 A/chicken/Iran/RZ37/2008 100% 72,2% A/chicken/Iran/RZ36/2008 62,6% A/chicken/Iran/RZ53/2008 89,5% A/chicken/Iran/133/2004 A/chicken/Iran/B102/2005 88,7% A/chicken/Iran/68/2006 A/chicken/Iran/SH2/2007 76,8% A/chicken/Iran/20/2006 76,4% 76,9% A/chicken/Iran/B11A/2005 87,2%A/chicken/Iran/B99/2005 100% 74,4% A/chicken/Iran/L248/2003 75,4% A/chicken/Iran/B326/2005 A/chicken/Iran/B76/2004 A/ck/Kurdistan/4693v09-88/2009 66,9% 99,5% A/ck/Kurdistan/4693v09-9/2009 99,6% A/chicken/Iran/10VIR854-4/2009 20,2% 99,9% A/chicken/Iraq/EKI_14/2008 0% A/chicken/Iran/B308B/2004 A/chicken/Iran/320/2003 A/chicken/G-Karachi/2003 A/ck/Afghanistan/329v09-3AFG-Khost2/2008 0% A/ck/Afghanistan/329v09-5AFG-Khost4/2008 0% A/ck/Afghanistan/329v09-6AFG-Khost9/2008 0% A/ck/Afghanistan/329v09-2AFG-Khost1/2008 99,7% A/ck/Afghanistan/329v09-4AFG-Khost3/2008 100% A/chicken/Pakistan/UDL-02/2008 100% A/chicken/Pakistan/UDL-03/2008 A/chicken/Bangladesh/VP01/2007 100% A/chicken/Bangladesh/627/2007 50,9% A/chicken/Uchal/8286/2006 67,7% 99,2% A/chicken/India/3/2003 A/chicken/Bangladesh/FDIL/112/2007 81,8%
97,5%
98,5%
99,4%
96,2%
72,5% 97,9% 79,2% 31,3%
Iran Afghanistan Pakistan
A/chicken/Iran/THLBM862/2007
Iran 2003-2008 Iraq
G1 India Bangladesh Afghanistan Pakistan
A/chicken/Iran/SS2/2008 A/chicken/Iran/THLBM864/2007 54,4%
96,5% 94,3%
100% A/Chicken/Pakistan/5/1999 99,2% A/Chicken/Pakistan/2/1999 A/Chicken/Pakistan/4/1999
90,3%
A/chicken/Dubai/383/2002 78,6% A/chicken/Dubai/339/2001 99,8% A/chicken/Dubai/338/2001 A/chicken/Emirates/R66/2002 A/chicken/Dubai/463/2003
0%
94,5%
99,7%
0% A/HongKong/1074/1997 89,2% 0% A/HongKong/1074/1999 95,3% A/HongKong/1073/1999 A/guineafowl/HongKong/WF10/1999 A/Quail/HongKong/G1/1997
99,7% 99,9%
A/Chicken/HongKong/G9/97 42,8% A/Chicken/HongKong/G23/97
C
Pakistan 1999
A Israel Introduction III
58,5% 82,5% 0% 99,2% 0% chicken/Maale_HaHamisha/90658/2000_Maale_Hahamisha chicken/Israel/90658/2000_Maale_Hahamisha A/chicken/Israel/1990658/2000 A/chicken/Middle_East/ED-1/1999 A/chicken/Saudi/Arabia/EPD-22-01/2002 A/chicken/Iran/10VIR854-1/2002 76,5% 0% A/garganey/Iran/K8/2003 A/chicken/Iran/B314/2003 85,1% A/chicken/Iran/119/2005 69,7% A/chicken/Iran/284/2002 100% A/chicken/Iran/B322/2004 0% A/Chicken/Iran/TH78/1999 74,8% A/Chicken/Iran/TH79/2000 49,7% A/chicken/Iran/705/1999 77,9% A/chicken/Iran/B263/2003 100% 76,2% A/chicken/Iran/152/2004 100% A/chicken/Iran/B308A/2004 99,3% 74% 38%A/chicken/Iran/772/1999 A/chicken/Iran/233/2001 46,8% A/chicken/Iran/565/2000 86,3% A/Chicken/Iran/11T/1999 A/chicken/Iran/698/1998 83,3% A/chicken/Iran/SH1/2007 0% A/chicken/Iran/450/2001 96,6% A/chicken/Iran/86/2005 100% 90,2% A/chicken/Iran/L252/2003 A/Chicken/Iran/TH77/1998 49,5% 92,1% A/chicken/Iran/661/1998 86,8% A/chicken/Iran/SS1/1998 82,4% A/chicken/Saudi/Arabia/CP7/1998 A/parakeet/Narita/1992A/1998 96,5%
2003-2008 A/chicken/Pakistan/NCVI-09/2010
UAE 2000-2002
A/Iraq/579v09-8/2005 79,3% A/avian/Jordan/7-Y1/2004 77,4% 0% turkey/Nahalel/1547/2004_Nahalal 0% chicken/Tel_Adashim/1469/2003_Tel_Adashim chicken/Alonim/1552/2003_Alonim 0% 83,5% chicken/Tel_Adashim/1506/03_Tel_Adashim turkey/Beit_HaLevi/1566/04_Bet_Halevi 0% A/ck/Jordan/1436v04-1451/2004 84,3% 90,5% chicken/Neve_Ilan/1504/03_Neve_Ilan chicken/Pardes_Hana_Carcur/1475/2003_Pardes_Hanna 0% chicken/Israel/1475/2003_Pardes_Hanna chicken/Israel/1966/2004_Kalanit 0% chicken/Beit_Aran/29/2005_Beit_Aran 0% 0% chicken/Israel/29/2005_Kalanit 95,8% chicken/Klanit/1966/2004_Kalanit 0% turkey/Israel/747/2005_Neot_Golan 100% 97,2% turkey/Israel/884/2005_Ramot 0% A/chicken/Lebanon/1080/2004 chicken/Gan_Shomron/1465/03_Gan_Shomron 83,7% chicken/Israel/909/2005_Hadera 0% chicken/Israel/853/2005_Maanit 97,8% chicken/Israel/793/2005_Maanit 73,8% A/ck/Jordan/579v09-4/2007 99,7% 100% chicken/Israel/114/2007_Gevim 0% chicken/Israel/375/2007_Shazor 0% chicken/Israel/1953/2004_Alonim 0% chicken/Alonim/1953/2004_Alonim 93,9% 0% turkey/Israel/425/2005_Mishmar_Hasharon 91,2% chicken/Amioz/1527/03_Amioz chicken/Iarah/1376/03_Iarah chicken/Tel_Adashim/1321/2003_Tel_Adashim avian/Israel/584/2005_Sahnin 99,7% chicken/Israel/178/2006_Dalton 0% chicken/Israel/282/2005_Goren 43,7% A/chicken/Israel/421201/2004 0% 98% turkey/Tzur_Moshe/1565/2004_Tzur_Moshe chicken/Israel/554/2005_Elipelet 99,4% turkey/Bazat/89/2005_Bezet 0% 0% A/ck/Jordan/579v09-1/2007 93,7% turkey/Beit_HaLevi/1562/04_Beit_Halevi 100% turkey/Gyvat_Haim_Ehud/1544/03_Gyvat_Haim 81,1% chicken/Tzrofa/1568/2004_Tzrofa 0% A/ck/Jordan/1436v04-997/2004 0% A/ck/Jordan/1436v04-643/2004 0% 91,6% A/duck/Jordan/1436v04-890/2004 A/ck/Jordan/579v09-2/2007 0% 90,4% A/ck/Jordan/579v09-3/2007 86,1% turkey/Ein_Tzurim/1738/2004_Ein_Tzurim chicken/Ysodot/1362/2003_Yesodot 0% 0% chicken/Beit_Alfa/1282/2003_Beit_Alpha 0% turkey/Beit_Herut/1267/2003_Bet_Herut 0% chicken/Uzziyyahu/1651/2004_Uziyahu 0% chicken/Netohah/1373/03__Neohah 0% chicken/Talmei_Elazar/1304/03_Talmei_Elazar chicken/Telmond/1308/03_Tel_Mond turkey/Qevuzat_Yavne/1242/2003_Yavne 0% turkey/Avigdor/1209/2003_Avigdor 0% turkey/Israel/1209/2003_Avigdor 99,4% turkey/Brosh/1276/2003_Brosh 74,3% turkey/Israel/1013/2002_Naharia 0% 85,8% turkey/Hod_Ezyon/699/2002_Hod_Ezyon 0% turkey/Givat_Haim/965/2002_Givat_Haim turkey/Mishmar_Hasharon/619/2002_Mishmar_Hasharon 39,9% turkey/Yedidia/625/02_Yedidia 91,6% ostrich/Eshkol/1436/03_Eshkol 0% turkey/Kfar_Vitkin/616/2002_Kfar_Vitkin 84,6% turkey/Kfar_Vitkin/615/2002_Kfar_Vitkin 91,2% 0% chicken/Kfar_Monash/636/2002_Kfar_Monash turkey/Givat_Haim/810/01_Givat_Haim 0%0% chicken/Tel_Adashim/812/2001_Tel_Adashim 99,9% chicken/Tel_Adashim/811/2001_Tel_Adashim chicken/Tel_Adashim/786/2001_Tel_Adashim turkey/Neve_Ilan/90710/2000_Neve_Ilan
86,1%
99,7%
91,8%
A/quail/Dubai/301/2000 88,6% 90,3%A/quail/Dubai/302/2000 A/quail/Dubai/303/2000
2005-2010
Israel Introduction I+II A/quail/SaudiArabia/3489v08-46as104/2006
Iran 1998-2005 Saudi Arabia
A/HongKong/33982/2009 100% A/HongKong/35820/2009
D
Hong Kong 1997-2009 A/mallard/Iran/T366/2007 0% A/mallard/Iran/C370/2007 0% A/mallard/Iran/T370/2007 100% A/mallard/Iran/C364/2007
A/Duck/HongKong/Y280/97
0.04
Fig. 1 Phylogenetic tree created by the ML algorithm by PhyML of GenBank AIV H9N2 HA complete sequences—Israel until 2012 and Eastern countries, including isolates included Blast similar and
isolates of Fusaro et al. [4]. Colours and classification A–D were by Fusaro et al. [4]. Blue—Israeli H9N2 isolates, Black—global H9N2 isolates (Color figure online)
Phylogenetic analysis
Substitution rates and mean time to the most recent common ancestor (tMRCA)
The maximum likelihood (ML) tree (Fig. 1) was estimated by the PhyML software using the general time-reversible (GTR) model of nucleotide substitution with gamma-distributed rate variation among sites, and a heuristic SPR branch swapping search.
Rates of nucleotide substitution per site per year and the tMRCA of the sampled data were estimated as before [4], using the BEAST program version 1.7 [8], which employs a Bayesian Markov chain Monte Carlo (MCMC) approach.
123
Virus Genes
A
A/chicken/Israel/330/2008#2008.163_Gilat 14,88% A/turkey/Israel/900/2007#2007.874_Zarchia 7,91% A/chicken/Israel/869/2007#2007.847_EinGev 90,62% A/chicken/Israel/760/2008#2008.34_Goren 99,92% A/chicken/Israel/884/2008#2008.453_Avdon 24,38% A/chicken/Israel/524/2008#2008.192_Azrikam 10,53% A/chicken/Israel/292/2008#2008.068_SdeDavid 27,42% A/avian/Israel/314/2008#2008.086_BatYam 98,92% 90,92%A/chicken/Israel/310/2008#2008.074_Gilat A/turkey/Israel/689/2008#2008.292_EinHashlosha 97,82%
A/chicken/Israel/1167/2010#2010.893_Mehola 33,01% 96,7% A/chicken/Israel/1293/2010#2010.958_Zipori A/chicken/Israel/1184/2010#2010.901_Elipelet 100% A/chicken/Israel/1164/2011#2011.96_PardesHanna A/chicken/Israel/694/2008#2008.3_EvenMenahem A/chicken/Israel/702/2008#2008.308_Kabri A/turkey/Israel/775/2009#2009.927_MeiAmi 100% A/chicken/Israel/712/2009#2009.877_Nahalal 93,06% A/chicken/Israel/1256/2008#2008.96_Afula 100% 99,99% 98,39% A/chicken/Israel/184/2009#2009.125_Gilat A/chicken/Israel/883/2008#2008.453_Elipelet A/chicken/Israel/32/2009#2009.025_TelKazir 100% A/chicken/Israel/1195/2008#2008.901_RamatZvi 79% 99,96% A/chicken/Israel/43/2009#2009.033_Ziveon 93,96% A/chicken/Israel/1205/2008#2008.91_Eilon 100% A/chicken/Israel/1018/2008#2008.774_Goren A/turkey/Israel/1223/2008#2008.927_EinZurim A/chicken/Israel/1302/2010#2010.958_Shuval 12,47% A/chicken/Israel/742/2009#2009.896_SdeEliahu 9,01% A/chicken/Israel/724/2009#2009.89_Mezer 100% A/turkey/Israel/746/2009#2009.904_EinYron 100% A/chicken/Israel/58/2012#2012.03_TalmeBilu 100%A/chicken/Israel/50/2012#2012.027_Klahim 99,97% A/chicken/Israel/3/2012#2012.003_Kalia 100% A/chicken/Israel/1067/2010#2010.821_Haifa 83,72% A/chicken/Israel/794/2009#2009.941_Bizaron 100% A/chicken/Israel/663/2009#2009.838_MishmarHanegev 88,77% A/turkey/Israel/311/2009#2009.127_KfarEzion 31,47% 100% A/turkey/Israel/1257/2008#2008.944_RoshZurim 99,67% A/turkey/Israel/117/2009#2009.079_SdeYoav 6,72%A/chicken/Israel/386/2007#2007.186_Dovev A/chicken/Israel/449/2007#2007.262_Goren 29,15% A/chicken/Israel/728/2007#2007.573_Afek 2,49% 60,09% A/chicken/Israel/402/2007#2007.205_Goren A/chicken/Israel/1525/2006#2006.947_Gshor 9,12% 7,58% A/chicken/Israel/215/2007#2007.086_MidrachOz A/chicken/Israel/182/2008#2008.027_Gilat 100% A/chicken/Israel/54/2008#2008.003_Gilat 100%49,02% A/chicken/Israel/1548/2006#2006.955_Metzer 3,82% A/turkey/Israel/1608/2006#2006.977_Nahalal A/chicken/Israel/1638/2006#2006.99_GivatHen A/chicken/Israel/1089/2011#2011.941_Bezet 32,8% A/chicken/Israel/51/2012#2012.027_Nahalal 96,59% A/chicken/Israel/1115/2011#2011.949_Avital 100% A/chicken/Israel/46/2012#2012.016_Nahalal 18,68% A/chicken/Israel/11/2012#2012.008_UmElPhahem 38,04% A/chicken/Israel/1163/2011#2011.971_Avital 28,75% 99,76%22,83%
99,86%
100%
100%
A/chicken/Alonim/1552/2003#2004.004_Alonim 40,18% A/chicken/Tel_Adashim/1506/03#2003.93_TelAdashim 24,33% A/turkey/Beit_HaLevi/1566/04#2004.038_BetHalevi 95,11% 97,26% A/turkey/Nahalel/1547/2004#2004.033_Nahalal 65,28%A/chicken/Tel_Adashim/1469/2003#2003.918_TelAdashim A/chicken/Israel/1475/2003#2003.941_PardesHanna 40,54% A/chicken/Pardes_Hana_Carcur/1475/2003#2003.941_PardesHanna A/chicken/Israel/793/2005#2005.91_Maanit 5,37% 32,97% A/chicken/Israel/909/2005#2005.996_Hadera 100% A/chicken/Israel/853/2005#2005.936_Maanit 82,34% A/chicken/Israel/114/2007#2007.03_Gevim 100% A/chicken/Israel/375/2007#2007.184_Shazor 1,43% A/turkey/Israel/425/2005#2005.596_MishmarHasharon 29,29% 99,6% 100%A/chicken/Alonim/1953/2004#2004.879_Alonim A/chicken/Israel/1953/2004#2004.879_Alonim 99,96% 6,57% A/chicken/Amioz/1527/03#2003.985_Amioz A/chicken/Gan_Shomron/1465/03#2003.922_GanShomron 15,78% A/chicken/Israel/1966/2004#2004.936_Kalanit 19,82% A/chicken/Beit_Aran/29/2005#2005.005_BeitAran 100% A/chicken/Klanit/1966/2004#2004.936_Kalanit 17,9% A/chicken/Israel/29/2005#2005.011_Kalanit 100% 2,29% A/turkey/Israel/884/2005#2005.985_Ramot 11,45% 100% A/turkey/Israel/747/2005#2005.912_NeotGolan 1,52% A/chicken/Uzziyyahu/1651/2004#2004.175_Uziyahu A/chicken/Talmei_Elazar/1304/03#2003.348_TalmeiElazar A/chicken/Iarah/1376/03#2003.54_Iarah 13,28% A/chicken/Telmond/1308/03#2003.367_TelMond 100%A/avian/Israel/584/2005#2005.807_Sahnin 48,85% A/chicken/Israel/178/2006#2006.085_Dalton 72,84% A/turkey/Bazat/89/2005#2005.063_Bezet 1,13% 100% 94,58% A/chicken/Israel/554/2005#2005.804_Elipelet 99% A/chicken/Israel/282/2005#2005.378_Goren A/turkey/Tzur_Moshe/1565/2004#2004.036_TzurMoshe 79,86% A/turkey/Gyvat_Haim_Ehud/1544/03#2003.996_GyvatHaim 47,19% A/turkey/Beit_HaLevi/1562/04#2004.996_BeitHalevi 59,09% 3,2% A/chicken/Tzrofa/1568/2004#2004.025_Tzrofa 32,55% 40,02% A/chicken/Ysodot/1362/2003#2003.529_Yesodot 28,84% A/turkey/Ein_Tzurim/1738/2004#2004.337_EinTzurim 29,22% A/chicken/Neve_Ilan/1504/03#2003.979_NeveIlan 100% 18,98% A/chicken/Beit_Alfa/1282/2003#2003.286_BeitAlpha A/chicken/Tel_Adashim/1321/2003#2003.405_TelAdashim A/chicken/Netohah/1373/03_#2003.289_Neohah 7,19% A/turkey/Beit_Herut/1267/2003#2003.267_BetHerut A/chicken/Israel/90658/2000#2000.416_MaaleHahamisha A/chicken/Maale_HaHamisha/90658/2000#2000.397_MaaleHahamisha A/turkey/Brosh/1276/2003#2003.256_Brosh 47,36% A/turkey/Israel/1209/2003#2005.014_Avigdor 23,16% A/turkey/Avigdor/1209/2003#2003.014_Avigdor 100% A/turkey/Qevuzat_Yavne/1242/2003#2003.127_Yavne 87,37% 30,27% 18,04% A/turkey/Hod_Ezyon/699/2002#2002.459_HodEzyon A/turkey/Israel/1013/2002#2002.303_Naharia 100% 97,23% A/turkey/Naharia/1013/2002#2002.303_Naharia A/turkey/Givat_Haim/965/2002#2002.214_GivatHaim 96,58% A/turkey/Yedidia/625/02#2002.111_Yedidia 99,88% A/ostrich/Eshkol/1436/03#2003.834_Eshkol 39,81% A/turkey/Mishmar_Hasharon/619/2002#2002.082_MishmarHasharon 99,36% A/turkey/Kfar_Vitkin/616/2002#2002.082_KfarVitkin 98,62% A/turkey/Kfar_Vitkin/615/2002#2002.082_KfarVitkin 20,18% A/chicken/Kfar_Monash/636/2002#2002.13_KfarMonash 19,23% 35,47% A/chicken/Tel_Adashim/812/2001#2001.971_TelAdashim 23,56% A/turkey/Givat_Haim/810/01#2001.988_GivatHaim 100% A/chicken/Tel_Adashim/811/2001#2001.938_TelAdashim A/chicken/Tel_Adashim/786/2001#2001.938_TelAdashim A/turkey/Neve_Ilan/90710/2000#2000.416_NeveIlan
2.0
2000,0
2005,0
2010,0
2015,0
1,E2
B
1,E1
1,E0
0 2002,5
2005
2007,5
Time
123
2010
Virus Genes b Fig. 2 a Phylogenetic tree of Israeli AIV H9N2 HA gene segments
(Table 1). b Bayesian Skyride plot of the HA gene showing changes in genetic diversity in H5N1 Israeli viruses (2000–2012). A measure of genetic diversity (Net) is given on the y-axis with 95 % HPD values shown in blue. Vertical blue shaded lines highlight peaks of drastic increase in genetic diversity (Color figure online)
The dataset was analysed with the codon-based SRD06 nucleotide substitution model and the Skyline coalescent prior. To reflect the population complexity and dynamics of the AIV H9N2 viruses in Israel, the Bayesian and skyline coalescent tree were used (Fig. 2). Two molecular clocks models—a strict (constant) and an uncorrelated lognormal relaxed clock—were compared by estimating the marginal likelihood implemented in the Tracer program [8]. In addition, an independent estimate of the nucleotide substitution rate was obtained using a rootto-tip regression method available in the Path-O-Gen program (kindly provided by Andrew Rambaut, University of Edinburgh) http://tree.bio.ed.ac.uk/software/pathogen), in which genetic distances are estimated from a maximum likelihood (ML) tree and are plotted against the day of sampling. Analysis of selection pressures Gene- and site-specific selection pressures for the AIV HA protein of the Israeli AIV H9N2 viruses were measured as the ratio of non-synonymous (dN) to synonymous (dS) nucleotide substitutions per site. In all the cases, dN/dS ratios were estimated using the single-likelihood ancestor counting (SLAC) and, fixed-effects likelihood (FEL). The codon-based maximum likelihood (IFEL) and FUBAR [9]. (http://mbe.oxfordjournals.org/content/early/2013/02/18/mol bev.mst030.short) methods were available at the DataMonkey online version of the HY-Phy package http://www. datamonkey.org [10].
Results As AIV H9N2 continuously evolve from a genetic point of view and considering that molecular characteristics are mainly described on a country basis, timely and continental surveillance are essential. A recent molecular portrayal of the AIV H9N2 viruses which had emerged in Israel until 2012 revealed the introduction of thea fifth subgroup. However, only a limited number of AIV H9N2 isolated until 2011 from Eastern countries were analysed [5]. Fusaro et. al. [4] focused on the phylogeography and molecular evolution of AIV H9N2 in the Middle East and in Central Asia countries, but included only a limited number of Israeli H9N2 isolates until 2006. In addition, Monne et al.
[11] further identified and characterised the AIV H9N2 cocirculating with the AIV H5N1 in the Egyptian poultry farms. Figure 1 illustrates the completion of the gap in the global perception regarding the possible precursor viruses of the AIV H9N2 isolated during the last 12 years in Israel. For the first time, the spatiotemporal phylogenetic analysis has revealed the most recent molecular evolution of H9N2 AIVs isolated in Israel through a comparison with a large collection of AIV H9N2 from surrounding countries (Table 1). Basically, the inclusion of all Israeli AIV H9N2 viruses into 5 phylogenetic clades [5] is also confirmed in Fig. 1, but innovatively, the inclusion of AIV H9N2 from other countries has enabled a more comprehensive evaluation regarding their origin. Moreover, Fig. 1 shows the classification into 4 divisions, A–D by their high bootstrap values and long branches between the clades. It is evident that Israeli groups I, II and III differ from groups IV and V, as they belong to classes A and B, respectively [4]. According to the phylogenetic data illustrated in Fig. 1, the Hong Kong AIV H9N2 isolates of 1997 evolved into the viruses detected in Iran in 1998 and were the closest to the virus subclade of the Ist Introduction of the Israeli H9N2 between May 2000 and December 2001. The IInd group of viruses was isolated until April 2003, and the IIIrd group until December 2006. AIV H9N2 belonging to groups I–III cladded together with AIV H9N2 isolated from Jordan and Lebanon. In a separate clade, the AIV H9N2 isolated from the more distant Central Asian countries, such as Pakistan, Kurdistan, Iran and Saudi Arabia, (1999–2009) cladded with the Vth group of Israeli H9 viruses, identified starting from November 2011. The Egyptian H9N2 AIV appeared between 2010 and 2012 [11] and shared a common clade with the Israeli H9N2 group IV viruses, which had been isolated between December 2006 and early 2013. The present spatiotemporal phylogenetic analysis redefines the presence of successive, separate H9N2 virus introductions and provides evidence that each virus group introduced in Israel shared distinct ancestors with viruses which had been circulating in different geographical areas, thus suggesting potential epidemiological links. For example, viruses of groups I, II and III originated in the Far East, sharing common ancestors with Iranian isolates and had co-populated Israel, Jordan and Lebanon. Differently, group IV viruses are linked to the H9N2 strains recently described in Egypt, also circulating in the region, as demonstrated by similar viruses in Jordan and Lebanon. Israeli group V viruses are linked to viruses circulating in Saudi Arabia and in other Persian Gulf countries, confirming the previous findings [4]. Furthermore, we intended to analyse the Israeli H9N2 AIV molecular evolution, evolutionary rates and population dynamics, as represented by the HA gene segment. The Bayesian coalescent approach was applied using 65
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complete HA gene sequences and their collection date (Table 1; Fig. 2). Figure 2a shows the Maximum Clade Credibility (MCC) tree of the Israeli AIV H9N2 isolates. The five genetic groups of viruses are distinctly separated on different nodes, consistent with the previous phylogenetic tree obtained by the maximum likelihood (ML) algorithm [5]. However, a more robust dissection was obtained now by the Bayesian algorithm, as compared with the previous tree obtained by the ML algorithm [12]. In addition, while genetic groups I and II had previously been associated to two separate introductions [5], the molecular epidemiological evidence from the present investigation indicates that groups I and II likely represent one single introduction that re-emerged subsequent to their first appearance in the country. Based on the phylogenetic tree (Fig. 2a), the virus population dynamics Skyline was calculated (Fig. 2b). The similar range of variability throughout the 12 year Skyline molecular evolution is indicative of its robustness. The analysis of virus population genetic diversity revealed two distinct remarkable increases, the first between the years 2003–2004 and the second at the beginning of 2008. Between 2004 and 2007 no increase in population size was detected. The two peaks in the Skyline genetic variability of the Israeli isolates reflect the sharp increase of the population genetic diversity, and are determined by both the number of isolates tested and by their genetic diversity. Interestingly, the first peak corresponds to the appearance of AIV H9N2 belonging to group III, while the second to the detection of several H9N2 outbreaks caused by group IV viruses. These two peaks corresponded in time also to the introduction of the two vaccines; the first consisting of isolate A/ty/Israel/965/02, which had been used starting from 2003, one year before the genetic variability of the viruses peaked. The second vaccine, containing the virus A/ck/Israel/215/07, was adopted at the end of 2008, the same year when the second dramatic increase in the virus population diversity occurred. The H9N2 vaccine introductions might be considered as important factors to keep in mind [13], but cannot be considered alone as responsible for the increased evolutionary rate of the AIV H9N2 in Israel. Besides, the selection pressures of the Israeli AIV H9N2 were estimated by assessing the overall number of non-synonymous to synonymous substitutions (dN/dS) The same two sites in the HA gene were under a positive selection pressure by 4 computational models with significant statistical values (http://www.datamonkey.org), FUBAR (posterior probability 0.99), FEL, IFEL and SLAC (p value for all 0.1–0.0006). The two amino acids which were found to be prone to changes were 198 and 234, corresponding to positions 190 and 226 by the AIV H3 nomenclature. These
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two positions have been previously shown to be the place for amino acid substitutions in H9N2 viruses circulating in poultry [8] and responsible for potential antigenic and adaptive changes. Additionally, substitutions E190 and L226 together with H183 and A189 at the receptor binding domain of the HA molecule are essential for the horizontal spread in mammals of reassortant viruses carrying the surface proteins of avian AIV H9N2 in a human H3N2 backbone. Furthermore, position 189, located on the tip of the globular head of HA1 is considered a critical antigenic site and has been implicated in the H9 escape mutants [14]. Similar evolutionary rate for the Israeli HA-H9N2 genes was found by two methods, Beast and Path-O-Gen, thus a double confirmation was obtained. In fact, the mean rate of the nucleotide substitution of all the Israeli isolates collected between 2000 and 2012 obtained by the Beast method was 6.123 E-3, while by the Path-O-Gen method it was of 6.58 E-3 substitutions/site/year (correlation coefficient: 0.98, R squared–0.96). Interestingly, similar values were also obtained for the Egyptian and the Indonesian H5N1 viruses [13]. The time interval from the most recent common ancestor (tMRCA) was 12.3 years, as calculated by both molecular clock models, corresponding to the year 2000, when H9N2 virus was first isolated in Israel.
Discussion The present evaluation of the phylogenetic evolution of the AIV H9N2 HA segment has indeed provided novel information to better understand the molecular epidemiology of the H9N2 virus subtype in Israel and in the region. On the basis of the results of the tMRCA presented herein, the first detection of H9N2 in Israel occurred soon after its introduction in the country. However, the early detection in year 2000 did not prevent the virus to circulate, evolve and reemerge in subsequent years (2001–2003). As for other infectious diseases, the H9N2 infection control, mainly based on vaccination alone, limited the disease and the associated economic losses but probably l impacted only slightly in limiting the virus circulation and preventing further re-introductions. In the present study, the comparison of Israeli H9N2 viruses with the strains circulating at a continental level has allowed to better infer the epidemiology of H9N2 in Israel and in the region. It is interesting to note that both in the case of the recent H9N2 epidemic (caused by group IV) and the H5N1 incursions the strains isolated in Israel are about the same as the viruses circulating in Egypt and in other bordering countries, confirming the transboundary spread of the infections and the need to increase the efficacy of the control measures at a transnational level.
Virus Genes Acknowledgements We wish to thank Drs. A. Panshin, S. Perk, A. Lublin, E. Rosenbluth, Y. Paranoushi, L. Simanov, Mrs. E. Lapin, Mrs. I. Shkoda, Mrs. A. Altouri and Ms. I. Reibshtein, involved in the AIV diagnosis and sequencing at the Division of Avian Diseases at KVI, Israel, for the facilitation of the Gene Bank Published sequences. This study was supported by Grant US-4379-11 from the USAIsrael Agricultural Research and Development Fund (BARD).
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