Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China

Veterinary Microbiology 183 (2016) 85–91 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate...

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Veterinary Microbiology 183 (2016) 85–91

Contents lists available at ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Short communication

Novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China Ying-Feng Suna,1, Xiu-Hui Wangb,1, Xiu-Li Lia , Li Zhanga , Hai-Hua Lia , Chao Lua , Chun-Lei Yanga , Jing Fenga , Wei Hana , Wei-Ke Rena , Xiang-Xue Tiana , Guang-Zhi Tongb , Feng Wenb , Ze-Jun Lib , Xiao-Qian Gongb , Xiao-Min Liub , Bao-Yang Ruanb , Ming-Hua Yana,**, Hai Yub,* a b

Tianjin Animal Husbandry and Veterinary Research Institute, Tianjin 300381,China Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 September 2015 Received in revised form 4 December 2015 Accepted 10 December 2015

Pigs are susceptible to both human and avian influenza viruses and therefore have been proposed to be mixing vessels for the generation of pandemic influenza viruses through reassortment. In this study, for the first time, we report the isolation and genetic analyses of three novel triple-reassortant H1N1 swine influenza viruses from pigs in Tianjin, Northern China. Phylogenetic analysis showed that these novel viruses contained genes from the 2009 pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. This indicated that the reassortment among the 2009 pandemic H1N1, Eurasian swine and triple-reassortant swine influenza viruses had taken place in pigs in Tianjin and resulted in the generation of new viruses. Furthermore, three human-like H1N1, two classical swine H1N1 and two Eurasian swine H1N1 viruses were also isolated during the swine influenza virus surveillance from 2009 to 2013, which indicated that multiple genetic lineages of swine H1N1 viruses were co-circulating in the swine population in Tianjin, China. The emergence of novel triple-reassortant H1N1 swine influenza viruses may be a potential threat to human health and emphasizes the importance of further continuous surveillance. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Swine influenza Novel ressortant H1N1 influenza virus Phylogenetic analysis

1. Introduction Swine influenza (SI) is a highly contagious respiratory disease caused by influenza A virus, within the Orthomyxoviridae family. The primary clinical manifestations of viral infection are coughing, sneezing, nasal discharge, elevated rectal temperatures, lethargy, difficult breathing and reduced appetite (Kothalawala et al., 2006). Currently, three predominant subtypes of influenza viruses are circulating in the swine population throughout the world; subtypes H1N1, H3N2 and H1N2 (Brown, 2000). The circulation of swine influenza viruses (SIV) is not only an economic burden on the swine industry globally, but also a

* Corresponding author at: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 518, Ziyue Road, Minhang District, Shanghai 200241, China. Fax: +86 21 54081818. ** Corresponding author. E-mail addresses: [email protected] (M.-H. Yan), [email protected] (H. Yu). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.vetmic.2015.12.006 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.

potential threat to human health. In 1976, an H1N1 virus closely related to SIV, (A/Swine/Iowa/15/30), caused an influenza outbreak at an American military facility (Hodder et al., 1977). In 2009, the emergence of a novel pandemic influenza A/H1N1 (pH1N1) widely raised public concern about SI, particularly as the gene constellation of the pH1N1 is a unique combination of SIV from North American and Eurasian lineages (Novel Swine-Origin Influenza et al., 2009). In 2011, a variant H3N2 influenza virus, which caused the influenza outbreaks in multiple U.S. states, contained the M gene from pH1N1 and the other seven segments from swine triplereassortant H3N2 viruses (Centers for Disease and Prevention, 2011, 2012). In addition, there have also been many incidents of human infection with SIV, which further emphasizes the significance of SIV public health (Claas et al., 1994; Gregory et al., 2003; Myers et al., 2007; Shinde et al., 2009). China is thought to be the epicenter for the human influenza pandemics throughout history (Shortridge and Stuart-Harris, 1982). The tracheal epithelium in pigs expresses receptors for both human and avian influenza viruses, which provides a biological basis for the susceptibility of pigs to both avian and

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A/swine /Tian jin/6/2011 (H1N1) A/swine /Tian jin/7/2012 (H1N1) A/swine /Jiang su/s15 /201 1(H1N1) A/swine /Jiang su/zg6 /2010 (H1N1) A/swine /Sha ndong /275 /2009 (H1N1) A/swine /Shand ong /327 /2009 (H1N1) A/swine/Zhe jian g/1/200 7(H1N1) A/swine /Hube i/101 /2009 (H1N1) A/swine/Beijing/26/20 08(H1N1) A/swine/Fujian /58 /2008 (H1N1) A/swine/Sha ndong /62 /2008 (H1N1) A/swine/Sha ndong /692 /200 8(H1N1) A/swine /Jiang su/40/20 11(H1N1) 94 A/swine /Shaa nxi/s2/20 12(H1N1) 99 A/swine /Guan gdong /1605 /20 10(H1N1) 99 A/swine /Guan gdong /1623 /20 10(H1N1) A/swine/Belgium/WVL2 /198 3(H1N1) 99 A/swine/Eng lan d/WVL7 /199 2(H1N1) A/swine/Tianjin/1/2009 (H1N1) A/swine/Tianjin/3/2009 (H1N1) 99 A/swine/Tianjin/2/2009 (H1N1) A/swine/Fujian /0325 /200 8(H1N1) A/Denm ark/11 /200 1(H1N1) 95 A/swine/Tianjin/01 /2004 (H1N1) A/Mem phis/12 /19 86(H1N1) A/swine /Tianjin/4/2010 (H1N1) A/swine /Tianjin/5/2010 (H1N1) A/swine /Guang dong /322 /2006 (H1N1) A/swine /Shangha i/1/2005 (H1N1) A/swine/Shangha i/2/2005 (H1N1) A/swine /Guan gdong /33 /2006 (H1N1) A/swine /Sha ngha i/3/20 05(H1N1) A/swine /Guan gdong /01 /2008 (H1N1) A/swine /Sha ndong /1112 /200 8(H1N1) 98 A/swine /Guan gdong /11 /2009 (H1N1) A/swine /Guang dong /109 /2006 (H1N1) A/swine /Guangdo ng/L6/2009 (H1N1) A/swine/Beijing/156/1991 (H1N1) 97 99 A/swine /Hube i/02 /2008 (H1N1) A/swine/Wiscon sin/1/1967 (H1N1) A/swine /Tian jin/9/201 3(H1N1) 95 A/swine /Tian jin/10/20 13(H1N1) A/swine /Tian jin/8/2013 (H1N1) A/swine/Gua ngxi/BB1/2013 (H1N1) A/Finland /553 /2009 (H1N1) A/swine /Guangdong/213/2009 (H1N1) A/swine /Shandon g/731/2009 (H1N1 A/swine /Nanchan g/3/20 10(H1N1) A/swine /Jang su/504/2010 (H1N1) A/swine /Heilong jian g/105/2009 (H1N1) A/California/04 /20 09(H1N1) A/swine /Yunnan /74 /201 0(H1N1) A/swine /Guangdong/106/2009 (H1N1) A/Singapo re/ON803 /2009 (H1N1) A/swine /Shandon g/811/2009 (H1N1) A/swine /Nanchan g/5/20 10(H1N1) 95 A/swine /Jang su/49/20 10(H1N1) A/swine /Shandon g/94/2010 (H1N1) A/swine /Guangdong/1361/2010 (H1N1) A/swine /Guangdong/94/2009(H1N1) A/swine /Guangdong/1397/2010 (H1N1) A/swine /Guangdong/286/2010 (H1N1) A/swine /Guan gxi/1/201 1(H1N1) A/swine /Shandon g/327/2010 (H1N1) A/swine /Shandon g/854/2009 (H1N1) A/swine /Heilongjiang /44 /2009 (H1N1) A/swine /Guangdong/114/2009 (H1N1) A/swine /Guangdong/50/2010(H1N1) A/Swine /Minnesota/593 /1999 (H3N2) A/swine /Texas/4199 -2/1998 (H3N2) A/Swine/Ind iana /P124 39/2000 (H1N2) A/swine/Heilong jiang /10/2007 (H3N2) A/swine /Guangdong/1/20 10(H1N1) A/swine /Sha ngha i/1/20 07(H1N2) 99 A/du ck/NY/1315 2-13 /19 94(H1N1)

PB2

PB1

Classical Swine

Pandemic H1N1

99

Triple Reassortant Swine

99

Pandemic H1N1

Triple Reassortant Swine

Eurasian Swine

Human Seasonal

Classical Swine

0.02

A/swine/Tian jin/8/2013(H1N1) A/swine/Tianjin/9/2013(H1N1) A/swine/Tianjin/10/2013(H1N1) A/swine /Guangxi/BB1/2013 (H1N1) A/swine/Guangdong /286/2010(H1N1) A/swine /Guangdong/176/2009 (H1N1) 100 A/swine /Guangdo ng/213/2009 (H1N1) A/swine /Guangxi/1/2011 (H1N1) A/swine /Nan chang/6/2010(H1N1) A/swine /Yunnan/74 /201 0(H1N1) A/swine /Guangdong/94/20 09(H1N1) A/swine /Shando ng/94/20 10(H1N1) A/swine /Heilongjiang /44 /2009(H1N1) A/swine/Shando ng/327/2010 (H1N1) A/swine/Guangdong/50/2010(H1N1) A/swine /Jang su/504/2010 (H1N1) A/swine /Jang su/49/20 10(H1N1) A/swine /Guangdong/114/2009 (H1N1) 100 A/swine/Shando ng/811/2009 (H1N1) A/swine/Shando ng/854/2009 (H1N1) A/swine /Guangdong/106/2009 (H1N1) A/swine /Heilongjiang/105 /200 9(H1N1) A/swine/Gua ngxi/S2/20 13(H1N1) A/swine/Guangdong/1361 /2010(H1N1) A/swine/Guangdong/1367 /2010(H1N1) A/Reunion /269 2-2-M2E/2009 (H1N1) A/Canada -MB/RV2018 -10 /2009 (H1N1) A/California/04/2009(H1N1) 94 A/swine /Guangdong /1/2010(H1N1) 95 A/swine /Guangxi/13 /2006 (H1N2) A/swine/Sha ngha i/1/2007 (H1N2) A/Swine/Ind iana /P1243 9/20 00(H1N2) 97 A/Swine/Minne sota/593 /1999 (H3N2) 96 A/swine /Texas/4199 -2/1998 (H3N2) A/du ck/NY/131 52-13/19 94(H1N1) 96 A/swine /Tian jin/7/2012 (H1N1) A/swine/Tianjin/6/2011(H1N1) A/swine/Shaan xi/s2/2012 (H1N1) A/swine /Jiangsu/zg6 /2010(H1N1) 95 A/swine /Jiang su/40 /2011 (H1N1) A/swine/Jiangsu/zg15/2011(H1N1) 100 A/swine/Guangdong /1605 /201 0(H1N1) A/swine/Guangdong/1623/201 0(H1N1) A/swine /Zhe jian g/1/2007(H1N1) A/swine /Shandong /275 /200 9(H1N1) A/swine/Beijing/26/20 08(H1N1) 98 A/swine/Fujian/58/2008 (H1N1) 97 A/swine/Shandong /62 /2008 (H1N1) A/swine/Hubei/101 /2009(H1N1) A/swine/Spa in/51915 /2003 (H1N1) 100 A/swine /Eng land /WVL7 /199 2(H1N1) A/swine/Belgium/WVL2 /1983 (H1N1) 100 A/swine/Tianjin/1/20 09(H1N1) A/swine/Tianjin/2/20 09(H1N1) 98 A/swine/Tianjin/3/2009(H1N1) 100 A/Dunedin/2/2000 (H1N1) A/swine/Fujian /0325 /2008(H1N1) 99 A/swine /Tianjin/01 /2004(H1N1) A/Singapo re/6/1986 (H1N1) A/swine/Tianjin/4/201 0(H1N1) A/swine /Guangdong /L6/2009(H1N1) A/swine/Tian jin/5/2010 (H1N1) A/swine /Guangdong /11/2009(H1N1) 100 A/swine /Shang hai/1/2005 (H1N1) A/swine /Guangdong /33/2006(H1N1) A/swine /Guangdong /01/2008(H1N1) A/swine /Guangdong /09/2009(H1N1) A/swine/Shandong /1112 /200 8(H1N1) 92 A/swine/Guangdong/322 /2006 (H1N1) A/swine/Shangha i/3/20 05(H1N1) A/swine/Shangha i/2/20 05(H1N1) A/swine /Guangdong/109/2006 (H1N1) 100 A/swine/Beijing/156/1991 (H1N1) A/swine/Wisconsin/1/1967 (H1N1) 0.02

Pandemic H1N1

Triple Reassortant Swine

100

Eurasian Swine

Human Seasonal

Classical Swine

A/swine/Tian jin/9/201 3(H1N1) A/swine /Tian jin/8/2013 (H1N1) A/swine/Tianjin/10 /2013 (H1N1) A/swine /Guang xi/BB1/2013 (H1N1) A/swine /Taiwan /CH-120 4/20 09(H1N1) A/Maryland /05 /201 0(H1N1) A/swine /Guangdo ng/213/2009 (H1N1) A/swine /Guang xi/1/2011 (H1N1) A/swine /Heilon gjiang /44 /2009 (H1N1) A/swine/Jang su/504 /20 10(H1N1) A/swine /Shandon g/81 1/2009 (H1N1) A/swine /Yunnan /74 /201 0(H1N1) A/swine /Jang su/49 /201 0(H1N1) A/swine/Guangdong /50 /201 0(H1N1) A/swine /Heilong jian g/10 5/20 09(H1N1) A/swine/Shandong /327 /201 0(H1N1) A/swine /Guangdon g/114/2009 (H1N1) A/swine /Guangdon g/106/2009 (H1N1) 98 A/swine /Guangdon g/94 /20 09(H1N1) A/swine /Guangdon g/1361/2010(H1N1) A/swine /Guangdon g/1397/2010(H1N1) 10099 A/swine /Guangdon g/286/2010 (H1N1) A/swine /Guang xi/S2/201 3(H1N1) A/California/04 /2009 (H1N1) 97 100 A/swine/Gua ngdong /1/2010 (H1N1) 98 A/swine /Guang xi/13 /2006 (H1N2) A/swine /Shan gha i/1/200 7(H1N2) A/Swine /Indian a/P12439 /2000 (H1N2) 98 A/Swine/Minne sota/593 /1999 (H3N2) A/swine /Texas/4199 -2/1998 (H3N2) A/swine /Tian jin/5/2010 (H1N1) A/swine /Guangdo ng/322/2006 (H1N1) 99 A/swine /Guangd ong /L6/2009 (H1N1) A/swine /Guangd ong /11/2009 (H1N1) A/swine /Guangd ong /33/2006 (H1N1) A/swine/Guan gdong /07 /2009 (H1N1) A/swine /Guangdo ng/01/2008 (H1N1) 100 A/swine /Guangdon g/10 9/2006 (H1N1) A/swine /Tian jin/4/201 0(H1N1) 97 A/swine/Shangha i/1/2005 (H1N1) A/swine /Beijing /156 /199 1(H1N1) 98 A/swine/Hub ei/02 /2008 (H1N1) A/swine /Wisconsin/1/196 7(H1N1) A/swine /Tian jin/1/2009 (H1N1) A/swine /Tian jin/2/2009 (H1N1) 100 A/Dune din/2/2000 (H1N1) A/swine/Tian jin/3/200 9(H1N1) A/swine/Fujian /0325 /2008 (H1N1) 100 100 A/swine /Hena n/01 /20 06(H1N1) A/swine /Tianjin/01 /2004 (H1N1) 100 A/Singa pore/6/1986 (H1N1) 100 A/swine/Tian jin/7/201 2(H1N1) A/swine /Tian jin/6/201 1(H1N1) A/swine/Jiangsu/zg6 /201 0(H1N1) A/swine /Jiang su/zg15/2011 (H1N1) A/swine /Jiang su/40 /2011 (H1N1) 100 A/swine/Sha anxi/s2/2012 (H1N1) 100 A/swine/Beijing/26/20 08(H1N1) A/swine/Shandong /62 /2008 (H1N1) A/swine/Fujian /58 /2008 (H1N1) 95 A/swine /Shand ong /275 /2009 (H1N1) A/swine /Zhejiang /1/2007 (H1N1) A/swine/Hub ei/101 /2009 (H1N1) A/swine/Gua ngdong /162 3/20 10(H1N1) A/swine /Spa in/51915 /2003 (H1N1) 100 A/swine /Eng land /WVL7/1992 (H1N1) A/swine /Belgium/WVL2 /1983 (H1N1) 93 100 100

NP

94 99

95

99

Human Seasonal

0.02

PA

A/swine /Tianjin/10 /2013 (H1N1) A/swine /Tianjin/8/2013 (H1N1) A/swine/Tianjin/9/20 13(H1N1) A/swine/Gua ngxi/BB1/20 13(H1N1) A/Mississ ipp i/01/20 09(H1N1) A/swine /Guangdon g/1361 /2010 (H1N1) A/swine /Jang su/49/2010 (H1N1) A/swine /Guangdon g/50 /2010(H1N1) A/swine /Guangdo ng/106 /2009 (H1N1) A/swine /Guangdo ng/114 /2009 (H1N1) A/swine /Guangdo ng/94/2009 (H1N1) A/swine /Guang xi/1/2011 (H1N1) A/swine/Gua ngdong /213 /200 9(H1N1) A/swine/Jang su/504 /201 0(H1N1) A/New York/96 /2009 (H1N1) 99 A/swine /Heilongjiang /44 /2009 (H1N1) A/swine /Yunna n/74 /20 10(H1N1) A/swine /Heilongjiang /105 /2009 (H1N1) A/swine/Gua ngdong /286 /201 0(H1N1) A/swine /Shan dong /811 /2009 (H1N1) A/California/04/20 09(H1N1) 97 A/swine /Guang dong /1/2010 (H1N1) A/swine /Guangxi/13 /2006 (H1N2) A/swine /Shangha i/1/2007 (H1N2) A/Swine /Ind iana /P12 439 /2000 (H1N2) A/Swine /Minne sota/593 /1999 (H3N2) A/swine/Texas/4199 -2/1998 (H3N2) A/Wisconsin/67 /200 5(H3N2) A/Memph is/1/199 0(H3N2) A/swine /Tian jin/6/2011 (H1N1) A/swine/Jiangsu/s15 /2011 (H1N1) A/swine/Jiangsu/40 /2011 (H1N1) 94 A/swine /Shaan xi/s2/2012 (H1N1) A/swine /Hube i/101 /20 09(H1N1) A/swine/Zhejiang /1/2007 (H1N1) A/swine /Jiang su/zg6 /201 0(H1N1) A/swine/Shandong /327 /200 9(H1N1) A/swine /Beijing /26 /2008 (H1N1) A/swine /Fujian/58/20 08(H1N1) A/swine /Shand ong /62/2008 (H1N1) A/swine /Tian jin/7/2012 (H1N1) A/swine /Guangdo ng/1605 /2010 (H1N1) 99 A/swine /Guangdo ng/1623 /2010 (H1N1) A/swine /Spa in/5191 5/20 03(H1N1) 99 A/swine /Eng land /WVL7 /1992 (H1N1) A/swine /Fujian/03 25/2008 (H1N1) A/swine /Tianjin/3/2009 (H1N1) 99 A/swine /Tian jin/2/2009 (H1N1) A/swine /Tian jin/1/2009 (H1N1) 99 A/Dune din/2/2000 (H1N1) A/swine /Tian jin/01 /2004 (H1N1) 99 A/Memph is/4/1987 (H1N1) A/swine /Tian jin/4/2010 (H1N1) A/swine /Guangdo ng/33/2006 (H1N1) A/swine /Tian jin/5/201 0(H1N1) A/swine /Guang dong /L6 /2009 (H1N1) A/swine /Guangdo ng/01/2008 (H1N1) A/swine /Guangdo ng/07/2009 (H1N1) A/swine /Shando ng/1112 /2008 (H1N1) 99 A/swine /Guangdon g/11/20 09(H1N1) A/swine/Guan gdong /109 /2006 (H1N1) 97 A/swine /Shangha i/1/2005 (H1N1) A/swine /Shangha i/2/2005 (H1N1) A/swine /Guangd ong /322 /2006 (H1N1) 99 A/swine/Hube i/02 /2008 (H1N1) A/swine/Beijing /15 6/19 91(H1N1) A/swine /Guan gxi/12 /20 05(H1N1) 99 A/swine/Wiscon sin/1/1967 (H1N1) 99

Eurasian Swine

Pandemic H1N1

Triple Reassortant Swine

Classical Swine

Human Seasonal

Eurasian Swine

0.02

Fig. 1. Phylogenetic trees of the PB2, PB1, PA and NP genes of the swine H1N1 influenza viruses. The unrooted phylogenetic tree was generated by the distance-based neighbor-joining method using MEGA 3.1. Reliability of the tree was assessed by bootstrap analysis with 1000 replications. Our swine influenza viruses in the different lineages are indicated by different markers (black diamond, circle, triangle and square).

Y.-F. Sun et al. / Veterinary Microbiology 183 (2016) 85–91

human influenza viruses. Pigs can therefore function as intermediate hosts or “mixing vessels” in establishing new influenza virus lineages by supporting coinfection, replication, and reassortment among human, avian, and swine influenza viruses. Therefore, carrying out serological and virological surveillance of swine influenza viruses in China is of great significance, and could provide necessary information for swine influenza control, and useful data for the prediction and preparedness of future human influenza pandemics. In the past, a number of influenza viruses were isolated from pigs in China, including classical swine H1N1 viruses, avian-like H1N1 viruses, pH1N1 viruses, humanlike H1N1 viruses, reassortant H1N1 viruses, reassortant H1N2 viruses, human-like H3N2 viruses, reassortant H3N2 viruses, avian-like H5N1 viruses and avian-like H9N2 viruses (Fan et al., 2012; He et al., 2013; Liang et al., 2014; Liu et al., 2009; Yu et al., 2007, 2008a,b,2009a,b, 2011; Zhao et al., 2012; Zhou et al., 2011).

2. Materials and methods 2.1. Sampling of pigs From 2009 to 2013, we carried out swine influenza virus surveillance in Tianjin, Northern China. A total of 1238 nasal swabs were collected from pigs showing clinical symptoms (coughing, sneezing, nasal discharge, difficult breathing and reduced appetite) at 67 pig farms across 8 districts (Baodi, Ninghe, Dongli, Jixian, Xiqing, Wuqing, Jinghai and Beichen) in Tianjin and 9–30 nasal swabs were collected from each farm.

2.2. Virus isolation and identification For virus isolation, strict measures were followed to prevent aberrant contamination. Gloves and injection syringes were sterile, work surfaces were sterilized with ultraviolet light, and 10-day-old SPF embryonated chicken eggs were handled by using iodine tincture and alcohol. Samples were inoculated into amnionic and allantoic cavities of eggs and the eggs were incubated for 48–72 h at 35  C. Virus isolates were passaged and identified by a hemagglutination inhibition (HI) test and a neuraminidase inhibition (NI) test, using a panel of reference sera. 2.3. Viral gene sequencing and analysis Viral gene sequencing and analyses of the ten swine influenza isolates were carried out as follows. Briefly, viral RNA was directly extracted from infected allantoic fluids using an RNeasy Mini Kit (Qiagen, Chatsworth, CA) and RT was carried out under standard conditions using Uni12 primer. PCR was performed using specific primers for eight genes (primer sequences are available on request). PCR products were purified with the QIAquick PCR purification Kit (Qiagen, Inc.) and cloned into pMD18-T vector, and sequenced using synthetic oligonucleotides (Invitrogen). Sequence data were edited and analyzed using Bioedit software. The phylogenetic trees were generated with the MEGALIGN program (DNASTAR, Madison, WI), using the Clustal alignment algorithm. Sequence data of the swine influenza viruses were compiled and edited with the Lasergene sequence analysis software package (DNASTAR Inc., Madison, WI). Multiple sequence alignment was carried out with CLUSTAL W, and the unrooted phylogenetic trees

NA HA

99

99

0.05

A/swine /Heilongjiang /105 /2009 (H1N1) A/swine/Jang su/49 /2010 (H1N1) A/swine /Heilongjiang /44 /2009 (H1N1) A/swine /Yun nan /74/2010 (H1N1) A/swine /Shand ong /811 /2009 (H1N1) A/swine /Jang su/504/2010 (H1N1) A/swine /Shan dong /327 /2010 (H1N1) A/swine/Guan gdong /50 /2010 (H1N1) A/swine/Gua ngdong /106 /200 9(H1N1) A/swine/Gua ngdong /114 /200 9(H1N1) 95 A/swine/Gua ngdong /94 /2009 (H1N1) A/swine/Gua ngdong /286 /201 0(H1N1) A/swine /Guang xi/1/2011 (H1N1) A/swine /Guang dong /176 /2009 (H1N1) 99 A/swine /Guang dong /213 /2009 (H1N1) A/swine/Nanchang /3/2010 (H1N1) A/swine /Nan cha ng/5/20 10(H1N1) A/California/04 /200 9 A(H1N1) A/swine /Tianjin/4/2010 (H1N1) A/swine/Tianjin/5/20 10(H1N1) A/swine/Gua ngdong /L6 /2009 (H1N1) A/swine/Shandong /1112 /20 08(H1N1) 99 A/swine/Gua ngdong /2/2009 (H1N1) A/swine/Gua ngdong /2/2001 (H1N1) A/swine /Guangdo ng/11/20 09(H1N1) A/swine /Guang dong /1/2005 (H1N1) A/swine/Sha ngha i/1/200 5(H1N1) A/swine /Guangdong/322/2006 (H1N1) 99 A/swine /Guang dong /33 /2006 (H1N1) A/swine /Shan gha i/2/2005 (H1N1) A/swine /Guangd ong /01/2008 (H1N1) A/swine/Guangdong /109 /20 06(H1N1) 99 A/swine/Beijing/156 /1991 (H1N1) A/swine /Wisconsin/1/1967 (H1N1) A/swine /Tian jin/2/2009 (H1N1) 99 A/swine /Tianjin/1/2009 (H1N1) A/swine /Fujian/0325 /2008 (H1N1) A/swine /Tianjin/3/2009 (H1N1) A/Nanchang /17 /199 6(H1N1) A/Florida /3/2006 (H1N1) A/Washing ton /10/2008 (H1N1) A/Brisbane /59 /2007 (H1N1) 99 A/swine /Tian jin/01 /2004 (H1N1) A/Baylor/1173 5/19 82(H1N1) 99 A/swine/Tianjin/6/2011 (H1N1) 97 A/swine/Tianjin/7/2012 (H1N1) A/swine /Jiang su/s16 /2011 (H1N1) A/swine /Tian jin/10 /201 3(H1N1) 99 A/swine /Tian jin/8/2013 (H1N1) A/swine /Tian jin/9/2013 (H1N1) A/swine /Hong Kong /1780 /2008 (H1N1) A/swine /Jiang su/zg18 /2011 (H1N1) A/Jiang su/ALS1/2011 (H1N1) A/swine /Jiang su/zg7 /201 0(H1N1) A/swine /Shaan xi/s2/2012 (H1N1) A/swine/Beijing/26 /20 08(H1N1) 98 A/swine /Sha ndong /275 /2009 (H1N1) A/swine/Fujian /58 /2008 (H1N1) A/swine/Shandong /62 /2008 (H1N1) A/swine /Liaoning/32/2006 (H1N1) A/swine /Hube i/101/2009 (H1N1) A/swine/Zhejiang /1/2007 (H1N1) A/swine /Guangd ong /1361 /2010 (H1N1) A/swine /Hong Kong /8512 /200 1(H1N1) 95 A/swine /Guang dong /1/2010 (H1N1) 98 A/swine /Guang xi/S2/2013 (H1N1) A/swine/Guan gdong /1605 /201 0(H1N1) 99 99 A/swine/Guan gdong /1623 /201 0(H1N1) A/Swine/Spain/500 47/2003 (H1N1) 99 A/swine /Hunga ry/19774 /2006 (H1N1) A/swine/Englan d/WVL14 /199 6(H1N1) A/swine /Belgium/1/1983 (H1N1) A/chicken /Hong Kong /14 /1976 (H1N1)

87

Pandemic H1N1

Classical Swine

Human Seasonal

Eurasian Swine

A/swine /Shan dong /94 /2010 (H1N1) A/swine /Guang xi/1/2011 (H1N1) A/swine /Yunna n/74 /201 0(H1N1) A/swine /Heilongjiang /105 /2009 (H1N1) A/swine /Guangdo ng/286/2010 (H1N1) A/swine /Guangdo ng/213/2009 (H1N1) A/swine /Guangdo ng/106/2009 (H1N1) A/swine /Guangdo ng/114/2009 (H1N1) A/swine /Guangdo ng/94/2009 (H1N1) A/swine /Heilongjiang /44 /2009 (H1N1) A/swine /Shando ng/327 /2010 (H1N1) A/swine /Shandon g/36 1/2010 (H1N1) 100 A/swine /Jan gsu/504 /2010 (H1N1) A/swine /Guangdong/50/20 10(H1N1) A/swine/Nan chang /3/2010 (H1N1) A/swine/Nan chang /5/2010 (H1N1) A/swine/Jang su/49 /2010 (H1N1) A/swine /Shand ong /731 /2009 (H1N1) A/swine /Shandon g/81 1/2009 (H1N1) A/swine /Shand ong /854 /2009 (H1N1) A/swine/Shandong /N1/2009 (H1N1) A/California/04 /200 9(H1N1) 99 A/swine /Tian jin/6/2011 (H1N1) 98 A/swine /Tian jin/7/2012 (H1N1) A/swine /Jiang su/s15/20 11(H1N1) A/swine/Tianjin/8/2013 (H1N1) 100 A/swine /Tian jin/9/2013 (H1N1) 99 A/swine /Tianjin/10 /2013 (H1N1) A/swine/Gua ngxi/BB1/20 13(H1N1) A/swine/Jiangsu/40 /2011 (H1N1) 97 100 A/swine /Sha anxi/s2/2012 (H1N1) 98 A/swine /Jiang su/zg5 /2010 (H1N1) A/swine /Jiang su/zg7/2010 (H1N1) A/swine /Shan dong /275 /2009 (H1N1) A/swine /Shan dong /327 /2009 (H1N1) 100 A/swine /Shan dong /187 /2008 (H1N1) A/swine /Beijing /26 /2008 (H1N1) A/swine /Shand ong /692 /2008 (H1N1) 100 A/swine/Shandong /128 /200 8(H1N1) 94 A/swine/Shandong /443 /200 8(H1N1) A/swine /Fujian/58/2008 (H1N1) 96 A/swine /Shand ong /62 /2008 (H1N1) A/swine /Guang dong /1361 /2010 (H1N1) 90 A/swine /Zhe jiang/1/20 07(H1N1) A/swine /Hube i/101 /2009 (H1N1) 98 A/swine /Jiang su/zg6 /201 0(H1N1) A/swine /Guangdo ng/1/20 10(H1N1) A/swine /Guangd ong /1605 /2010 (H1N1) 100 100 A/swine /Guangd ong /1623 /2010 (H1N1) A/swine /Spa in/50047 /2003 (H1N1) A/swine/Belgium/1/198 3(H1N1) A/swine/England/WVL1 4/1996 (H1N1) A/swine /Tianjin/1/2009 (H1N1) A/swine/Tianjin/2/20 09(H1N1) A/swine /Tian jin/3/2009 (H1N1) A/swine /Fujian /0325 /2008 (H1N1) A/Duned in/2/2000 (H1N1) 100 A/Florida/3/200 6(H1N1) 100 A/Washing ton /10 /2008 (H1N1) A/swine/Tianjin/01/2004 (H1N1) 100 A/Neimen ggu /63/1997 (H1N1) A/swine /Tian jin/5/2010 (H1N1) A/swine /Tianjin/4/2010 (H1N1) A/swine /Guan gdong /11 /2009 (H1N1) A/swine /Guang dong /33 /2006 (H1N1) A/swine /Shangha i/1/2005 (H1N1) A/swine /Shangha i/2/2005 (H1N1) A/swine/Guan gdong /09 /2009 (H1N1) A/swine /Shando ng/1112 /2008 (H1N1) 99 A/swine /Sha ndong /1123 /200 8(H1N1) A/swine/Shangha i/3/20 05(H1N1) A/swine/Guan gdong /01 /2008 (H1N1) A/swine /Guang dong /109 /2006 (H1N1) 100 A/swine /Guang dong /322 /2006 (H1N1) A/swine /Guang dong /2/2009 (H1N1) 100 A/swine /Guang xi/12 /2005 (H1N1) A/swine /Beijing /156 /199 1(H1N1) 100 A/swine /Wisconsin/1/19 67(H1N1)

Pandemic H1N1

Eurasian Swine

Human Seasonal

Classical Swine

0.02

Fig. 2. Phylogenetic trees of the HA, NA and M genes of the swine H1N1 influenza viruses. The method used is as given in the legend to Fig. 1. Our swine influenza viruses in the different lineages are indicated by different markers (black diamond, circle, triangle and square).

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M

A/swine /Guangdon g/13 61/2010 (H1N1) A/swine /Guangdon g/13 97/2010 (H1N1) A/swine/Guangdong /50 /201 0(H1N1) A/swine/Gua ngdong /106 /200 9(H1N1) A/swine/Gua ngdong /94 /2009 (H1N1) A/swine/Gua ngdong /114 /200 9(H1N1) A/swine/Jang su/49 /201 0(H1N1) A/swine /Nan cha ng/3/20 10(H1N1) A/swine /Nan cha ng/5/20 10(H1N1) A/swine /Shan dong /327 /2010 (H1N1) A/swine /Yunn an/74/2010 (H1N1) A/swine /Guan gxi/1/201 1(H1N1) A/swine /Heilongjiang /105 /200 9(H1N1) A/swine /Guan gdong /213 /2009 (H1N1) A/swine /Sha ndong /811 /2009 (H1N1) A/swine /Guan gdong /286 /2010 (H1N1) A/swine /Heilong jian g/44 /20 09(H1N1) A/swine /Jan gsu/504 /2010 (H1N1) A/California/04 /2009 (H1N1) A/swine/Tian jin/8/201 3(H1N1) 98 A/swine /Tian jin/10 /2013 (H1N1) 99 A/swine/Tianjin/9/2013 (H1N1) A/swine /Guang xi/BB1/2013 (H1N1) A/swine /Guang xi/G2/2013 (H1N1) A/Jiang su/ALS1/20 11(H1N1) A/swine/Tianjin/6/2011 (H1N1) A/swine/Tianjin/7/2012 (H1N1) A/swine/Shaan xi/s2/2012 (H1N1) A/swine /Zhe jian g/1/200 7(H1N1) A/swine /Jiang su/zg15 /2011 (H1N1) A/swine /Hube i/101 /2009 (H1N1) A/swine /Jiang su/40/2011 (H1N1) A/swine/Beijing/26/2008 (H1N1) A/swine /Fujian/58/20 08(H1N1) 92 A/swine /Shand ong /275 /2009 (H1N1) A/swine /Shand ong /62/2008 (H1N1) 99 A/swine /Jiang su/zg6/2010 (H1N1) A/swine /Guangdo ng/1/20 10(H1N1) A/swine /Guang xi/S2/2013 (H1N1) A/swine /Guan gdong /1605 /201 0(H1N1) 99 A/swine /Guan gdong /1623 /201 0(H1N1) A/swine/England/WVL1 4/1996 (H1N1) A/swine /Den mark/WVL9 /199 3(H1N1) 99 A/swine /Eng land /WVL7 /1992 (H1N1) A/swine/Tianjin/3/2009 (H1N1) A/swine /Tianjin/2/2009 (H1N1) 99 A/swine /Tian jin/1/2009 (H1N1) A/swine /Fujian/03 25/2008 (H1N1) A/Canterbu ry/79 /2000 (H1N1) 99 A/swine/Hena n/01/20 06(H1N1) A/Memph is/4/1987 (H1N1) A/swine/Tianjin/01/2004 (H1N1) A/swine /Guan gdong /07 /2009 (H1N1) A/swine /Tianjin/5/2010 (H1N1) A/swine /Tian jin/4/201 0(H1N1) A/swine/Guangdong /33 /200 6(H1N1) A/swine/Guangdong /01 /200 8(H1N1) A/swine /Guangdon g/11/20 09(H1N1) A/swine /Shangh ai/1/2005 (H1N1) A/swine /Shangh ai/2/2005 (H1N1) A/swine /Guangdo ng/L6/2009 (H1N1) A/swine /Shan dong /1112 /2008 (H1N1) A/swine /Guangd ong /109 /2006 (H1N1) A/swine/Guangdong /32 2/20 06(H1N1) A/swine /Beijing /156 /1991 (H1N1) 99 A/swine /Hube i/02 /2008 (H1N1) 97 A/Swine /Minnesota/593 /1999 (H3N2) A/swine/Texas/419 9-2/1998 (H3N2) A/swine/Heilong jiang/10/2007 (H3N2) A/swine/Guang xi/13 /2006 (H1N2) 96 A/swine /Sha ngha i/1/200 7(H1N2) 99 A/Swine/Ind iana /P12439 /200 0(H1N2) A/swine /Wisconsin/1/1967 (H1N1) 99

Pandemic H1N1

Eurasian Swine

Human Seasonal

Classical Swine

0.01

Fig. 2. (Continued)

were generated by the distance-based neighbor-joining method using MEGA 3.1. Bootstrap values were calculated on 1000 replicates of the alignment. 3. Results and discussion 3.1. Virus isolation and identification Ten influenza A viruses were isolated using 10-day-old SPF chicken eggs and identified by a hemagglutination inhibition (HI) test and neuraminidase inhibition (NI) test, using a panel of reference sera. Three swine influenza viruses from a pig farm in Dongli district were designated as A/swine/Tianjin/1/2009 (H1N1), A/swine/Tianjin/2/2009 (H1N1) and A/swine/Tianjin/3/ 2009 (H1N1). Two isolates from a pig farm in the Beichen district were named as A/swine/Tianjin/4/2010 (H1N1) and A/swine/ Tianjin/5/2010 (H1N1). Two isolates from a farm in the Ninghe district were designated as A/swine/Tianjin/6/2011 (H1N1) and A/ swine/Tianjin/7/2012 (H1N1). Three isolates from a pig farm in Jinghai district were named as A/swine/Tianjin/8/2013 (H1N1), A/swine/Tianjin/9/2013 (H1N1) and A/swine/Tianjin/10/2013 (H1N1).

3.2. Phylogenetic analysis of swine H1N1 influenza viruses isolated in Tianjin from 2009 to 2013 The eight gene segments of the swine influenza virus isolates were sequenced, and the phylogenetic trees were constructed using the nucleotide sequences of the PB2, PB1, PA, HA, NP, NA, M and NS genes available in GenBank. Phylogenetic analyses of PB2, PB1, PA and NP genes (Fig. 1) revealed that our ten isolates were located in four different lineages, including the Eurasian swine lineage, human seasonal lineage, classical swine lineage and pandemic H1N1 lineage. A/swine/Tianjin/1/2009, A/swine/Tianjin/2/ 2009 and A/swine/Tianjin/3/2009 belonged to the human seasonal lineage. A/swine/Tianjin/4/2010 and A/swine/Tianjin/5/2010 were grouped in the classical swine lineage. A/swine/Tianjin/6/2011 and A/swine/Tianjin/7/2012 were incorporated into the Eurasian swine lineage. A/swine/Tianjin/8/2013, A/swine/Tianjin/9/2013 and A/ swine/Tianjin/10/2013 were included in the pandemic H1N1 lineage. Phylogenetic analyses of HA, NA and M genes (Fig. 2) showed that our ten isolates were located into three distinct lineages. A/ swine/Tianjin/1/2009, A/swine/Tianjin/2/2009 and A/swine/Tianjin/3/2009 were closely related to the human seasonal lineage. A/ swine/Tianjin/4/2010 and A/swine/Tianjin/5/2010 belonged to the classical swine lineage. A/swine/Tianjin/6/2011, A/swine/Tianjin/7/ 2012, A/swine/Tianjin/8/2013, A/swine/Tianjin/9/2013 and A/ swine/Tianjin/10/2013 were all grouped in the Eurasian swine lineage.

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A/swine /Shandong/327/20 10(H1N1) A/swine /Guangdong /114 /200 9(H1N1) A/swine /Guangdong /94 /2009 (H1N1) A/swine /Guangdong/10 6/2009 (H1N1) A/swine /Guangdong /50/2010 (H1N1) A/swine /Nanchang /3/2010 (H1N1) A/swine /Nan chan g/5/2010 (H1N1) 100 A/swine /Guangdong /286 /201 0(H1N1) A/swine /Guangdong/13 61/2010(H1N1) A/swine /Guangdong/13 97/2010(H1N1) A/swine /Jan gsu/49/2010 (H1N1) A/swine /Guangdong/21 3/2009 (H1N1) A/swine /Guang xi/1/20 11(H1N1) A/swine /Shandong /811 /2009 (H1N1) A/swine /Yunnan/74 /2010 (H1N1) A/swine /Heilongjiang/105/2009 (H1N1) A/swine /Heilongjiang/44 /2009 (H1N1) A/swine/Jang su/504 /2010 (H1N1) A/California/04/2009 (H1N1) A/swine /Tianjin/8/2013 (H1N1) 95 A/swine /Tianjin/9/2013 (H1N1) 100 A/swine /Tianjin/10 /2013 (H1N1) A/swine/Guangxi/BB1/2013 (H1N1) 100 A/swine /Guangdong /1605 /20 10(H1N1) A/swine /Guangdong /1623 /20 10(H1N1) 99 A/swine /Hong Kong /1422 /2009 (H1N1) A/swine/Gua ngdong/1/2010 (H1N1) A/swine/Guan gxi/S2/2013 (H1N1) A/swine /Hong Kong /72 /2007 (H1N1) A/swine /Hong Kong/729/2005 (H1N2) A/swine/Sha ngha i/1/2007 (H1N2) 95 A/swine/Guang xi/13/2006 (H1N2) A/Swine /Minne sota/593 /199 9(H3N2) 100 A/swine/Tianjin/4/20 10(H1N1) A/swine/Guangdong /109 /2006 (H1N1) A/swine /Tian jin/5/2010 (H1N1) 100 A/swine /Guang dong /322 /2006 (H1N1) A/swine /Guangdong/07/2009(H1N1) A/swine /Guan gdong/11 /2009 (H1N1) A/swine /Shan dong /1112/2008 (H1N1) 100 A/swine /Guangdong/01 /200 8(H1N1) A/swine /Guangdong/L6 /2009 (H1N1) A/swine /Shanghai/2/2005 (H1N1) A/swine /Guangdong/33 /2006 (H1N1) A/swine /Shanghai/1/2005 (H1N1) A/swine /Beijing /156 /1991 (H1N1) 98 A/swine /Hube i/02 /2008 (H1N1) A/swine /Wiscon sin/1/1967 (H1N1) A/swine /Tian jin/3/2009(H1N1) A/swine /Tianjin/2/2009 (H1N1) 93 A/swine /Fujian /032 5/20 08(H1N1) 100 A/swine/Tian jin/1/2009 (H1N1) 96 A/Nethe rland s/26 /2007 (H1N1) A/swine /Hena n/01 /200 6(H1N1) 98 A/swine /Tianjin/01 /2004(H1N1) 100 A/Mem phis/1/1987 (H1N1) A/New York/35 9/2005(H3N2) 100 A/England/198 /2002 (H3N2) 95 A/swine/Tianjin/6/201 1(H1N1) A/swine /Tianjin/7/2012 (H1N1) A/swine /Jiang su/40 /2011 (H1N1) A/swine/Jiang su/zg15 /2011 (H1N1) A/swine /Shaanxi/s2/2012 (H1N1) A/swine /Jiang su/zg6 /201 0(H1N1) A/swine/Fujian /58 /2008 (H1N1) A/swine /Shandong/275/2009 (H1N1) 10090 A/swine /Shandong /62 /2008 (H1N1) A/swine /Beijing /26 /2008 (H1N1) A/swine /Zhejiang/1/2007 (H1N1) A/swine /Hube i/101/2009 (H1N1) 100 A/swine/Englan d/WVL7/1992 (H1N1) A/swine /Spa in/51915 /2003 (H1N1)

89

NS

Pandemic H1N1

Triple reassortant Swine

Classical Swine

Human Seasonal

Eurasian Swine

0.02 Fig. 3. Phylogenetic tree of the NS gene of the swine H1N1 influenza viruses. The method used is as given in the legend to Fig. 1. Our swine influenza viruses in the different lineages are indicated by different markers (black diamond, circle, triangle and square).

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Table 1 Genetic origin of swine influenza viruses isolated from pigs in Tianjin, Northern China. Isolate name

Lineage assigned to gene segment PB2

PB1

PA

HA

NP

NA

M

NS

A/swine/Tianjin/1/2009(H1N1) A/swine/Tianjin/2/2009(H1N1) A/swine/Tianjin/3/2009(H1N1) A/swine/Tianjin/4/2010(H1N1) A/swine/Tianjin/5/2010(H1N1) A/swine/Tianjin/6/2011(H1N1) A/swine/Tianjin/7/2012(H1N1) A/swine/Tianjin/8/2013(H1N1) A/swine/Tianjin/9/2013(H1N1) A/swine/Tianjin/10/2013(H1N1)

HU HU HU CS CS EA EA pdm/09 pdm/09 pdm/09

HU HU HU CS CS EA EA pdm/09 pdm/09 pdm/09

HU HU HU CS CS EA EA pdm/09 pdm/09 pdm/09

HU HU HU CS CS EA EA EA EA EA

HU HU HU CS CS EA EA pdm/09 pdm/09 pdm/09

HU HU HU CS CS EA EA EA EA EA

HU HU HU CS CS EA EA EA EA EA

HU HU HU CS CS EA EA TR TR TR

PB, polymerase basic protein; PA, polymerase acidic protein; HA, hemagglutinin; NP, nucleocapsid protein; NA, neuraminidase; M, matrix; NS, nonstructural; HU, genes with closest homology to human seasonal influenza viruses; CS, genes with closest homology to classical swine H1N1 viruses; EA, genes with closest homology to Eurasian swine H1N1 viruses; pdm/09, genes with closest homology to pandemic (H1N1) 2009 virus; TR, genes with closest homology to triple-reassortant swine influenza viruses.

Phylogenetic analysis of the NS gene (Fig. 3) indicated that our ten swine viruses were located in four lineages (triple-reassortant swine lineage, classical swine lineage, human seasonal lineage and Eurasian swine lineage). A/swine/Tianjin/1/2009, A/swine/ Tianjin/2/2009 and A/swine/Tianjin/3/2009 were incorporated into the human seasonal lineage. A/swine/Tianjin/4/2010 and A/ swine/Tianjin/5/2010 belonged to the classical swine lineage. A/ swine/Tianjin/6/2011 and A/swine/Tianjin/7/2012 were located in the Eurasian swine lineage. A/swine/Tianjin/8/2013, A/swine/ Tianjin/9/2013 and A/swine/Tianjin/10/2013 were clustered into the triple-reassortant swine lineage. Based on the phylogenetic trees of the gene segments from the ten swine viruses (Table 1), A/swine/Tianjin/1/2009, A/swine/ Tianjin/2/2009 and A/swine/Tianjin/3/2009 seemed to be descendants of human H1N1 viruses circulating in the 2000s. A/swine/Tianjin/4/2010 and A/swine/Tianjin/5/2010 were derived from the classical swine H1N1 influenza viruses. A/ swine/Tianjin/6/2011 and A/swine/Tianjin/7/2012 had close genetic relationships with the Eurasian swine H1N1 influenza viruses. A/swine/Tianjin/8/2013, A/swine/Tianjin/9/2013 and A/ swine/Tianjin/10/2013 were novel triple-ressortant H1N1 influenza viruses from pigs in China containing genes from the pandemic H1N1 (PB2, PB1, PA and NP), Eurasian swine (HA, NA and M) and triple-reassortant swine (NS) lineages. Since the first report on infection of pigs with the 2009 pandemic H1N1 virus (pH1N1) in Canada in 2009 (Howden et al., 2009), pH1N1 has been isolated from pigs all over the world, including China (Zhao et al., 2012). Introduction of pH1N1 into the swine population has raised concerns about the generation of novel reassortant viruses in pigs, which could have pandemic potential. The first reassortant H1N1 virus was found in Hong Kong, southern China, in 2009, and contained NA from pH1N1, HA from the Eurasian avian-like H1, and six internal genes from triple-reassortant SIVs (Vijaykrishna et al., 2010). Subsequently, many double-reassortant viruses were reported worldwide (Fan et al., 2012; Howard et al., 2011; Kitikoon et al., 2011; Liang et al., 2014; Liu et al., 2012; Moreno et al., 2011; Starick et al., 2011). Previously, in Tianjin, Northern China, only a human-like swine influenza virus (A/swine/Tianjin/1/2004(H1N1)) isolated from a pig was reported by us, and the virus seemed to be descendants of human H1N1 viruses circulating in 1980s (Yu et al., 2009b). In this study, three novel triple-reassortant H1N1 viruses were isolated from pigs in Tianjin in 2013 and contained genes from pH1N1 (PB2, PB1, PA and NP), Eurasian avian-like swine H1N1 (HA, NA and M) and triple-reassortant SIVs (NS). The emergence of these three novel triple-reassortant H1N1 swine influenza viruses in pigs in Tianjin, Northern China provides further evidence that

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