Virus Genes DOI 10.1007/s11262-014-1092-6

Identification and genetic characterization of avian-origin H3N2 canine influenza viruses isolated from the Liaoning province of China in 2012 Xinyan Yang • Chunguo Liu • Fei Liu • Dafei Liu • Yan Chen • Haifeng Zhang • Liandong Qu • Yijing Li Donghua Xia • Ming Liu



Received: 26 February 2014 / Accepted: 27 May 2014 Ó Springer Science+Business Media New York 2014

Abstract A total of 158 serum samples and 510 nasal swab specimens were collected between September 2010 and May 2012, from dogs exhibiting respiratory symptoms, in order to investigate the epidemiology of H3N2 canine influenza viruses (CIVs) in the Liaoning province of China. Serological surveillance demonstrated that 10.8 % (17/ 158) of serum samples were positive for H3N2 canine influenza. Two H3N2 influenza viruses, A/canine/Liaoning/27/2012 and A/canine/Liaoning/H6/2012, were isolated from pet dogs in 2012. Phylogenetic analysis indicated that the genes from these two viruses were closely related to those of avian-origin, H3N2 subtype CIVs from China and Thailand. Genetic analysis of eight genes revealed that these two H3N2 canine influenza isolates were highly similar (99.2–99.8 %) to the current common strains in Asia. Analysis of the genotype demonstrated that each gene of the two strains in this study had the same genotype (K, G, E, 3B, F, 2D, F, 1E) as those prevalent in Xinyan Yang, Chunguo Liu and Fei Liu contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1092-6) contains supplementary material, which is available to authorized users.

H3N2 CIVs. Our findings further confirm that avian-origin H3N2 canine influenza has become established in China. Conducting extensive serological and epidemiological surveillance is necessary to develop an effective vaccine against this disease. Keywords Canine influenza virus  H3N2  Isolation  Molecular characterization

Introduction Canine influenza is a recently emergent respiratory disease that occurs in dogs and is caused by a variety of influenza A viruses in the Orthomyxoviridae family. The first case of canine influenza was reported in 2004, in racing greyhounds that became infected with the H3N8 subtype equine influenza virus, in Florida [1]. In the following years, the H3N8 subtype canine influenza viruses (CIVs) were the only known influenza viruses circulating in the canine population. In recent years, many other subtype influenza viruses have been isolated from dogs, including H1N1 [2], H5N1 [3], H5N2 [4, 5], H9N2 [6], H3N1 [7], and H3N2 [8,

X. Yang Haikou Center for Disease Control & Prevention, Haikou 570102, China

F. Liu Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China

C. Liu  D. Liu  Y. Chen  L. Qu  M. Liu (&) State Key Lab of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China e-mail: [email protected]

H. Zhang Heilongjiang Animal Husbandry Research Institute, Qiqihar 160001, China

C. Liu  Y. Li College of Veterinary Medicine, Northeast of Agricultural University, Harbin 150030, China

D. Xia (&) Luoniushan Animal Husbandry Co. Ltd., Haikou 570102, China e-mail: [email protected]

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9]. However, except for H3N2, the other subtype CIVs did not lead to the spread of disease among dogs [10, 11]. The first case of H3N2 subtype canine influenza occurred in the Guangdong province of China in 2006 and originated from the H3N2 subtype avian influenza viruses [9]. Soon after, South Korea also reported this incident. Until recently, the H3N2 subtype canine influenza has been confined to Asian countries, including South Korea, Thailand [12], and China. Among these, China has been the hardest hit with many provinces and cities, including Guangdong, Zhejiang, Jiangsu, Liaoning, and Beijing, reporting outbreaks of H3N2 subtype canine influenza [2, 13–15]. Thus, it is necessary to conduct extensive serological and epidemiological surveillance to lay the foundation for effective prevention of this disease. In this study, 158 serum samples were gathered from pet dogs (59) in 10 pet clinics, from farm dogs (53), and from stray dogs (46) between September 2010 and May 2012. Sera antibody titers were detected using hemagglutination inhibition (HAI) tests according to procedures recommended by the World Organization for Animal Health (OIE). In brief, the sera were initially treated with receptor destroying enzyme (Sigma, St. Louis, MO, USA) overnight and inactivated H3N2 subtype avian influenza virus A/duck/Hebei/wy03/2012 (H3N2) (Animal Influenza Center of Harbin Veterinary Research Institute, Harbin, China) was then used as diagnostic antigen to conduct the HAI test (HAI cut-off value = 1:16). The results showed that 10.8 % (17/158) of the total sera samples were positive for H3N2. The positive rate was highest, at 38.4 % (10/26), among stray dogs. The rates of positive serum samples from pet dogs and farm dogs were relatively lower compared to that of stray dogs [5.1 % (3/59) and 5.5 % (4/73), respectively]. The percentage of positive sera samples was higher among female dogs compared with male dogs [14.8 % (9/61) and 8.2 % (8/97), respectively] (Table 1). In addition to the serum samples, 510 nasal swab samples were also collected from dogs that were in the pet clinic due to respiratory symptoms, in order to isolate the H3N2 canine influenza virus. Two influenza viruses, A/canine/Liaoning/ 27/2012 (LN-27) and A/canine/Liaoning/H6/2012 (LN-H6), were obtained via inoculating 9-day-old specific-pathogenfree (SPF) embryonated chicken eggs with samples from the nasal swabs. Titers from the hemagglutination test (HA) of the allantoic fluid were 1:256 and 1:512, respectively and the 50 % embryo infective doses (EID50) were 4 9 107.1/ml and 5 9 106.8/ml, respectively. The H3 subtype of these two isolates was identified via HAI with the reference positive chicken sera against the avian influenza virus A/duck/Hebei/ wy03/2012 (H3N2). The allantoic fluid that contained the viruses was stored at -70 °C until being used for RNA extraction.

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Table 1 Seroprevalence studies of H3N2 canine influenza Source of dog

Number of HAI antibody sera positive for H3N2 canine influenza/total Male

Female

Total

Positive rate (%)

Pet dog

2/38

1/21

3/59

5.1

Farm dog

2/52

2/21

4/73

5.5

Stray dog

4/7

6/19

10/26

38.4

Total

8/97 (8.2 %)

9/61 (14.8 %)

17/158

10.8

The viral RNA of the isolates was extracted from the allantoic fluid with a commercial Viral RNA Mini Kit (Axygen Scientific Inc., Hangzhou, China). RT-PCR was performed on total RNA to amplify each gene segment of the two viruses, LN-27 and LN-H6, with the specific primers for each gene (primers available from the authors upon request). The products of RT-PCR were extracted from agarose gels using a DNA Gel Extraction kit (Axygen Scientific Inc., Hangzhou, China) and all genes were subcloned into pMD18-T vector (TaKaRa, Dalian, China). Genes were then sequenced by Beijing Genomics Institute (Beijing, China), using the Sanger method. The nucleotide sequence of each gene in LN-27 and LN-H6 was edited using the Lasergene sequence analysis software package (DNASTAR, Madison, WI, USA) and submitted to the Genbank. Genbank accession numbers of the eight genes of the two viruses are numbered from KF042257 to KF042272. The most closely related reference viruses to LN-27 and LN-H6 viruses were looking for by BLAST analysis, and then the nucleotide sequences of the same gene of the isolates and the reference viruses were added into the MegAlign program and conducted the ClustalW analysis to generate the homologous rate between the isolates and the reference viruses. Phylogenetic analyses were performed using the neighbor-joining program MEGA4 with 1,000 bootstrap replicates [16]. The nucleotide sequence alignments indicated that the PB2, PB1, NP, M, and NS genes from LN-27 and LN-H6 were highly related to those of A/canine/Thailand/CU-DC5299/2012 (H3N2) (99.4, 99.4, 99.3, 99.8, and 99.6 % respectively). The PA gene shared 99.2 % identity with that of A/canine/Zhejiang/1/2010 (H3N2). The HA and NA genes showed the highest homology (up to 99.6 %) with the corresponding genes of A/canine/Guangdong/05/2011 (H3N2). Each corresponding gene of LN-27 and LN-H6 showed a homology of 99.8–100 % (Table 2). Phylogenetic analyses of each gene of LN-27 and LNH6 demonstrated that the HA and NA genes were located in the same cluster as those of A/canine/Guangdong/05/ 2011 (H3N2) and the six remaining internal genes were in the same cluster with those of A/canine/Thailand/

Virus Genes Table 2 Influenza A viruses with the highest nucleotide sequence identity to the isolates Gene

Nucleotide sequence compared

Identity (%)

Virus designation

Genbank accession no.

PB2

28–2307

99.4

A/canine/Thailand/CUDC5299/2012 (H3N2)

KC599547

PB1

25–2298

99.4

A/canine/Thailand/CUDC5299/2012 (H3N2)

KC599546

PA

25–2175

99.2

A/canine/Zhejiang/1/ 2010 (H3N2)

JF714151

HA

20–1730

99.6

A/canine/Guangdong/ 05/2011 (H3N2)

JX414244

NP

46–1542

99.3

A/canine/Thailand/CUDC5299/2012 (H3N2)

KC599552

NA

21–1437

99.4

A/canine/Guangdong/ 05/2011 (H3N2)

JX414246

M

26–1008

99.8

A/canine/Thailand/CUDC5299/2012 (H3N2)

KC599545

NS

27–864

99.6

A/canine/Thailand/CUDC5299/2012 (H3N2)

KC599550

CU-DC5299/2012 (H3N2). Results of phylogenetic analyses demonstrated that LN-27 and LN-H6 were closely related to H3N2 CIVs coming from China and Thailand. All H3N2 CIVs were highly related to H3N2 avian influenza viruses, suggesting that the two CIVs isolated in this study also originated from H3N2 avian influenza viruses (Fig. 1). To determine the genotypes of the LN-27 and LN-H6 CIVs, all of the gene fragments were entered into the influenza A virus genotype tool [17]. The results indicated that the genotypes of the PB2, PB1, PA, HA, NP, NA, M, and NS segments of these two isolates were defined as K, G, E, 3B, F, 2D, F, and 1E, respectively; the same as those of the H3N2 CIVs listed in Genbank and H3N2 avian influenza viruses used in this study, which suggested that these two isolates were not the recombinant viruses. Molecular analysis of the deduced amino acid sequence showed that the HA cleavage site of all viruses in this study was PEK/RQTRGL with low pathogenic characterization. The HA of the isolates had Q226 and G228 on its receptor binding site, indicating that these two viruses preferentially bind to avian-like NeuAca2, 3-Gal receptors [18] (Table 3). The high virus titers in the lungs of dogs that have been artificially infected (data not shown) suggest that the isolates can efficiently replicate in dogs, which in accord with the characteristic of avian-like NeuAca2, 3-Gal receptors expression in the respiratory tract of dogs [19]. To analyze the antigenic site of HA genes, the HA numbering and HA structure (1HGD) of H3N2 subtype human virus was used in this article [20]. The results showed that all antigenic sites are conserved among the CIVs, but differentiate among the avian H3N2 influenza viruses at positions 81 (D/E to N) and 160 (S/A/T to T) (Table 3).

Compared with H3N2 avian influenza viruses, the HA proteins of all CIVs lost the N-link glycosylation site at positions 2, 6, and 8, but gained an N-link glycosylation site due to the mutation of D(E) to N at position 81 (Table S1). We found two amino acid insertions, at positions 76 and 77, when comparing the deduced amino acid sequences of the NA protein of the isolates with those of H3N2 avian influenza viruses. Most CIVs, except the viruses isolated from Korea and the Guangdong province (not including Ca/GD/05/2011), keep this insertion. The effect of these two amino acid insertions is unclear and requires further research (Table 3). The NS1 protein is known to play a key role in the virulence of several influenza virus subtypes in different hosts [21, 22]. The substitution at position E92D of the NS1 protein has been shown to increase virulence in pigs [23].The NS1 protein of the isolates possessed D at position 92, which was the same as the other viruses in this study, suggesting that the isolates may be virulent. However, up to now, except Seo et al. report [25], there was no other experimental evidence to prove the speculation about virulence, so the further study was needed. Large-scale genome sequence analysis of AIVs has shown that the PDZ-binding motif (PBM) in NS1 consists of four C-terminal residues with the characteristic X-S/T-X-V motif and may represent a virulence determinant [21, 24]. The NS1 C-terminal PBM of avian influenza viruses is ESEV, suggesting that these viruses might have high virulence. However, the PBM of CIVs, including the viruses isolated in this study, are ESEI and the exact role of ESEI requires further study. The amino acid substitutions at positions E627K and D701N in the PB2 protein have been correlated to the increased virulence of H7N7 and H5N1 viruses in humans and mice, respectively, however, these substitutions were not found in the CIVs that were isolated in this study. Influenza A virus is an important pathogen that causes respiratory disease and can infect many species, including humans, pigs, horses, birds, and sea mammals. Aquatic birds are considered to be the reservoir of influenza A viruses. The interspecies transmission of avian influenza viruses has been a common, and sometimes serious event, throughout history. Recently, the low pathogenic H7N9 avian influenza viruses have crossed species barriers, infecting mammals and causing infection and death in humans [25, 26]. This has also led to serious public health issues. Previous research, as well as our results, has demonstrated that the low pathogenic H3N2 avian influenza viruses infect, not only dogs, but also cats [27, 28], causing serious respiratory disease. Dogs and cats are important companion animals and closely associate with humans in daily life. Thus, there is an increased risk of humans becoming infected with H3N2 avian influenza viruses. Therefore, it is important to monitor avian-originated

123

Virus Genes

Fig. 1 Phylogenetic analysis of the HA and NA genes of H3N2 CIVs and the reference strains obtained from Genbank. The tree was generated by the neighbor-joining method using MEGA4. The

123

reliability of the tree was assessed by bootstrap analysis with 1,000 replicates. Virus strains obtained in this study are labeled with a triangle

Virus Genes Table 3 Comparison of HA, NA, and NS1 amino acid sequences from avian-origin H3N2 influenza viruses Virus

HA Cleavage site

Receptor binding site

Antigenic sites

226

81 (E)

228

133 (A)

160 (B)

NA

NS1

Deletion

92

PBM

80–84

Insertion 74–79

Dk/GD/W12/2011

PEKQTRGL

Q

G

D

N

S

TIASV

D

ESEV

EK**EP

Dk/SH/C84/2009

PERQTRGL

Q

G

D

N

S

TIASV

D

ESEV

EK**GP

Dk/JS/26/2004 Ab/KR/KN-2/2005

PEKQTRGL PEKQTRGL

Q Q

G G

D E

N N

A T

TIASV TIASV

D D

ESEV –a

EK**EL EK**EI

Dk/KR/JS53/2004

PEKQTRGL

Q

G

D

N

A

TIASV

D



EK**EI

Dk/KR/LPM66/2006

PEKQTRGL

Q

G

D

N

A

TIASV

D



EK**EI

CIV-27

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

CIV-H6

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/GD/1/2006

PEKQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EK**EL

Ca/GD/2/2007

PEKQTRGL

Q

G

N

N

T

*****

D

KSEI

EK**EI

Ca/GD/1/2011

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EK**EI

Ca/GD/05/2011

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/JS/01/2009

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/JS/02/2010

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/BJ/295/2009

PERQTRGL

Q

G

N

S

T

TIASV

D

ESEI

EKEKEI

Ca/BJ/305/2009

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/BJ/511/2010

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Ca/ZJ/1/2010 Ca/KR/01/2007

PERQTRGL PERQTRGL

Q Q

G G

N N

N N

T T

TIASV TIASV

D D

ESEI ESEI

EKEKEI EK**EI

Ca/KR/GCVP01/2007

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EK**EI

Ca/KR/KRIBB01/2011

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EK**EI

Ca/TH/CU/2012

PERQTRGL

Q

G

N

N

T

TIASV

D

ESEI

EKEKEI

Dk/GD/W12/2011: A/duck/Guangdong/W12/2011; Dk/SH/C84/2009: A/duck/Shanghai/C84/2009; Dk/JS/26/2004: A/duck/Jiangsu/26/2004; Ab/Korea/KN-2/2005: A/aquatic bird/Korea/KN-2/2005; Dk/Korea/JS53/2004: A/duck/Korea/JS53/2004; Dk/Korea/LPM66/2006: A/duck/ Korea/LPM66/2006; Ca/GD/1/2006: A/canine/Guangdong/1/2006; Ca/GD/2/2007: A/canine/Guangdong/2/2007; Ca/GD/1/2011: A/canine/ Guangdong/1/2011; Ca/GD/05/2011: A/canine/Guangdong/05/2011; Ca/JS/01/2009: A/canine/Jiangsu/01/2009; Ca/JS/02/2010: A/canine/Jiangsu/02/2010; Ca/BJ/295/2009: A/canine/Beijing/295/2009; Ca/BJ/305/2009: A/canine/Beijing/305/2009; Ca/BJ/511/2010: A/canine/Beijing/ 511/2010; Ca/ZJ/1/2010: A/canine/Zhejiang/1/2010; Ca/Korea/01/2007: A/canine/Korea/01/2007; Ca/Korea/GCVP01/2007: A/canine/Korea/ GCVP01/2007; Ca/Korea/KRIBB01/2011: A/canine/Korea/KRIBB01/2011; Ca/Thailand/CU/2012: A/canine/Thailand/CU-DC5299/2012 a

Not detect

H3N2 influenza viruses in various animals, particularly animals that are in close contact with humans, such as pets.

2.

Acknowledgments This study was partly supported by the Heilongjiang Postdoctoral Financial Assistance (LBH-Z13043), the Chinese Special Fund for Agro-scientific Research in the Public Interest (201303046), the Open Fund of State Key Laboratory of Veterinary Biotechnology of Harbin veterinary research institute (SKLVBF201404), and the Basic Scientific Research Operation Cost of State-leveled Public Welfare Scientific Research Courtyard (0302014014).

5.

The authors have declared that they have no

6.

Conflict of interests competing interests.

References 1. P.C. Crawford, E.J. Dubovi, W.L. Castleman, I. Stephenson, E.P. Gibbs, L. Chen, C. Smith, R.C. Hill, P. Ferro, J. Pompey, R.A.

3.

4.

7.

8.

Bright, M.J. Medina, C.M. Johnson, C.W. Olsen, N.J. Cox, A.I. Klimov, J.M. Katz, R.O. Donis, Science 310, 482–485 (2005) D.G. Lin, S.S. Sun, L.J. Du, J.J. Ma, L.H. Fan, J. Pu, Y.P. Sun, J.Y. Zhao, H.L. Sun, J.H. Liu, J. Gen. Virol. 93, 119–123 (2012) T. Songserm, A. Amonsin, R. Jam-on, N. Sae-Heng, N. Pariyothorn, S. Payungporn, A. Theamboonlers, S. Chutinimitkul, R. Thanawongnuwech, Y. Poovorawan, Emerg. Infect. Dis. 12, 1744–1747 (2006) Q.Q. Song, F.X. Zhang, J.J. Liu, Z.S. Ling, Y.L. Zhu, S.J. Jiang, Z.J. Xie, Vet. Microbiol. 161, 331–333 (2013) G.J. Zhan, Z.S. Ling, Y.L. Zhu, S.J. Jiang, Z.J. Xie, Vet. Microbiol. 155, 409–416 (2012) X.X. Sun, X.K. Xu, Q. Liu, D.J. Liang, C.Y. Li, Q.S. He, J.X. Jiang, Y.M. Cui, J. Li, L.F. Zheng, J.G. Guo, Y. Xiong, J.H. Yan, Infect. Genet. Evol. 2, 471–475 (2013) D. Song, H.J. Moon, D.J. An, H.Y. Jeoung, H. Kim, M.J. Yeom, M. Hong, J.H. Nam, S.J. Park, B.K. Park, J.S. Oh, M. Song, R.G. Webster, J.K. Kim, B.K. Kang, J. Gen. Virol. 93, 551–554 (2012) S.J. Park, H.J. Moon, B.K. Kang, M. Hong, W. Na, J.K. Kim, H. Poo, B.K. Park, D.S. Song, J. Virol. 86, 9548–9549 (2012)

123

Virus Genes 9. H. Wang, K. Jia, W.B. Qi, M.Z. Zhang, L. Sun, H.B. Liang, G.H. Du, L.K. Tan, Z.W. Shao, J.H. Ye, L.S. Sun, Z.P. Cao, Y. Chen, P. Zhou, S. Su, S.J. Li, Virus Genes 46, 558–562 (2013) 10. Y.B. Zhang, J.D. Chen, J.X. Xie, W.J. Zhu, C.Y. Wei, L.K. Tan, N. Cao, Y. Chen, M.Z. Zhang, G.H. Zhang, S.J. Li, J. Vet. Med. Sci. 75, 1061–1062 (2013) 11. C. Lee, D. Song, B. Kang, D. Kang, J. Yoo, K. Jung, G. Na, K. Lee, B. Park, J. Oh, Vet. Microbiol. 137, 359–362 (2009) 12. N. Bunpapong, N. Nonthabenjawan, S. Chaiwong, R. Tangwangvivat, S. Boonyapisitsopa, W. Jairak, R. Tuanudom, D. Prakairungnamthip, S. Suradhat, R. Thanawongnuwech, A. Amonsin, Virus Genes 48, 56–63 (2014) 13. Y. Lin, Y. Zhao, X. Zeng, C. Lu, Y. Liu, Vet. Microbiol. 158, 247–258 (2012) 14. Y. Sun, S. Sun, J. Ma, Y. Tan, L. Du, Y. Shen, Q. Mu, J. Pu, D. Lin, J. Liu, Virology 435, 301–307 (2013) 15. S. Su, Z.G. Yuan, J.D. Chen, J.X. Xie, H.T. Li, Z. Huang, M.Z. Zhang, G.H. Du, Z.M. Chen, L.Q. Tu, Y.F. Zou, J.H. Miao, H. Wang, K. Jia, S.J. Li, Virus Genes 46, 554–557 (2013) 16. K. Tamura, J. Dudley, M. Nei, S. Kumar, Mol. Biol. Evol. 24, 1596–1599 (2007) 17. G. Lu, T. Rowley, R. Garten, R.O. Donis, Nucleic Acids Res. 35, 275–279 (2007) 18. M. Matrosovich, N. Zhou, Y. Kawaoka, R. Webster, J. Virol. 73, 1146–1155 (1999) 19. M. Muranaka, T. Yamanaka, Y. Katayama, K. Hidari, H. Kanazawa, T. Suzuki, K. Oku, T. Oyamada, J. Vet. Med. Sci. 73, 125–127 (2011)

123

20. N.K. Sauter, J.E. Hanson, G.D. Glick, J.H. Brown, R.L. Crowther, S.J. Park, J.J. Skehel, D.C. Wiley, Biochemistry 31, 9609–9621 (1992) 21. C.F. Basler, A.H. Reid, J.K. Dybing, T.A. Janczewski, T.G. Fanning, H. Zheng, M. Salvatore, M.L. Perdue, D.E. Swayne, A. Garcia-Sastre, P. Palese, J.K. Taubenberger, Proc. Natl. Acad. Sci. USA 98, 2746–2751 (2001) 22. J.C. Obenauer, J. Denson, P.K. Mehta, X. Su, S. Mukatira, D.B. Finkelstein, X. Xu, J. Wang, J. Ma, Y. Fan, K.M. Rakestraw, R.G. Webster, E. Hoffmann, S. Krauss, J. Zheng, Z. Zhang, C.W. Naeve, Science 311, 1576–1580 (2006) 23. S.H. Seo, E. Hoffmann, R.G. Webster, Virus Res. 103, 107–113 (2004) 24. D. Jackson, M.J. Hossain, D. Hickman, D.R. Perez, R.A. Lamb, Proc. Natl. Acad. Sci. USA 105, 4381–4386 (2008) 25. R. Gao, B. Cao, Y. Hu, Z. Feng, D. Wang, W. Hu, J. Chen, Z. Jie, H. Qiu, K. Xu, X. Xu, H. Lu, W. Zhu, Z. Gao, N. Xiang, Y. Shen, Z. He, Y. Gu, Z. Zhang, Y. Yang, X. Zhao, L. Zhou, X. Li, S. Zou, Y. Zhang, L. Yang, J. Guo, J. Dong, Q. Li, L. Dong, Y. Zhu, T. Bai, S. Wang, P. Hao, W. Yang, J. Han, H. Yu, D. Li, G.F. Gao, G. Wu, Y. Wang, Z. Yuan, Y. Shu, N. Engl. J. Med. 368, 1888–1897 (2013) 26. T.M. Uyeki, N.J. Cox, N. Engl. J. Med. 368, 1862–1864 (2013) 27. H. Kim, D. Song, H. Moon, M. Yeom, S. Park, M. Hong, W. Na, R.J. Webby, R.G. Webster, B. Park, J.K. Kim, B. Kang, Influenza Other Respi. Viruses 7, 265–270 (2012) 28. N. Lei, Z.G. Yuan, S.F. Huang, D.W. Zhang, A.G. Zhang, B.H. Huang, G.H. Zhang, S.J. Li, Vet. Microbiol. 160, 481–483 (2012)

Identification and genetic characterization of avian-origin H3N2 canine influenza viruses isolated from the Liaoning province of China in 2012.

A total of 158 serum samples and 510 nasal swab specimens were collected between September 2010 and May 2012, from dogs exhibiting respiratory symptom...
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