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Journal of Clinical Microbiology, August 2009, p. 2643-2646, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00262-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Emergence of European Avian Influenza Virus-Like H1N1 Swine Influenza A Viruses in China{triangledown}

Jinhua Liu,1,3,{dagger}* Yuhai Bi,1,{dagger} Kun Qin,2 Guanghua Fu,1 Jun Yang,1 Jinshan Peng,1 Guangpeng Ma,1 Qinfang Liu,1 Juan Pu,1 and Fulin Tian3

Laboratory of Infectious Diseases, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China,1 Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China,2 The Shandong Animal Disease Control Center, Jinan 250022, Shandong Province, People's Republic of China3

Received 6 February 2009/ Returned for modification 7 May 2009/ Accepted 13 June 2009


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ABSTRACT
 
During swine influenza surveillance from 2007 to 2008, 10 H1N1 viruses were isolated and analyzed for their antigenic and phylogenetic properties. Our study revealed the emergence of avian-origin European H1N1 swine influenza virus in China, which highlights the necessity of swine influenza surveillance for potential pandemic preparedness.


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TEXT
 
The pig possesses receptors for both {alpha}-sialic acid (2, 3) and {alpha}-Gal linkage (2, 6), which are well recognized by avian and human influenza viruses; this "mixing vessel" could possibly generate novel reassortant variants with pandemic potential in the human population (7). Cocirculation of nonswine origin and classical swine influenza viruses in the pig population emphasizes the role of the pig in the ecology of influenza viruses in this region. A regular swine influenza surveillance program was implemented in three provinces in China from 2007 to 2008.

A total of 1,344 nasal swab samples were collected from healthy pigs in Fujian (southeastern China), Shandong (eastern China), and Beijing (northern China) from October 2007 to September 2008. Swab samples were collected and put into viral transport medium containing antibiotics and kept at 4°C during transportation to the laboratory. Viral isolation and identification steps were performed, as described previously (3). Sequencing of the isolates was performed, as previously described (3).

Among the 14 influenza A virus isolates, 10 H1N1 and 4 H3N2 viruses were identified. The isolation rates of the H1N1 and H3N2 viruses were 0.74% and 0.30%, respectively. All the H3N2 viruses were obtained in the Shandong and Fujian provinces in 2007, and all the H1N1 isolates were found in the Fujian, Shandong, and Beijing provinces in 2007 and 2008. The results indicated that H1N1 subtype influenza viruses were more dominant in the pig population than the H3N2 subtype in China during this period.

A/Dk/HB/843/05 (H1N2) and A/Sw/GD/1/05 (H1N1) were previously documented and classified as Eurasian avian-origin influenza virus and classical swine influenza virus (Fig. 1), respectively. To analyze the antigenic properties of the H1N1 swine influenza isolates, a panel of the hyperimmune rabbit antisera against the contemporary H1N1 isolates A/Sw/FJ/204/07, A/Sw/BJ/21/08, A/Sw/GD/1/05, and A/Dk/HB/843/05 was raised in our laboratory. The hemagglutinin (HA) inhibition (HI) test results showed that the 10 H1N1 viruses isolated from 2007 to 2008 reacted well with the antisera against A/Sw/BJ/21/08 and A/Sw/FJ/204/07 (Table 1). These viruses had moderate reactivity to anti-A/Dk/HB/843/05 serum while having relatively lower or no reactivity to anti-A/Sw/GD/1/05 serum, which demonstrated that the antigenic analysis in HI testing was generally consistent with their phylogenetic relationships (Fig. 1).


Figure 1
Figure 1
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  		FIG. 1. Phylogenetic trees of the HA (positions 84 to 1052) (A), NA (positions 21 to 1429) (B), PB2 (positions 1303 to 2207) (C), PB1 (positions 1117 to 1813) (D), PA (positions 747 to 1243) (E), NP (positions 46 to 1542) (F), M (positions 26 to 1007) (G), and NS (positions 27 to 866) (H) genes of the H1N1 influenza A viruses. The trees were generated by the distance-based neighbor-joining method with MEGA 4.1 software. The reliability of the trees was assessed by bootstrap analysis with 1,000 replications, and only bootstrap values of ≥90% are shown. The HA tree was rooted to A/South Carolina/1/18, the NP, NA, M, NS, PB2, and PB1 trees were rooted to A/Brevig Mission/1/1918, and the PA tree was rooted to A/swine/Iowa/15/30. Horizontal distances are proportional to genetic distance. The isolated viruses are in italics, and the viruses in boldface were used to prepare polyclonal antisera in this study. Avian-like, avian influenza virus-like.


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  		TABLE 1. Antigenic characterization of H1N1 influenza viruses isolated from pigs

The eight gene segments of the 10 viruses were sequenced and phylogenetically characterized, and the homology of those H1N1 isolates in this study was 99.0 to 100%. A/Sw/FJ/204/07 was selected as a reference strain for analysis, which had high homology (96.3% to 98.7%) to the A/swine/Cotes d'Armor/1488/99 (H1N1) virus currently circulating in European swine populations (10). However, A/Sw/FJ/204/07 had lower homology with the classical swine virus A/swine/Shanghai/1/05 prevailing in China, with identity ranging from 75.3% to 87.4%.

Phylogenetic analysis of the HA genes revealed that H1N1 viruses can be divided into human, classical swine, Asian avian influenza virus-like swine, and avian-origin European swine influenza virus lineages. As shown in Fig. 1A, all of the H1N1 viruses isolated in this study fall into the avian-origin European swine influenza sublineage. Most of the H1N1 viruses isolated previously in China are grouped into the classical swine influenza lineage. Like HA genes, the other seven genes also showed the same evolutionary pattern (Fig. 1B to H). All eight gene segments of the 10 isolates obtained formed the same group, and no reassortment was observed. These findings strongly suggested that the H1N1 viruses in this present study were avian-origin European swine influenza viruses and may be prevailing in China.

Previous studies of the influenza viruses from pigs in China revealed that the classical H1N1 swine influenza viruses, cocirculating with H3N2 swine influenza viruses, were most prevalent in the swine population (2, 8). The Asian avian influenza virus-like H1N1 influenza viruses in pigs appeared in the 1990s and are cocirculating with the classical swine influenza virus in China (5). However, the Asian avian influenza virus-like viruses were not found in the swine populations in this region since the discovery. In contrast, the avian-origin European H1N1 swine influenza viruses were prevalent in European pig populations in the late 1970s (13) and appeared to have a selective advantage over classical H1N1 viruses (1). Since the human population has weak immunity to the European swine influenza viruses, which are antigenically and genetically distinct from the H1N1 human influenza viruses (4), human infection with swine influenza viruses was occasionally reported worldwide (4, 11, 12). It is reasonable to postulate that current seasonal H1N1 human influenza vaccine will confer little or no protection against avian-origin European swine influenza virus-like viruses. Thus, the emergence of avian-origin European H1N1 swine influenza virus in China will inevitably pose a great threat not only to the pig industry but also to public health.

In addition to the well-adapted H1 and H3 viruses in the pig population in China, H5 and H9 avian influenza viruses and H1 and H3 human influenza viruses have been detected in the swine population (3, 9, 14). These scenarios increased the possibility that novel reassortant variants with pandemic potential could be generated from the swine in this region. Therefore, an intensified surveillance program for swine influenza should be implemented for a larger-scale geographic region in China for human pandemic preparedness.

Nucleotide sequence accession numbers. The nucleotide sequences obtained in this study have been deposited in the GenBank database (accession numbers FJ536762 to FJ536843).


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ACKNOWLEDGMENTS
 
This work was supported by the National Natural Scientific Foundation (grant 30599431), the National Key Technologies R&D Program (grant 2006BAD06A01), the National Basic Research Program (973) (grant 2005CB523003), the 863 program (grant 2006AA10A205), and European Union AIV project FLUINNATE (grant SP5B-CT-2006-044161). J.L. was funded by the Taishan Scholar Foundation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Infectious Disease, College of Veterinary Medicine, China Agricultural University, Beijing 100193, People's Republic of China. Phone and fax: 0086-10-62733837. E-mail: ljh{at}cau.edu.cn Back

{triangledown} Published ahead of print on 24 June 2009. Back

{dagger} These authors contributed equally to this work. Back


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Journal of Clinical Microbiology, August 2009, p. 2643-2646, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00262-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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