Ahead of Print -Novel Avian Influenza A(H7N9) Virus in Tree Sparrow, Shanghai, China, 2013 - Volume 20, Number 5—May 2014 - Emerging Infectious Disease journal - CDC
Volume 20, Number 5—May 2014
Dispatch
Novel Avian Influenza A(H7N9) Virus in Tree Sparrow, Shanghai, China, 2013
Baihui Zhao1, Xi Zhang1, Wenfei Zhu1, Zheng Teng, Xuelian Yu, Ye Gao, Di Wu, Enle Pei, Zhengan Yuan, Lei Yang, Dayan Wang, Yuelong Shu, and Fan Wu
Author affiliations: Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China (B. Zhao, X. Zhang, Z. Teng, X. Yu, Y. Gao, Z. Yuan, F. Wu); National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China (W. Zhu, L. Yang, D. Wang, Y. Shu);Shanghai Wildlife Conservation and Management Center, Shanghai (D. Wu, E. Pei)
Abstract
In spring 2013, influenza A(H7N9) virus was isolated from an apparently healthy tree sparrow in Chongming Dongping National Forest Park, Shanghai City, China. The entire gene constellation of the virus is similar to that of isolates from humans, highlighting the need to monitor influenza A(H7N9) viruses in different species.
Since its emergence in China in February 2013, avian influenza A(H7N9) virus has resulted in 217 human infections and 57 deaths (1). The biological features of the virus and its pandemic potential have caused global concern (2). Although the epidemic declined quickly after the closure of live poultry markets in China in April 2013, new cases in humans have reemerged since October 2013. The number of new cases has increased sharply since January 1, 2014, paralleling the peak of the first wave (1,3,4), indicating that subtype H7N9 viruses were circulating asymptomatically among natural hosts. Sequence data indicated that the hemagglutinin gene of this novel subtype H7N9 virus might originate from a subtype H7N3 virus in ducks and that the neuraminidase gene probably originated from a subtype H7N9 virus in wild birds (5) or ducks or chickens (6,7). These data suggest that wild birds might play a role in the emergence of subtype H7N9 viruses, similar to the role they played in the geographic spread of avian subtype H5N1 viruses (8). However, although avian influenza A(H7N9) viruses have been isolated from chickens and pigeons, to our knowledge, none have been isolated from wild birds. To better understand the role of wild birds in the emergence and potential dissemination of subtype H7N9 viruses, during spring of 2013, the Shanghai Municipal Center for Disease Control and Prevention, in collaboration with the Shanghai Wildlife Conservation and Management Center, investigated influenza A(H7N9) virus infection among wild birds in Shanghai.
The Study
Figure 1
Figure 1. . Location of tree sparrow from which novel avian influenza A(H7N9) virus was isolated: Chongming National Dongping Forest Park of Shanghai (yellow solid circle), which is located in the Australia–East Asia...
During April 10–May 15, a total of 2,198 fecal, tissue, cloacal swab, and tracheal swab samples were collected from wild birds in Shanghai. Trained staff captured healthy birds with an approved trapping method, collected samples, and released the birds. Tissue samples were collected from naturally dead wild birds. Information on bird species and sampling places are listed in Technical Appendix 
[PDF - 520 KB - 7 pages] Tables 1 and 2). RNA was extracted from each sample and tested by using influenza A universal real-time PCR according to the standard operating procedure of the World Health Organization (9). Influenza A virus–positive specimens were further subtyped by reverse transcription PCR with an avian influenza A virus subtype primer set reported previously (10). Of the 2,198 samples, 28 were positive for influenza A virus. One tracheal sample from an apparently healthy tree sparrow was positive for the novel subtype H7N9 virus, whereas the cloacal swab samples from this bird were negative. The positive sample was inoculated into 11-day-old specific pathogen free embryonated chicken eggs for virus isolation. The isolated virus was termed A/tree sparrow/Shanghai/01/2013 (H7N9). The tree sparrow had been collected from a forest on Chongming Dongping Forest Park, which is 47 km from Dongtan National Nature Reserve, a winter habitat for wild migratory birds (Figure 1).
[PDF - 520 KB - 7 pages] Tables 1 and 2). RNA was extracted from each sample and tested by using influenza A universal real-time PCR according to the standard operating procedure of the World Health Organization (9). Influenza A virus–positive specimens were further subtyped by reverse transcription PCR with an avian influenza A virus subtype primer set reported previously (10). Of the 2,198 samples, 28 were positive for influenza A virus. One tracheal sample from an apparently healthy tree sparrow was positive for the novel subtype H7N9 virus, whereas the cloacal swab samples from this bird were negative. The positive sample was inoculated into 11-day-old specific pathogen free embryonated chicken eggs for virus isolation. The isolated virus was termed A/tree sparrow/Shanghai/01/2013 (H7N9). The tree sparrow had been collected from a forest on Chongming Dongping Forest Park, which is 47 km from Dongtan National Nature Reserve, a winter habitat for wild migratory birds (Figure 1).To explore the genetic relationships between this sparrow-derived influenza A(H7N9) virus and other viruses from humans and poultry, we amplified total genomic segments by using viral RNA directly isolated from the original specimen with the primer sets listed in Technical Appendix [PDF - 520 KB - 7 pages] Table 3 and sequenced by Sunny Biotech Co., Ltd. (Shanghai, China). The Chinese National Influenza Center performed the sequencing by using RNA from chicken embryonated cultured viruses in an ABI 3730xl automatic DNA analyzer (Life Technologies, Foster City, CA, USA). Full-genome sequences from the original sample and the embryonated chicken eggs isolation were deposited in GenBank under accession nos. KF609524–KF609531 and KJ508887-KJ508894, respectively. To facilitate the phylogenetic analysis, we downloaded sequences of the novel subtype H7N9 viruses from 2013 and the avian subtype H7N9 viruses from before 2013 from the Global Initiative on Sharing Avian Influenza Data (http://platform.gisaid.org/epi3/frontend#46b284
). Sequence alignments were performed by using the MegAlign method of Lagergene 7.01 software (www.dnastar.com/t-megalign.aspx). Phylogenetic analysis was analyzed by using the neighbor-joining method in MEGA software version 5.10 (www.megasoftware.net).
[PDF - 520 KB - 7 pages] Table 3 and sequenced by Sunny Biotech Co., Ltd. (Shanghai, China). The Chinese National Influenza Center performed the sequencing by using RNA from chicken embryonated cultured viruses in an ABI 3730xl automatic DNA analyzer (Life Technologies, Foster City, CA, USA). Full-genome sequences from the original sample and the embryonated chicken eggs isolation were deposited in GenBank under accession nos. KF609524–KF609531 and KJ508887-KJ508894, respectively. To facilitate the phylogenetic analysis, we downloaded sequences of the novel subtype H7N9 viruses from 2013 and the avian subtype H7N9 viruses from before 2013 from the Global Initiative on Sharing Avian Influenza Data (http://platform.gisaid.org/epi3/frontend#46b284). Sequence alignments were performed by using the MegAlign method of Lagergene 7.01 software (www.dnastar.com/t-megalign.aspx). Phylogenetic analysis was analyzed by using the neighbor-joining method in MEGA software version 5.10 (www.megasoftware.net).
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