Microbes and Infection 17 (2015) 48e53 www.elsevier.com/locate/micinf
Original article
Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City, China Xiansheng Ni a,1, Fenglan He a,*,1, Maohong Hu a,1, Xianfeng Zhou a, Bin Wang a, Changhua Feng a, Yumei Wu b, Youxing Li c, Junling Tu a, Hui Li a, Mingbin Liu a, Haiying Chen a, Shengen Chen a,* a
b
Nanchang Center for Disease Control and Prevention, Nanchang 330038, China Qingshanhu District Center for Disease Control and Prevention, Nanchang 330038, China c Xinjian County Center for Disease Control and Prevention, Nanchang 330038, China Received 23 June 2014; accepted 19 September 2014 Available online 2 October 2014
Abstract Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Avian influenza virus; H10N8; H7N9; Avian flu
1. Introduction The outbreak of the H7N9 influenza virus in human in China has raised justifiable global concern in the spring of 2013 that a new influenza pandemic could occur [1].At the end of 2013, another novel avian influenza A(H10N8) virus has also been reported to cause human death but no apparent outbreaks in poultry has emerged in Nanchang City, China
* Corresponding authors. No 833, Lijing Road, Honggutan District, Nanchang City, Jiangxi Province 330038, China. Tel./fax: þ86 791 86363255. E-mail addresses: [email protected] (F. He), [email protected] (S. Chen). 1 These authors contributed equally to this work.
[2].To our knowledge, it's the first time that H10N8 virus has infected people and caused fatal case. Recently, the highly dangerous bird flu has caused 3 human cases of infection in Nanchang, resulting in 2 fatalities according to the data of the laboratory-based influenza virological surveillance system and the surveillance system for pneumonia of unknown etiology [3]. Although the novel H10N8 influenza virus has caused severe illness and death among individuals, thus far there has been no evidence of sustained person-to-person transmission [4]. The sources of H10N8 human infections remain elusive. Field investigations show that all confirmed cases infected with avian influenza virus had a history of exposure to poultry before illness onset from Nanchang [2,5]. And H10N8 virus has been isolated from live poultry market (LPM) where the
http://dx.doi.org/10.1016/j.micinf.2014.09.007 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
first H10N8 human infection case visited [6]. H10N8 virus might be carried by poultry, in their secretions or excretions. Transmission can occur through direct or close contact with poultry or through exposure to environments that are contaminated with poultry. It is at present not known what extent the activity of influenza virus in poultry contributes to human infection in Nanchang and what and how the underlying risk factors are involved in the cross-species transmission. Lack of such knowledge has made it difficult to refine prevention. Influenza A virus belongs to the family Orthomyxoviridae. Based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, influenza A viruses are categorized into different subtypes. Currently, 18 HA subtypes and 11 NA subtypes have been identified. All subtypes were identified initially from avian species, except for H17N10 and H18N11 subtype found in bats [7,8]. Among these subtypes, high pathogenic avian influenza (HPAI) is characterized by systemic infections, high mortality and morbidity. Unlike the HPAI viruses such as H5N1, in which outbreaks in poultry precede human infections and imply where the public health threat lies, the novel H10N8 virus seems to be asymptomatic or cause mild disease in poultry and wild bird populations as well as H7N9 [1,2,9]. This means that the virus is likely to spread silently in birds or other animal reservoirs. Some low pathogenic avian influenza (LPAI) viruses, such as H9N2, can break species barriers and provide genes to other influenza virus, which could present a risk for severe human infection [10]. Notably, H9N2 virus provides the internal genes not only for the H10N8 virus, but also for H7N9 and H5N1 viruses [6]. In view of all reported human infections with avian influenza A virus caused by H5, H7, H9 and H10 subtypes, these subtypes have infected more than 1 human case and pose great threats to public health [11]. The epidemiological data of these subtypes will be useful to plan strategies for preventing human infections. Most human infections with H7N9 virus were concentrated at the Yangtze River delta on China's eastern seaboard (Fig. 1B), sporadic cases were distributed in large areas of adjacent provinces [12]. Nanchang City, capital of Jiangxi province (28 410 N, 115 530 E, Southeastern China), where Poyang Lake was located, very close to the Yangtze River
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Delta and Dongting Lake (Fig. 1C), is the only city in which two novel avian-origin influenza viruses appeared in mainland China. Migratory birds stop over each year for watering and breeding in Poyang Lake and Dongting Lake, where the subtypes of influenza viruses were prosperous [13,14]. For a long time, southern China has been considered a hypothetical epicenter for facilitating the emergence of pandemic influenza viruses in the world [15]. In recent years, several subtypes of avain influenza virus (AIV) have been circulating and evolving in southern China [16e19]. Thus, epidemiological surveillance in poultry and wild birds is of vital importance for investigating the evolution of known and unknown AIVs. In December 2013, a study was initiated to survey AIVs in Nanchang City. The objectives of this study therefore are to conducted epidemiological surveillance of H5, H7, H9, H10 and N8 subtypes in poultry and wild birds of Nanchang region during the emergence of human infection with H10N8 virus. The results from this surveillance may be helpful in influencing worldwide surveillance of AIVs, given that this region is on the route of bird immigration. 2. Materials and methods 2.1. Ethics statement This study was approved by the institutional review board of Nanchang Center for Disease Control and Prevention (CDC), which prepared protocol for conducting sample collection. The institute did not issue a number or ID to this animal study, because the studied poultry were not an endangered or protected species and birds were not sacrificed for sampling. Briefly, with verbal permission from the poultry owners, biological samples were gently collected from cloacal sample, faecal sample, contaminated environmental and water of healthy chickens, ducks, pigeons, gooses, and wild birds without being anesthetized before sampling. 2.2. Field work The investigation in poultry and wild birds was conducted in 28 randomly selected LBMs, 18 local poultry yards, over 6 wild-bird habitats and delivery vehicles in Nanchang during
Fig. 1. Geographic location of Nanchang in China (A) and Yangtze River delta (represented in green round plot) on China's eastern seaboard (B) and Poyang Lake and Dongting Lake (C).
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X. Ni et al. / Microbes and Infection 17 (2015) 48e53
the period from December 2013 to February 2014. Oropharyngeal, cloacal, faecal, contaminated environmental samples were collected from poultry using cotton swabs. The cotton swabs were then suspended in 3 ml storage medium that was transported in 10 ml finger tubes on ice. According to the protocol of the world organization for animal health (OIE), the storage medium contained Penicillin (10,000 unit/ml), Streptomycin (10 mg/ml), Gentamycin (10,000 unit/ml), Kanamycin (10,000 unit/ml) and 5% fetal calf serum in sterile PBS (pH ¼ 7.2). Water samples didn't do any preliminary processing, directly into 15/50 ml sterile centrifuge tube on ice. Samples collected were sealed, packed and transported immediately to the laboratory while maintaining the cold chain and stored at 80 C until testing in accordance with national biosafety regulations. 2.3. Viral analysis The collected samples were centrifuged at 3000 rpm for 10 min at 4 C, while still in the storage medium. Viral RNA was extracted from biological samples with QIAamp Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Real-time RT-PCR were used for influenza typing and subtyping. The samples were identified as containing influenza A on the basis of the M gene, but could not be subtyped. Specific real-time RT-PCR assays for avian influenza H5, H9, H7, (Liveriver, shanghai, China) and H10 subtypes were done to verify the viral subtypes from nucleic acids positive to influenza A virus in M gene. Original biological samples obtained from poultry were further performed with self-designed specific primer and probe sets for detecting H5, H9, H7 and H10 by the national institute for viral disease control and prevention, China CDC, by means of real-time RTPCR, to be positive for influenza A virus. 2.4. Statistical analysis We initially entered our data into Excel and then transferred it to SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) version 17.0 for statistical analysis. Differences between proportions were analyzed using the X2 test. A difference was considered statistically significant when p < 0.05.
Fig. 2. Poultry and wild birds density map of AIVs during the emergence of H10N8 in Nanchang.
positive samples, 242 (97.98%) were from LPMs that were experiencing the occurrence of H10N8. LPM had primary responsibility for positive rate of AIV. Whereas, only 1/129 (0.78%) viruses were detected in poultry countyards. Moreover, none of the positive sample was detected in wild-bird habitats. Interestingly, four samples were detected positive from 25 in the transport links (Fig. 3). 3.2. The distribution of AIVs and HA subtypes identified in different poultry species All samples for AIVs detection were comprised of 29 oropharyngeal, 443 cloacal, 131 faecal, 110 contaminated environmental samples and 214 drinking or slaughter water, which were collected from more than ten kinds of poutry. A small portion of biological samples were paired samples. As processing AIVs testing, 161 (26.05%) were positive in healthy chickens, and 110 (30.81%) in ducks. Positive rate between goose and pigeon was very similar. By contrast, all wild birds were tested negative for AIVs. There was a significantly higher positive rate of AIVs in drinking or
3. Results 3.1. Positive rate of AIVs in different sites from four county and five districts of Nanchang During the period from December 2013 to February 2014, a total of 1036 samples were collected from LPMs, poultry countyards, delivery vehicles, and wild-bird habitats at four county and five districts of Nanchang. 23.84% of 1036 samples tested positive for AIVs. The majority (74.49%) of positive samples occurred in the metropolitan area, Donghu, Xihu, Qingshanhu, Qingyunpu and Wanli district. Two suburban county, Xinjian and Anyi county, showed a higher positive rate compared to other two county (Fig. 2). Of the 247
Fig. 3. Positive rate of AIVs in different sites during the emergence of H10N8 in Nanchang (LPM: live poultry market; PC: poultry countyard; DV: delivery vehicle; WBH: wild-bird habitat).
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
slaughter water from pigeons (51.85%) ( p < 0.05), and oropharyngeal samples from chickens (61.54%) ( p < 0.05) (Table 1). In our analysis, H5, H7, H9, H10 and other A untypable were all detected in chickens, ducks, gooses, pigeons. The H5 subtype was dominant in ducks and the H9 subtype in chickens. Of the 247 positive samples, 21 were tested positive for untypable HA type (Fig. 4A). The distribution of different HA subtypes in chickens and ducks was not meaningful different ( p < 0.05). 3.3. Mixed infection of different HA subtypes Out of the 247 samples positive for AIVs, almost one sixth poultry samples showed mixed infections of different HA subtypes of AIVs. As shown in Fig. 4B, mixed infection of H5 and H9 subtypes was the most commonly mixed infection, followed by H5, H9 and H10 subtypes. Among these concomitant infections, 33 were mixes of two different HA subtypes, 8 were mixes of three different HA subtypes. Additional, concurrent infection was more frequent in ducks and chickens than other terrestrial poultry. 3.4. Coexistence of different HA subtypes with N8 In the present study, 54 samples were tested positive for N8 type. H10 subtypes coexistence with N8, the most prevalent agent, was present in 38.89% (21/54) samples, H5 and N8 in 9.26% (5/54)samples, H7 and N8 in 1.85% (1/54)samples, H9 and N8 in 12.96% (7/54) samples, and untypable HA and N8 in 33.88% (18/54) sample (Fig. 5). 4. Discussion The two novel influenza viruses in human, H7N9 and H10N8, had similar genetic lineage: the hemagglutinin (H) gene originated from ducks and the neuraminidase (N) gene from wild birds [2,20]. H10N8 virus causing human infections without preceding outbreak in poultry was quite unexpected. The apparently low pathogenicity in poultry could help the H10N8 virus to circulate in poultry without detection. Another two human cases of infection with H10N8 were reported in Nanchang City in early 2014. The widespread detection of AIV from poultry and wild birds due to the emergence of A(H10N8) virus in Nanchang city in a relatively short time period indicated that several subtypes of AIVs transmit
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efficiently among local poultry, in particular within domestic ducks, which further supported a report proposing the major role of domestic ducks as mixing vessels of influenza viruses [21].Our results were consistent with a study, which reported that there was 23 different HA-NA subtype combinations detected mainly in domestic ducks in eastern China [22]. The association between occurrence of human cases and proximity of irrigated lands indicates that ducks may play a role in the transmission of H7N9, as they do for other AIVs [21,23]. On the other hand, a similar situation also prevailed in chickens in our study. Chickens might serve as the intermediate host and thus might be the source of transmission of the influenza virus to humans [24]. Therefore, as more was learned about the role of the chicken in the evolution and ecology of the influenza virus. LPMs were known to be a high-risk environment for pathogen transmission between birds and for zoonotic transfer to people [25]. The warm, humid climate in this region might prompt long-term survival and proliferation of the virus. Our results ruled out that there was a higher prevalence of AIVs in the poultry at LPMs than other sites. And AIVs infected chicken, duck, goose and pigeon with asymptomatic or mild disease in this area. If birds spent a sufficient amount of time in market to become infected and transmitted the virus to other susceptible birds, sustained virus circulation in LPMs could occur [26]. In addition, live poultry trade created networks of close contacts between poultry and human populations, thereby facilitating transmission of AIVs. Apart from birds and the wet environments at the LPMs, kinds of vehicles associated with poultry transport were another risk factor, including car, motorbike and so on, as investigated in the study. The same kind of vehicles sustained carrying different poultry, such environments contaminated by virus might not only increase transmission of AIVs in birds, but also likely increase the number of human infections. Geographically, although the viruses were detected in different regions of Nanchang, most positive samples occurred in the metropolitan area. In modern times, geographical distribution of three influenza (H10N8) confirmed cases didn't give us any clue to their relationships. We inferred that the regional trade played a key role transmission of AIVs in domestic birds in Nanchang. Of note, various subtypes of AIVs were circulating among poultry in Nanchang: H5 (n ¼ 98), H7 (n ¼ 6), H9 (n ¼ 92), H10 (n ¼ 56), and untypable HA (n ¼ 21). We discovered that mixed infection of different HA subtypes was very common in
Table 1 Positive detection rate of AIVs in different poultry species during the emergence of H10N8 in Nanchang. Type
O E C F W Total
Chicken
Duck
Goose
Pigeon
Wild bird
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
13 85 279 128 107 618
61.54 28.24 20.784 21.094 40.19 26.05
10 49 102 94 86 357
30 34.69 35.29 21.27 36.05 30.81
0 2 3 2 3 16
0 50 0 50 33.33 18.75
6 42 59 53 27 187
0 30.95 13.56 7.55 51.85 20.86
0 0 2 32 49 83
0 0 0 0 0 0
Sample type: O (oropharyngeal), E (contaminated environmental samples), C (cloacal), F (faecal) and W (drinking or washing water).
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X. Ni et al. / Microbes and Infection 17 (2015) 48e53
Fig. 4. HA subtypes distribution of positive samples (A) and mixed infection (B) in different poultry species during the emergence of H10N8 in Nanchang (The ratio of positive samples or mixed infection is the total number of H5, H7, H9 and H10 subtypes in different poultry species).
ducks in Nanchang, which postulated that ducks were the main virus reservoir and promote mixed infections of different HA subtypes of AIVs [27].Concurrent infection of viruses in birds or other animals provided the possibility for the emergence of a new reassortant virus [28e30]. The reassortant among different influenza viruses was considered as the main mechanism for the emergence of novel virus, which could lead to the influenza pandemic, as preliminarily confirmed by H7N9 and H10N8 viruses [6]. A(H10N8) was the first influenza virus affecting humans that carry N8. What we knew about H10N8 so far was that only two H10N8 viruses had been reported in China: one was from a water sample taken from Dongting Lake in Hunan province in 2007 [31], and the other from LPM in Guangdong province in 2012 [19]. As H10N8 virus possessed internal gene cassettes recruited from poultry, we sought to find some correlations between the emerging H10N8 virus and N8 in local poultry. 21 samples with the N8 and H10 coexistence, were the leading agent among all mixed infection. In recent years, continued AIV surveillance in poultry was carried out in Nanchang, as one of the important control measures for
fighting against avian influenza, but few H5 subtypes were detected. Furthermore, 6 H5N1 and 4 H7N9 of 61 poultry samples were positive during the outbreak of H7N9 in 2013. No H10 subtype were tested for positive according to our AIV surveillance datas before 2013 in Nanchang. Our results could serve as a useful resource for further functional experiments of the sources of H10N8 human infections. Poyang Lake was located in the East Asian-Australian migratory birds' flyway and it was the stopover and winter site for migratory birds [32]. Therefore, investigation of water for AIVs was of greater significance and convenience for understanding the route and mechanism of virus transmission between domestic fowl and migratory birds in this lake area. We collected tens of drinking water and faecal sample about wild bird categorized into Xinjian County, which adjoined Poyang Lake. Unfortunately, none of the positive sample was detected in this area. A novel genotype influenza virus was isolated from drinking Water near Poyang Lake in 2011 [33], which highlighted the importance of strengthening the surveillance of avian influenza in this region. Taken together, our results could be used as a reference for targeted surveillance and control efforts in both human and animal populations to reduce the risk of future human infections. Since we were currently outside the northern hemisphere influenza season, any upsurge in cases detected by routine surveillance should be a cause for concern. Thus, a more rigorous and long-term surveillance remained essential for early warning of novel reassortant viruses and interspecies transmission events. Conflict of interest None of the authors has any conflict of interest. Acknowledgments
Fig. 5. Distribution of coexistence of different HA subtypes with N8 during the emergence of H10N8 in Nanchang.
We acknowledge the contributions of Donghu, Xihu, Qingyunpu and Wanli district CDC andNanchang, Jinxian and Anyi county CDC for the field work and sample collection.
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
The contents of this manuscript are solely the responsibility of the authors and not necessarily represent the official views of Nanchang Centers for Disease Control and Prevention. This work was supported by National Natural Science Foundation of China (No. 81460302) and Major Science and Technological Project of Jiangxi Province (No. 20143ACG70004). References [1] Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 2013;368:1888e97. [2] Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, et al. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet 2014;383:714e21. [3] http://www.jxwst.gov.cn/gzdt/201402/t20140213_308109.htm. [4] Parry J. H10N8 avian flu virus claims its first known human casualty. BMJ 2014;348:g1360. [5] Zhou X, Li H, Ni X, Liu M, Hu M, Wu J, et al. Laboratory diagnosis and epidemiology of avian influenza A (H7N9) virus infection in humans in Nanchang City, China. Jpn J Infect Dis 2013;66:558e60. [6] Shi W, Li W, Li X, Haywood J, Ma J, Gao GF, et al. Phylogenetics of varied subtypes of avian influenza viruses in China: potential threat to humans. Protein Cell 2014;5:253e7. [7] Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS Pathog 2013;9:e1003657. [8] Schrauwen EJA, Fouchier RAM. Host adaptation and transmission of influenza A viruses in mammals. Emerg Microbes Infect 2014;3:e9. [9] Pei ris JS, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human health. Clin Microbiol 2007;20:243e67. [10] The SJCEIRS H9 Working Group. Assessing the fitness of distinct clades of influenza A (H9N2) viruses. Emerg Microbes Infect 2013;2:e75. [11] To KK, Tsang AK, Chan JF, Cheng VC, Chen H, Yuen KY. Emergence in China of human disease due to avian influenza A (H10N8)—cause for concern? J Infect 2014;68:205e15. [12] Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of the avian influenza A (H7N9) outbreak in China. N Engl J Med 2014;370:520e32. [13] Shi J, Gao L, Zhu Y, Chen T, Liu Y, Dong L, et al. Investigation of avian influenza infections in wild birds, poultry and humans in Eastern Dongting Lake, China. PLoS One 2014;9:e95685. [14] Duan L, Zhu H, Wang J, Huang K, Cheung CL, Peiris JS, et al. Influenza virus surveillance in migratory ducks and sentinel ducks at Poyang Lake, China. Influenza Other Respir Viruses 2011;5:65e8. [15] Shortridge KF, Stuart-Harris CH. An influenza epicentre? Lancet 1982;2:812e3. [16] Webster RG, Guan Y, Peiris M, Walker D, Krauss S, Zhou NN, et al. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J Virol 2002;76:118e26. [17] Choi YK, Ozaki H, Webby RJ, Webster RG, Peiris JS, Poon L, et al. Continuing evolution of H9N2 influenza viruses in Southeastern China. J Virol 2004;78:8609e14.
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[18] Huang K, Bahl J, Fan XH, Vijaykrishna D, Cheung CL, Webby RJ, et al. Establishment of an H6N2 influenza virus lineage in domestic ducks in Southern China. J Virol 2010;84:6978e86. [19] Jiao P, Cao L, Yuan R, Wei L, Song Y, Shen D, et al. Complete genome sequence of an H10N8 avian influenza virus isolated from a live bird market in Southern China. J Virol 2012;86:7716. [20] Lam TT, Wang J, Shen Y, Zhou B, Duan L, Cheung CL, et al. The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature 2013;502:241e4. [21] Kim JK, Negovetich NJ, Forrest HL, Webster RG. Ducks: the “Trojan horses” of H5N1 influenza. Influenza Other Respir Viruses 2009;3:121e8. [22] Zhao G, Pan J, Gu X, Zhao K, Chen H, Chen S, et al. Prevalence of low pathogenic avian influenza virus in one live bird market in Eastern China from 2002-2011. Avian Dis 2013;57:155e8. [23] Zhao G, Lu X, Gu X, Zhao K, Song Q, Pan J, et al. Molecular evolution of the H6 subtype influenza A viruses from poultry in Eastern China from 2002 to 2010. Virol J 2011;8:470. [24] Guo CT, Takahashi N, Yagi H, Kato K, Takahashi T, Yi SQ, et al. The quail and chicken intestine have sialyl-galactose sugar chains responsible for the binding of influenza A viruses to human type receptors. Glycobiology 2007;17:713e24. [25] Chen Y, Liang W, Yang S, Wu N, Gao H, Sheng J, et al. Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 2013;381:1916e25. [26] Fournie G, Guitian FJ, Mangtani P, Ghani AC. Impact of the implementation of rest days in live bird markets on the dynamics of H5N1 highly pathogenic avian influenza. J R Soc Interface 2011;8:1079e89. [27] Hulse-Post DJ, Sturm-Ramirez KM, Humberd J, Seiler P, Govorkova EA, Krauss S, et al. Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia. Proc Natl Acad Sci U S A 2005;102:10682e7. [28] Sharp GB, Kawaoka Y, Jones DJ, Bean WJ, Pryor SP, Hinshaw V, et al. Coinfection of wild ducks by influenza A viruses: distribution patterns and biological significance. J Virol 1997;71:6128e35. [29] Yu X, Jin T, Cui Y, Pu X, Li J, Xu J, et al. Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics. J Virol 2014;88:3423e31. [30] Liu Z, Xu B, Chen Q, Chen Z. Complete genome sequence of a mixedsubtype (H5N1 and H6N6) avian influenza virus isolated from a duck in Hunan Province, China. Genome Announc 2014;2:e00310e4. [31] Zhang H, Xu B, Chen Q, Chen J, Chen Z. Characterization of an H10N8 influenza virus isolated from Dongting Lake wetland. Virol J 2011;8:42. [32] Ji W, Zeng N, Wang Y, Gong P, Xu B, Bao S. Analysis on the waterbirds community survey of Poyang Lake in winter. Ann GIS 2007;13:51e64. [33] Dong BB, Xu CL, Dong LB, Cheng HJ, Yang L, Zou SM, et al. A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic duck farm in Poyang Lake area. Biomed Environ Sci 2013;26:546e51.
Original article
Investigation of avian influenza virus in poultry and wild birds due to novel avian-origin influenza A(H10N8) in Nanchang City, China Xiansheng Ni a,1, Fenglan He a,*,1, Maohong Hu a,1, Xianfeng Zhou a, Bin Wang a, Changhua Feng a, Yumei Wu b, Youxing Li c, Junling Tu a, Hui Li a, Mingbin Liu a, Haiying Chen a, Shengen Chen a,* a
b
Nanchang Center for Disease Control and Prevention, Nanchang 330038, China Qingshanhu District Center for Disease Control and Prevention, Nanchang 330038, China c Xinjian County Center for Disease Control and Prevention, Nanchang 330038, China Received 23 June 2014; accepted 19 September 2014 Available online 2 October 2014
Abstract Multiple reassortment events within poultry and wild birds had resulted in the establishment of another novel avian influenza A(H10N8) virus, and finally resulted in human death in Nanchang, China. However, there was a paucity of information on the prevalence of avian influenza virus in poultry and wild birds in Nanchang area. We investigated avian influenza virus in poultry and wild birds from live poultry markets, poultry countyards, delivery vehicles, and wild-bird habitats in Nanchang. We analyzed 1036 samples from wild birds and domestic poultry collected from December 2013 to February 2014. Original biological samples were tested for the presence of avian influenza virus using specific primer and probe sets of H5, H7, H9, H10 and N8 subtypes by real-time RT-PCR. In our analysis, the majority (97.98%) of positive samples were from live poultry markets. Among the poultry samples from chickens and ducks, AIV prevalence was 26.05 and 30.81%, respectively. Mixed infection of different HA subtypes was very common. Additionally, H10 subtypes coexistence with N8 was the most prevalent agent during the emergence of H10N8. This event illustrated a long-term surveillance was so helpful for pandemic preparedness and response. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Avian influenza virus; H10N8; H7N9; Avian flu
1. Introduction The outbreak of the H7N9 influenza virus in human in China has raised justifiable global concern in the spring of 2013 that a new influenza pandemic could occur [1].At the end of 2013, another novel avian influenza A(H10N8) virus has also been reported to cause human death but no apparent outbreaks in poultry has emerged in Nanchang City, China
* Corresponding authors. No 833, Lijing Road, Honggutan District, Nanchang City, Jiangxi Province 330038, China. Tel./fax: þ86 791 86363255. E-mail addresses: [email protected] (F. He), [email protected] (S. Chen). 1 These authors contributed equally to this work.
[2].To our knowledge, it's the first time that H10N8 virus has infected people and caused fatal case. Recently, the highly dangerous bird flu has caused 3 human cases of infection in Nanchang, resulting in 2 fatalities according to the data of the laboratory-based influenza virological surveillance system and the surveillance system for pneumonia of unknown etiology [3]. Although the novel H10N8 influenza virus has caused severe illness and death among individuals, thus far there has been no evidence of sustained person-to-person transmission [4]. The sources of H10N8 human infections remain elusive. Field investigations show that all confirmed cases infected with avian influenza virus had a history of exposure to poultry before illness onset from Nanchang [2,5]. And H10N8 virus has been isolated from live poultry market (LPM) where the
http://dx.doi.org/10.1016/j.micinf.2014.09.007 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
first H10N8 human infection case visited [6]. H10N8 virus might be carried by poultry, in their secretions or excretions. Transmission can occur through direct or close contact with poultry or through exposure to environments that are contaminated with poultry. It is at present not known what extent the activity of influenza virus in poultry contributes to human infection in Nanchang and what and how the underlying risk factors are involved in the cross-species transmission. Lack of such knowledge has made it difficult to refine prevention. Influenza A virus belongs to the family Orthomyxoviridae. Based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, influenza A viruses are categorized into different subtypes. Currently, 18 HA subtypes and 11 NA subtypes have been identified. All subtypes were identified initially from avian species, except for H17N10 and H18N11 subtype found in bats [7,8]. Among these subtypes, high pathogenic avian influenza (HPAI) is characterized by systemic infections, high mortality and morbidity. Unlike the HPAI viruses such as H5N1, in which outbreaks in poultry precede human infections and imply where the public health threat lies, the novel H10N8 virus seems to be asymptomatic or cause mild disease in poultry and wild bird populations as well as H7N9 [1,2,9]. This means that the virus is likely to spread silently in birds or other animal reservoirs. Some low pathogenic avian influenza (LPAI) viruses, such as H9N2, can break species barriers and provide genes to other influenza virus, which could present a risk for severe human infection [10]. Notably, H9N2 virus provides the internal genes not only for the H10N8 virus, but also for H7N9 and H5N1 viruses [6]. In view of all reported human infections with avian influenza A virus caused by H5, H7, H9 and H10 subtypes, these subtypes have infected more than 1 human case and pose great threats to public health [11]. The epidemiological data of these subtypes will be useful to plan strategies for preventing human infections. Most human infections with H7N9 virus were concentrated at the Yangtze River delta on China's eastern seaboard (Fig. 1B), sporadic cases were distributed in large areas of adjacent provinces [12]. Nanchang City, capital of Jiangxi province (28 410 N, 115 530 E, Southeastern China), where Poyang Lake was located, very close to the Yangtze River
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Delta and Dongting Lake (Fig. 1C), is the only city in which two novel avian-origin influenza viruses appeared in mainland China. Migratory birds stop over each year for watering and breeding in Poyang Lake and Dongting Lake, where the subtypes of influenza viruses were prosperous [13,14]. For a long time, southern China has been considered a hypothetical epicenter for facilitating the emergence of pandemic influenza viruses in the world [15]. In recent years, several subtypes of avain influenza virus (AIV) have been circulating and evolving in southern China [16e19]. Thus, epidemiological surveillance in poultry and wild birds is of vital importance for investigating the evolution of known and unknown AIVs. In December 2013, a study was initiated to survey AIVs in Nanchang City. The objectives of this study therefore are to conducted epidemiological surveillance of H5, H7, H9, H10 and N8 subtypes in poultry and wild birds of Nanchang region during the emergence of human infection with H10N8 virus. The results from this surveillance may be helpful in influencing worldwide surveillance of AIVs, given that this region is on the route of bird immigration. 2. Materials and methods 2.1. Ethics statement This study was approved by the institutional review board of Nanchang Center for Disease Control and Prevention (CDC), which prepared protocol for conducting sample collection. The institute did not issue a number or ID to this animal study, because the studied poultry were not an endangered or protected species and birds were not sacrificed for sampling. Briefly, with verbal permission from the poultry owners, biological samples were gently collected from cloacal sample, faecal sample, contaminated environmental and water of healthy chickens, ducks, pigeons, gooses, and wild birds without being anesthetized before sampling. 2.2. Field work The investigation in poultry and wild birds was conducted in 28 randomly selected LBMs, 18 local poultry yards, over 6 wild-bird habitats and delivery vehicles in Nanchang during
Fig. 1. Geographic location of Nanchang in China (A) and Yangtze River delta (represented in green round plot) on China's eastern seaboard (B) and Poyang Lake and Dongting Lake (C).
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X. Ni et al. / Microbes and Infection 17 (2015) 48e53
the period from December 2013 to February 2014. Oropharyngeal, cloacal, faecal, contaminated environmental samples were collected from poultry using cotton swabs. The cotton swabs were then suspended in 3 ml storage medium that was transported in 10 ml finger tubes on ice. According to the protocol of the world organization for animal health (OIE), the storage medium contained Penicillin (10,000 unit/ml), Streptomycin (10 mg/ml), Gentamycin (10,000 unit/ml), Kanamycin (10,000 unit/ml) and 5% fetal calf serum in sterile PBS (pH ¼ 7.2). Water samples didn't do any preliminary processing, directly into 15/50 ml sterile centrifuge tube on ice. Samples collected were sealed, packed and transported immediately to the laboratory while maintaining the cold chain and stored at 80 C until testing in accordance with national biosafety regulations. 2.3. Viral analysis The collected samples were centrifuged at 3000 rpm for 10 min at 4 C, while still in the storage medium. Viral RNA was extracted from biological samples with QIAamp Viral RNA Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Real-time RT-PCR were used for influenza typing and subtyping. The samples were identified as containing influenza A on the basis of the M gene, but could not be subtyped. Specific real-time RT-PCR assays for avian influenza H5, H9, H7, (Liveriver, shanghai, China) and H10 subtypes were done to verify the viral subtypes from nucleic acids positive to influenza A virus in M gene. Original biological samples obtained from poultry were further performed with self-designed specific primer and probe sets for detecting H5, H9, H7 and H10 by the national institute for viral disease control and prevention, China CDC, by means of real-time RTPCR, to be positive for influenza A virus. 2.4. Statistical analysis We initially entered our data into Excel and then transferred it to SPSS (Statistical Package for Social Sciences, Chicago, IL, USA) version 17.0 for statistical analysis. Differences between proportions were analyzed using the X2 test. A difference was considered statistically significant when p < 0.05.
Fig. 2. Poultry and wild birds density map of AIVs during the emergence of H10N8 in Nanchang.
positive samples, 242 (97.98%) were from LPMs that were experiencing the occurrence of H10N8. LPM had primary responsibility for positive rate of AIV. Whereas, only 1/129 (0.78%) viruses were detected in poultry countyards. Moreover, none of the positive sample was detected in wild-bird habitats. Interestingly, four samples were detected positive from 25 in the transport links (Fig. 3). 3.2. The distribution of AIVs and HA subtypes identified in different poultry species All samples for AIVs detection were comprised of 29 oropharyngeal, 443 cloacal, 131 faecal, 110 contaminated environmental samples and 214 drinking or slaughter water, which were collected from more than ten kinds of poutry. A small portion of biological samples were paired samples. As processing AIVs testing, 161 (26.05%) were positive in healthy chickens, and 110 (30.81%) in ducks. Positive rate between goose and pigeon was very similar. By contrast, all wild birds were tested negative for AIVs. There was a significantly higher positive rate of AIVs in drinking or
3. Results 3.1. Positive rate of AIVs in different sites from four county and five districts of Nanchang During the period from December 2013 to February 2014, a total of 1036 samples were collected from LPMs, poultry countyards, delivery vehicles, and wild-bird habitats at four county and five districts of Nanchang. 23.84% of 1036 samples tested positive for AIVs. The majority (74.49%) of positive samples occurred in the metropolitan area, Donghu, Xihu, Qingshanhu, Qingyunpu and Wanli district. Two suburban county, Xinjian and Anyi county, showed a higher positive rate compared to other two county (Fig. 2). Of the 247
Fig. 3. Positive rate of AIVs in different sites during the emergence of H10N8 in Nanchang (LPM: live poultry market; PC: poultry countyard; DV: delivery vehicle; WBH: wild-bird habitat).
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
slaughter water from pigeons (51.85%) ( p < 0.05), and oropharyngeal samples from chickens (61.54%) ( p < 0.05) (Table 1). In our analysis, H5, H7, H9, H10 and other A untypable were all detected in chickens, ducks, gooses, pigeons. The H5 subtype was dominant in ducks and the H9 subtype in chickens. Of the 247 positive samples, 21 were tested positive for untypable HA type (Fig. 4A). The distribution of different HA subtypes in chickens and ducks was not meaningful different ( p < 0.05). 3.3. Mixed infection of different HA subtypes Out of the 247 samples positive for AIVs, almost one sixth poultry samples showed mixed infections of different HA subtypes of AIVs. As shown in Fig. 4B, mixed infection of H5 and H9 subtypes was the most commonly mixed infection, followed by H5, H9 and H10 subtypes. Among these concomitant infections, 33 were mixes of two different HA subtypes, 8 were mixes of three different HA subtypes. Additional, concurrent infection was more frequent in ducks and chickens than other terrestrial poultry. 3.4. Coexistence of different HA subtypes with N8 In the present study, 54 samples were tested positive for N8 type. H10 subtypes coexistence with N8, the most prevalent agent, was present in 38.89% (21/54) samples, H5 and N8 in 9.26% (5/54)samples, H7 and N8 in 1.85% (1/54)samples, H9 and N8 in 12.96% (7/54) samples, and untypable HA and N8 in 33.88% (18/54) sample (Fig. 5). 4. Discussion The two novel influenza viruses in human, H7N9 and H10N8, had similar genetic lineage: the hemagglutinin (H) gene originated from ducks and the neuraminidase (N) gene from wild birds [2,20]. H10N8 virus causing human infections without preceding outbreak in poultry was quite unexpected. The apparently low pathogenicity in poultry could help the H10N8 virus to circulate in poultry without detection. Another two human cases of infection with H10N8 were reported in Nanchang City in early 2014. The widespread detection of AIV from poultry and wild birds due to the emergence of A(H10N8) virus in Nanchang city in a relatively short time period indicated that several subtypes of AIVs transmit
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efficiently among local poultry, in particular within domestic ducks, which further supported a report proposing the major role of domestic ducks as mixing vessels of influenza viruses [21].Our results were consistent with a study, which reported that there was 23 different HA-NA subtype combinations detected mainly in domestic ducks in eastern China [22]. The association between occurrence of human cases and proximity of irrigated lands indicates that ducks may play a role in the transmission of H7N9, as they do for other AIVs [21,23]. On the other hand, a similar situation also prevailed in chickens in our study. Chickens might serve as the intermediate host and thus might be the source of transmission of the influenza virus to humans [24]. Therefore, as more was learned about the role of the chicken in the evolution and ecology of the influenza virus. LPMs were known to be a high-risk environment for pathogen transmission between birds and for zoonotic transfer to people [25]. The warm, humid climate in this region might prompt long-term survival and proliferation of the virus. Our results ruled out that there was a higher prevalence of AIVs in the poultry at LPMs than other sites. And AIVs infected chicken, duck, goose and pigeon with asymptomatic or mild disease in this area. If birds spent a sufficient amount of time in market to become infected and transmitted the virus to other susceptible birds, sustained virus circulation in LPMs could occur [26]. In addition, live poultry trade created networks of close contacts between poultry and human populations, thereby facilitating transmission of AIVs. Apart from birds and the wet environments at the LPMs, kinds of vehicles associated with poultry transport were another risk factor, including car, motorbike and so on, as investigated in the study. The same kind of vehicles sustained carrying different poultry, such environments contaminated by virus might not only increase transmission of AIVs in birds, but also likely increase the number of human infections. Geographically, although the viruses were detected in different regions of Nanchang, most positive samples occurred in the metropolitan area. In modern times, geographical distribution of three influenza (H10N8) confirmed cases didn't give us any clue to their relationships. We inferred that the regional trade played a key role transmission of AIVs in domestic birds in Nanchang. Of note, various subtypes of AIVs were circulating among poultry in Nanchang: H5 (n ¼ 98), H7 (n ¼ 6), H9 (n ¼ 92), H10 (n ¼ 56), and untypable HA (n ¼ 21). We discovered that mixed infection of different HA subtypes was very common in
Table 1 Positive detection rate of AIVs in different poultry species during the emergence of H10N8 in Nanchang. Type
O E C F W Total
Chicken
Duck
Goose
Pigeon
Wild bird
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
Number
Positive (%)
13 85 279 128 107 618
61.54 28.24 20.784 21.094 40.19 26.05
10 49 102 94 86 357
30 34.69 35.29 21.27 36.05 30.81
0 2 3 2 3 16
0 50 0 50 33.33 18.75
6 42 59 53 27 187
0 30.95 13.56 7.55 51.85 20.86
0 0 2 32 49 83
0 0 0 0 0 0
Sample type: O (oropharyngeal), E (contaminated environmental samples), C (cloacal), F (faecal) and W (drinking or washing water).
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X. Ni et al. / Microbes and Infection 17 (2015) 48e53
Fig. 4. HA subtypes distribution of positive samples (A) and mixed infection (B) in different poultry species during the emergence of H10N8 in Nanchang (The ratio of positive samples or mixed infection is the total number of H5, H7, H9 and H10 subtypes in different poultry species).
ducks in Nanchang, which postulated that ducks were the main virus reservoir and promote mixed infections of different HA subtypes of AIVs [27].Concurrent infection of viruses in birds or other animals provided the possibility for the emergence of a new reassortant virus [28e30]. The reassortant among different influenza viruses was considered as the main mechanism for the emergence of novel virus, which could lead to the influenza pandemic, as preliminarily confirmed by H7N9 and H10N8 viruses [6]. A(H10N8) was the first influenza virus affecting humans that carry N8. What we knew about H10N8 so far was that only two H10N8 viruses had been reported in China: one was from a water sample taken from Dongting Lake in Hunan province in 2007 [31], and the other from LPM in Guangdong province in 2012 [19]. As H10N8 virus possessed internal gene cassettes recruited from poultry, we sought to find some correlations between the emerging H10N8 virus and N8 in local poultry. 21 samples with the N8 and H10 coexistence, were the leading agent among all mixed infection. In recent years, continued AIV surveillance in poultry was carried out in Nanchang, as one of the important control measures for
fighting against avian influenza, but few H5 subtypes were detected. Furthermore, 6 H5N1 and 4 H7N9 of 61 poultry samples were positive during the outbreak of H7N9 in 2013. No H10 subtype were tested for positive according to our AIV surveillance datas before 2013 in Nanchang. Our results could serve as a useful resource for further functional experiments of the sources of H10N8 human infections. Poyang Lake was located in the East Asian-Australian migratory birds' flyway and it was the stopover and winter site for migratory birds [32]. Therefore, investigation of water for AIVs was of greater significance and convenience for understanding the route and mechanism of virus transmission between domestic fowl and migratory birds in this lake area. We collected tens of drinking water and faecal sample about wild bird categorized into Xinjian County, which adjoined Poyang Lake. Unfortunately, none of the positive sample was detected in this area. A novel genotype influenza virus was isolated from drinking Water near Poyang Lake in 2011 [33], which highlighted the importance of strengthening the surveillance of avian influenza in this region. Taken together, our results could be used as a reference for targeted surveillance and control efforts in both human and animal populations to reduce the risk of future human infections. Since we were currently outside the northern hemisphere influenza season, any upsurge in cases detected by routine surveillance should be a cause for concern. Thus, a more rigorous and long-term surveillance remained essential for early warning of novel reassortant viruses and interspecies transmission events. Conflict of interest None of the authors has any conflict of interest. Acknowledgments
Fig. 5. Distribution of coexistence of different HA subtypes with N8 during the emergence of H10N8 in Nanchang.
We acknowledge the contributions of Donghu, Xihu, Qingyunpu and Wanli district CDC andNanchang, Jinxian and Anyi county CDC for the field work and sample collection.
X. Ni et al. / Microbes and Infection 17 (2015) 48e53
The contents of this manuscript are solely the responsibility of the authors and not necessarily represent the official views of Nanchang Centers for Disease Control and Prevention. This work was supported by National Natural Science Foundation of China (No. 81460302) and Major Science and Technological Project of Jiangxi Province (No. 20143ACG70004). References [1] Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al. Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 2013;368:1888e97. [2] Chen H, Yuan H, Gao R, Zhang J, Wang D, Xiong Y, et al. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet 2014;383:714e21. [3] http://www.jxwst.gov.cn/gzdt/201402/t20140213_308109.htm. [4] Parry J. H10N8 avian flu virus claims its first known human casualty. BMJ 2014;348:g1360. [5] Zhou X, Li H, Ni X, Liu M, Hu M, Wu J, et al. Laboratory diagnosis and epidemiology of avian influenza A (H7N9) virus infection in humans in Nanchang City, China. Jpn J Infect Dis 2013;66:558e60. [6] Shi W, Li W, Li X, Haywood J, Ma J, Gao GF, et al. Phylogenetics of varied subtypes of avian influenza viruses in China: potential threat to humans. Protein Cell 2014;5:253e7. [7] Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS Pathog 2013;9:e1003657. [8] Schrauwen EJA, Fouchier RAM. Host adaptation and transmission of influenza A viruses in mammals. Emerg Microbes Infect 2014;3:e9. [9] Pei ris JS, de Jong MD, Guan Y. Avian influenza virus (H5N1): a threat to human health. Clin Microbiol 2007;20:243e67. [10] The SJCEIRS H9 Working Group. Assessing the fitness of distinct clades of influenza A (H9N2) viruses. Emerg Microbes Infect 2013;2:e75. [11] To KK, Tsang AK, Chan JF, Cheng VC, Chen H, Yuen KY. Emergence in China of human disease due to avian influenza A (H10N8)—cause for concern? J Infect 2014;68:205e15. [12] Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of the avian influenza A (H7N9) outbreak in China. N Engl J Med 2014;370:520e32. [13] Shi J, Gao L, Zhu Y, Chen T, Liu Y, Dong L, et al. Investigation of avian influenza infections in wild birds, poultry and humans in Eastern Dongting Lake, China. PLoS One 2014;9:e95685. [14] Duan L, Zhu H, Wang J, Huang K, Cheung CL, Peiris JS, et al. Influenza virus surveillance in migratory ducks and sentinel ducks at Poyang Lake, China. Influenza Other Respir Viruses 2011;5:65e8. [15] Shortridge KF, Stuart-Harris CH. An influenza epicentre? Lancet 1982;2:812e3. [16] Webster RG, Guan Y, Peiris M, Walker D, Krauss S, Zhou NN, et al. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J Virol 2002;76:118e26. [17] Choi YK, Ozaki H, Webby RJ, Webster RG, Peiris JS, Poon L, et al. Continuing evolution of H9N2 influenza viruses in Southeastern China. J Virol 2004;78:8609e14.
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