Isolation and characterization of H4N6 avian influenza viruses from mallard ducks in Beijing, China

RESEARCH ARTICLE Isolation and characterization of H4N6 avian influenza viruses from mallard ducks in Beijing, China Junyi Hu1, Xinyi Xu1, Chenxi Wang1, Guoxia Bing2, Honglei Sun1, Juan Pu1, Jinhua Liu1, Yipeng Sun1* 1 Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China, 2 China Animal Disease Control Center, Beijing, China a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Hu J, Xu X, Wang C, Bing G, Sun H, Pu J, et al. (2017) Isolation and characterization of H4N6 avian influenza viruses from mallard ducks in Beijing, China. PLoS ONE 12(9): e0184437. https:// doi.org/10.1371/journal.pone.0184437 Editor: Yongchang Cao, Sun Yat-Sen University, CHINA Received: May 29, 2017 Accepted: August 23, 2017 Published: September 6, 2017 Copyright: © 2017 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Key Research and Development Program of China (2016YFD0501601), National Natural Science Fund for Outstanding Young Scholars (31522058), Beijing New-star Plan of Science and Technology (Z161100004916115), and by grants from the Chang Jiang Scholars Program. Competing interests: The authors have declared that no competing interests exist. * Abstract The novel H7N9 influenza virus, which has caused severe disease in humans in China, is a reassortant with surface genes derived from influenza viruses in wild birds. This highlights the importance of monitoring influenza viruses in these hosts. However, surveillance of influenza virus in wild birds remains very limited in China. In this study, we isolated four H4N6 avian influenza viruses (AIVs) from mallard ducks in Beijing Wetland Park, which is located on the East Asia–Australasia migratory flyway. The gene segments of these Chinese H4N6 viruses were closest to AIVs in wild birds from Mongolia or the Republic of Georgia, indicating the interregional AIV gene flow among these countries. All of our isolates belonged to a novel genotype that was different from other H4N6 viruses isolated in China. We further evaluated the virulence and transmission of two representative H4N6 strains in mammalian models. We found that both of these H4N6 viruses replicated efficiently in mice without adaptation. Additionally, these two strains had a 100% transmission rate in guinea pigs via direct contact, but they had not acquired respiratory droplet transmissibility. These results reveal the potential threat to human health of H4N6 viruses in migratory birds and the need for enhanced surveillance of AIVs in wild birds. Introduction Influenza viruses circulating in animals are novel to the human immune system; these viruses therefore pose a potential threat to public health once they are transmitted to humans. Moreover, animal influenza viruses can even cause pandemics in a human population once they acquire efficient transmissibility between people. Historically, influenza pandemics have been caused by animal influenza viruses (as in the 1918 and 2009 pandemics) or avian influenza viruses reassorting with human influenza viruses (pandemics in 1968 and 1957) [1–2]. Recently, a novel H7N9 influenza virus caused a total of 1364 laboratory-confirmed cases of influenza, with a nearly 33% case-fatality rate reported to the World Health Organization since its first detection in February 2013 in China [3]. Genetic analyses have found that this virus is a reassortant whose surface genes are directly derived from H7 and N9 subtypes of the wild bird viral PLOS ONE | https://doi.org/10.1371/journal.pone.0184437 September 6, 2017 1 / 12 Isolation and characterization of H4N6 AIVs gene pool, and with internal genes from H9N2 avian influenza viruses (AIVs) in chickens [4]. The novel H7N9 outbreak reminds us that other AIV subtypes in wild birds also pose a threat to humans, either alone by reassortment with other influenza viruses. Therefore, it is important to rigorously monitor the prevalence and biological properties of AIVs in wild birds. The H4N6 AIV subtype circulates among waterfowl worldwide, including in Asian, European, African, and North American countries [5–6]. It has been noted that H4N6 viruses have been repeatedly found in mammalian species, such as humans and pigs [7–11]. H4N6 is frequently isolated from domestic ducks in China [12–14]. Genetic analysis indicates that these H4N6 viruses are undergoing complex and frequent reassortment events. In particular, they can efficiently replicate in the respiratory tissues of infected mice and spread among guinea pigs via direct transmission. Moreover, some strains have acquired respiratory droplet transmissibility in a guinea pig model [12]. Although the characteristics of H4N6 in domestic ducks in China have been well studied, the genetic and biological properties of H4N6 in wild birds of this region have not been reported. In April 2016, we isolated four H4N6 AIVs from mallard ducks in Beijing Wild Duck Lake Wetland Nature Reserve, which is located on the East Asia–Australasia migratory flyway. The genomes of these viruses were sequenced and analyzed against all H4N6 viruses in China, available from the NCBI Influenza Virus Sequence Database. To evaluate their potential threat to human health, we further determined the replication and transmission of two H4N6 strains in mouse and guinea pig models. Materials and methods Sampling methods and ethical compliance We collected the fresh fecal samples from the ground after the wild birds flew away without direct contact with any birds. Feces were collected from the mallard ducks in Beijing Wild Duck Lake Wetland Nature Reserve (40˚220 04"~40˚300 31"N, 115˚460 16"~115˚590 48"E). The study and sampling procedures were permitted by Management Office of Yanqing Country Wild Duck Lake Wetland Nature Reserve. All animal work was approved by the Beijing Association for Science and Technology (approval SYXK [Beijing] 2007–0023) and conducted in accordance with Beijing Laboratory Animal Welfare and Ethics guidelines, as issued by the Beijing Administration Committee of Laboratory Animals, and in accordance with China Agricultural University (CAU) Institutional Animal Care and Use Committee guidelines (SKLAB-B-2010-003). Mice and guinea pigs were humanely euthanized using anesthesia followed by cervical dislocation. All animal researches in this study adhere to the ARRIVE Guidelines. A completed ARRIVE guidelines checklist is included in S1 Checklist. Virus isolation We collected feces from mallard ducks in Beijing Wetland Park Randomly. Viruses were isolated by inoculation in 10-day old s (...truncated)