New H6 influenza virus reassortment strains isolated from Anser fabalis in Anhui Province, China
© The Author(s). 2017
Received: 10 October 2016
Accepted: 11 January 2017
Published: 21 February 2017
H6 subtype avian influenza viruses are globally distributed and, in recent years, have been isolated with increasing frequency from both domestic and wild bird species as well as infected humans. Many reports have examined the viruses in the context of poultry or several wild bird species, but there is less information regarding their presence in migratory birds.
Hemagglutination and hemagglutination inhibition tests were used to measure HA activity for different HA subtypes. Whole viral genomes were sequenced and analysed using DNAstar and MEGA 6 to understand their genetic evolution. Pathogenicity was evaluated using a mouse infection model.
We isolated 13 strains of H6 virus from faecal samples of migratory waterfowl in Anhui Province of China in 2014. Phylogenetic analysis showed gene reassortment between Eurasian and North American lineages. Five of the identified H6 strains had the ability to infect mice without adaptation.
Our findings suggest that regular surveillance of wild birds, especially migratory birds, is important for providing early warning and control of avian influenza outbreaks.
KeywordsAvian influenza virus Anser fabalis H6 subtype
Avian influenza virus (AIV) is an important zoonotic pathogen  and can be classified into 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes based on the antigenicity of these two surface glycoproteins. At least 136 species have been discovered in 26 different families of wild birds and harbour 144 subtypes of influenza A virus [1–4]. Most subtypes are found not only in common aquatic birds but also in lower mammals, such as bats, as has been demonstrated in recent reports [1, 5–7]. In Qinghai Lake, China in 2005, massive numbers of migrating birds died by H5N1, and this virus subsequently spread worldwide [8, 9]. In recent years, different influenza virus subtypes (such as H5, H6, H7, and H10) have emerged in succession in poultry, crossed the species barrier and caused frequent outbreaks of human infection [1, 10–15]. The continuous outbreaks of avian influenza in recent decades have alarmed and increased focus on the role of wild birds, as major reservoirs, that maintain the persistence and variation of AIV, facilitating viral spread and disease occurrence.
H6 AIV was first isolated from turkeys in Massachusetts, USA in 1965 [10, 16]. Currently, H6 AIVs has a worldwide distribution, and strains of the virus have been detected in various animal species . On June 21, 2013, the first case of a human infection by an avian-origin H6N1 influenza A virus (A/Taiwan/2/2013, Taiwan2) was reported by the Taiwan Centres for Disease Control [11, 18]. Human infection with H6 AIVs in mainland China has not been reported, but a serum antibody positive for the H6 virus was found in poultry workers . More than 30% of the H6 AIVs circulating in poultry in China have enhanced affinity to human-like receptors (ɑ-2, 6 NeuAcGal). Some H6 subtypes can also infect mice without prior adaptation, and some can be efficiently transmitted among guinea pigs . These events indicate that H6 AIV can cross species barriers and directly infect humans. As the natural host of AIVs, wild birds play an important role in virus reassortment as well as intra- and interspecies transmission [4, 5]. However, there is little research regarding the biological properties of H6 AIV strains from wild birds and their potential threat to mammals compared to the many studies of H6 AIV in poultry.
In the present study, we isolated 13 H6 AIV strains after surveying wild birds in the wetlands of the National Nature Reserves in Anhui Province, China. For all of these strains, each gene segment was sequenced to analyse phylogenetic and speculate its origin. We also preliminarily evaluated the potential threat of each strain to mice. Our study highlights the importance of performing regular surveillance of AIV strains in Anhui Province, China as an integral part of worldwide efforts to better understand AIV ecology and prevent the emergence of novel, potentially pandemic strains. Surveillance of avian influenza virus (AIVs) in wild birds for early warning, prevention, and control of viral outbreaks should be enhanced to reduce the risk of pathogen emergence from wildlife host reservoirs.
Virus isolation and identification
In spring and autumn of 2014, 2970 faecal samples were collected from wild birds in Anhui Province, China. Fresh, single faecal samples were collected with cotton swabs and placed into 5 ml EP tubes with 2 ml virus protection solution (phosphate-buffered solution (pH 7.2) supplemented with penicillin, streptomycin, and 10% glycerinum). The handheld end of each cotton swab was broken off, and the remainder was placed into the sample tube and covered tightly. The handheld ends of the swabs were inserted into the ground to designate collection locations to avoid repetition in sampling.
Primers in RT-PCR
Genetic and phylogenetic analyses
Nucleotide sequences were edited using the SeqMan module of the DNASTAR package, and multiple sequence alignments were compiled using Clustal W [22–24]. Phylogenetic analyses were performed with the neighbour-joining method with maximum likelihood trees using MEGA 6.0 software [25, 26]. Bootstrap values of 1,000 were used. The phylogenetic analyses were based on the following coding sequences (nucleotides): polymerase PB2 (PB2), 1 to 2280; polymerase PB1 (PB1), 1 to 2274; polymerase PBA (PA), 1 to 2151; hemagglutinin (HA), 1 to 1701; nucleoprotein (NP), 1 to 1497; neuraminidase (NA), 1 to 1410; matrix protein 1 (M), 1 to 982; and non-structural protein 1 (NS), 1 to 844.
The 50% egg infection dose (EID50) was calculated according to the protocol of Reed and Muench. Six-week-old female BALB/c mice (Experimental Animal Centre of Vital River, Beijing, China) were randomly divided into four infection groups (8 mice/group) and one control group (5 mice/group). Animals in the infection groups were mildly anesthetized with dry ice and infected by intranasal inoculation with a dose of 106 EID50 (50 μl/mouse). Three of the eight mice were randomly euthanized on day 3 post inoculation (p.i.) for titration of virus from the lungs, nasal turbinate, kidneys, spleen, and brain. The remaining five mice were monitored daily for 2 weeks for changes in body weight and mortality.
Influenza virus isolation and subtype identification
Information of the 13 avian influenza viruses
Phylogenetic analysis of whole genes
Preliminary analyses of the possible origins of the strains were conducted. There were three Genotype 1 viruses, which were tri-reassortment of H6N2, H5N1 and H3N6. There were three Genotype 2 viruses, which were tri-reassortment of H6N2, H9N2 and H3N6. There were two Genotype 3 viruses, which were bi-reassortment of H6N2 and H9N2. There was only one virus in Genotype 4 and one in Genotype 5: both were tri-reassortment of H6N2, H9N2 and H5N1. There was only one virus in Genotype 6, which was a bi-reassortment of H6N2 and H9N2. There was only one virus in Genotype 7 and one in Genotype 8: both were tri-reassortments of H6N2, H6N1 and H3N6 (Fig. 4).
Molecular characterization of viral genes
The isolated H6 strains, except for AH/L221, contained seven potential glycosylation sites according to analysis performed at http://www.cbs.dtu.dk/services/NetNGlyc; these included sites at positions 26 to 28, 27 to 29, 39 to 41, 306 to 308, and 311 to 313 on HA1 and at positions 498 to 500 and 557 to 559 on HA2 (H3 numbering, which is used throughout the manuscript). Strain AH/L221 contained eight potential glycosylation sites, including a glycosylation site on NNT at positions 182 to 184. All viruses except for strain AH/L144 contained the PQIETR/G motif at the cleavage site between HA1 and HA2. The cleavage site on AH/L144 contained the PLIETR/G motif, which includes an amino acid substitution from Q to L. None of the strains contained consecutive basic amino acids within the motif, and all conformed to the characteristics of low-pathogenicity AIVs. The amino acid substitutions A138S, E190V, P186L , Q226L, and G228S, as well as the absence of glycosylation at positions 158 to 160 in HA, have been reported to increase the affinity of influenza virus for human-type receptors . The above-mentioned changes were not detected in any of the 13 H6 avian viruses sequenced. Necklace deletion in the NA gene confers enhanced virus lethality in mice. In the present study, none of the 13 H6 AIVs studied had a necklace deletion in the NA gene . Some amino acid substitutions, including T271A, E627K, and D701N in PB2, contribute to increased virulence and transmission of influenza viruses in mammals. The H6 viruses sequenced in this study did not contain any of these changes either. No Y436H substitutions in the PB1 protein or T515A substitutions in the PA protein were found, which suggests that the studied viruses have low pathogenicity for mammalian and avian hosts. Additionally, no amino acid substitutions were found in the M2 transmembrane domain, suggesting that the viruses are sensitive to M2 ion channel inhibitors .
The N66S substitution in the PB1-F2 protein, which has been associated with the increased virulence of the 1918 pandemic virus and the high pathogenicity of the AI H5N1 virus in mice and ferrets [31, 32], was not found in any of the H6 strains examined in this study. Additionally, none of the viruses harboured the S31N substitution in the M2 protein, indicating that all are sensitive to amantadine inhibitors . The virulence of influenza virus in humans is related to resistance to the antiviral effects of cytokines, such as interferon (IFN). In particular, the D92E mutation in the NS1 protein increases resistance to IFN; this mutation was not observed in any of the strains evaluated in this study .
Replication of H6 viruses in mice
Based on body shape, physiological characteristics, and other features, wild birds have unique flight patterns, which has led to their wide geographical spread, strong regional activity and extended seasonal migration. Wild birds migrate from breeding to wintering places each year along unique migration routes. Wild aquatic birds are the natural host of influenza virus and can act as a reservoir for the transfer of genes between species, forming transient “genome constellations” that are continually reshuffled by reassortment [36, 37]. The birds’ migration behaviours expand the distribution of the virus and provide conditions for the cross-border spread of viruses. Many reports have analysed the biological and pathogenic characteristics of influenza virus in poultry in China; however, studies of influenza virus in wild birds are relatively rare.
In recent years, several AIV subtypes have been circulating and evolving in southern China; these include the H5, H9, and H6 subtypes as well as the newly emerged H7N9 and H10N8 viruses. This has led to frequent avian influenza outbreaks in poultry and humans. Anhui Province is located in south-eastern China (29°41′to 34°38′N, 114°54′to 119°37′E). Lying in the middle and lower valleys of the Yangtze and HuaiHe rivers, Anhui is abundant in wetlands, with wetland areas accounting for 21% of the total area of the province. These areas provide important stopover and wintering grounds for birds migrating along the East Asia-Australia migratory flyway. In the fall and winter of each year, a large number of migratory birds gather in the wetlands, resulting in extremely high population densities of the same or different species, which likely favours the spread and viral gene reassortment of the influenza virus. As an important agricultural province, Anhui is also home to a great number of poultry raised under different farming practices. These poultry often come into close contact with wild birds, creating the opportunity for cross-species AIV transmission.
In 2 years of surveillance work in Anhui, we obtained 31 AIV strains from 2970 faecal samples. Among these strains, eight subtype combinations, including H1N1, H1N2, H3N3, H3N8, H6N1, H6N2, H9N2, and H11N9, were identified using HA-HI tests and PCR. The H6 subtype accounted for the largest proportion of those identified, being found in approximately 41.9% (13/31) of the samples. H6N2 accounted for the majority of these H6 AIVs (10/13). Overall, the goal of this work was to obtain baseline information regarding the epidemiological and virological characteristics of H6 virus strains that exist in wild waterfowl. Toward this aim, we assessed 13 strains of H6 virus isolated from faecal samples of bean goose (Anser fabalis) in Anhui Province, China in 2014.
Gillim-Ross et al.  described 14 strains of H6 virus from poultry that could infect and replicate in the lungs of mice and showed that some of these strains are lethal. Wang et al. also reported that 37 of 38 tested H6 viruses from poultry could replicate efficiently in the lungs of mice ; however, none were lethal. In the present study, experimental infection in mice revealed that all but one of the studied H6 strains could replicate in the lungs and turbinate of mice without adaptation. Some of the viruses also induced weight loss in mice. These data support the idea that H6 AIVs from wild birds can replicate in BALB/c mice without adaptation and that mammals can be directly infected with wild-bird AIV. Following infection, the AH/S45 strain was not found in any organs, but this strain has high sequence identity with strain AH/S20: these two strains have no reported differences in amino acid sequence. Further research is required to determine whether differences in amino acids that influence the viral replication ability exist between these two viruses. Some studies have reported that red blood cells from turkeys, sheep, and horses possess a-2, 3 receptors; however, red blood cells in humans with Type O blood and those from guinea pigs possess a-2, 6 receptors. Interestingly, in the present study, the 13 H6 AIV strains studied all exhibited high HA activity in red blood cells from horses and sheep but low activity in red blood cells from Type O humans and from guinea pigs. These results coincide with the properties bestowed by amino acids 226 and 228 in the HA gene of the virus, which together form the characteristic poultry receptor-binding site on the virus .
H6 AIV has a worldwide distribution, similar to H5N1 and H9N2 [41, 42], and has caused considerable losses in poultry farming. In one report, out of a total of 15,689 serum specimens gathered from 22 provinces in mainland China, approximately 63 specimens were positive for H6 AIV . These cases of H6-infected humans serve as a warning. As the natural host of influenza virus, wild birds, especially migratory species, play an indispensable role in the spread and reassortment of AIV and provide unknown opportunities for the mutation and emergence of novel influenza viruses . To fully understand the ecology, biology, and potential hazards of influenza virus, regular surveillance of all influenza virus hosts, and especially wild birds and poultry, is necessary. The data generated from such efforts must be combined to accurately assess the potential threat. Notably, the present study provides only a general overview of the H6 virus strains that are currently circulating among Anseriformes in Anhui, China. More intensive sampling and sequencing of AIVs from wild birds is needed to help refine our understanding of host specificity and viral evolution , which will also reveal the processes driving viral adaptation and maintenance in alternative hosts.
The H5, H6, and H9 subtypes of influenza A virus are found worldwide. Although it is known that H6 AIV strains found in poultry pose a potential threat to humans, few studies have surveyed this subtype in infected wild birds, and its threat to humans is therefore not well understood. In the present study, we performed a risk assessment of H6 influenza A virus transmission using phylogenetic and pathogenic analyses to better understand the potential threat of wild birds, especially migratory birds, transmitting influenza virus to poultry and/or humans. In doing so, we identified 13 strains of H6 AIV in Anseriformes. Some of these strains were reassortment from the Eurasian and North American lineages. Overall, we conclude that H6 AIV strains isolated from wild birds have the potential to infect mammals and humans.
Influenza A virus
Polymerase basic 1
Polymerase basic 2
Reverse-transcription polymerase chain reaction
We are grateful for the technical support and staff of related laboratories in the Natural Conservation & Management Station of Anhui Province.
This research was supported by the National Key National Key Research and Development Program of China(2016YFD0500201) and the Surveillance of Wildlife Diseases Department from the State Forestry Administration of China.
Availability of data and materials
Data supporting the conclusions of this article are presented in the manuscript.
This study is a result of collaborative work. YG participated in data collection, conceiving the study design, and drafting the manuscript. QY and SC worked on isolating viruses. XW and ZF participated in sample collection. HChen provided equipment used for the study. JM worked on data analysis. HChai participated in discussions and modified the manuscript. GD provided equipment used for the study as well as funding support. YH participated in study design, made suggestions throughout the study, revised the manuscript and provided funding. All authors read and approved the final manuscript.
The authors declare that their work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Consent for publication
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. General animal care was provided as required by the Institutional Animal Care and Use Committee (approval number BRDW-XBS-14 for mice) [17, 45].
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