Co-circulation of H5N6, H3N2, H3N8, and Emergence of Novel Reassortant H3N6 in a Local Community in Hunan Province in China

Multiple infections of avian influenza viruses (AIVs) in poultry or wild birds contribute to the continued evolution of H5 subtype viruses in nature and provide potential recombination of AIVs of different origins. In this study, we carried out surveillance of AIVs in ducks, geese and the environment of a community in Hunan province, China, from 2014–2015. We isolated multiple co-circulated AIVs including H3N2, H3N8, and H5N6, and, most importantly, a novel reassortant: H3N6. Phylogenetic analyses suggest that H3N6 is highly likely derived from H5N6, which has recently been shown to have zoonotic potential with human infections. Studies with mammalian cell lines and a mouse model indicate that four selected AIVs of duck or goose origin can infect MDCK and A549 cells but have low pathogenicity in mice. We propose that a potential co-circulation of multiple subtypes including H5N6 in local area may result in the production of novel subtypes such as H3N6 by gene reassortment.


Multiple infections of avian influenza viruses (AIVs) in poultry or wild birds contribute to the continued evolution of H5 subtype viruses in nature and provide potential recombination of AIVs of different origins.
In this study, we carried out surveillance of AIVs in ducks, geese and the environment of a community in Hunan province, China, from 2014-2015. We isolated multiple co-circulated AIVs including H3N2, H3N8, and H5N6, and, most importantly, a novel reassortant: H3N6. Phylogenetic analyses suggest that H3N6 is highly likely derived from H5N6, which has recently been shown to have zoonotic potential with human infections. Studies with mammalian cell lines and a mouse model indicate that four selected AIVs of duck or goose origin can infect MDCK and A549 cells but have low pathogenicity in mice. We propose that a potential co-circulation of multiple subtypes including H5N6 in local area may result in the production of novel subtypes such as H3N6 by gene reassortment.
Waterfowl such as wild birds, ducks, and geese are natural reservoirs of avian influenza viruses (AIVs) globally, and most highly pathogenic human influenza viruses originate from waterfowl [1][2][3][4][5][6] . At this time, 16 hammagglutinins (HAs) and 9 Neuraminidases (NAs) of AIVs have been isolated from aquatic birds and among the AIVs capable of infecting humans, H5 and H7 (N9) are the most commonly reported [7][8][9][10][11] . Importantly, asymptomatic infection of waterfowl with most AIVs can promote evolution of influenza viruses 12 . Domestic ducks and geese are positioned at the interface between wild aquatic birds and terrestrial poultry due to free-range farming in southern China. For these farms, no biosecurity measures are used and open feeding, and lack of no disinfection plus close animal contact between migratory birds and domestic ducks and geese who share water, food, and housing create problems. Thus, AIVs of various sources co-exist in the same ecosystem, facilitating interspecies transmission and viral gene reassortment. Co-infection with different AIVs in waterfowl is generally considered a significant mechanism for promoting viral diversity by generating novel reassortants 1,4,13 .
The highly pathogenic H5N1 virus was first identified in sick geese in Guangdong Province in 1996 14 . In 1997, H5N1 reassortants, with the HA gene derived from A/goose/Guangdong/1/96-like viruses, caused lethal outbreaks in poultry and humans in Hongkong 15 . Since the beginning of 2004, significant outbreaks of H5N1 viral infection in poultry farms caused millions of domestic poultry deaths 3,6,16 . In 2005, thousands of migratory aquatic birds died in the Qinghai Lake area due to H5N1 viral infection 3,6 . Then, the Qinghai Lake H5N1 virus spread to Europe and Africa and exacerbated the global prevalence of the virus. Recently, a novel H5N6 virus was isolated in Asia, which was a reassortant of H5N1 and H6N6 viruses [17][18][19] . Such a novel virus had high pathogenicity in chickens and led to several human infections since 2014 in China 20,21 . Moreover, the H5N6 virus has also been isolated from domestic cats and wild birds in China 22 , suggesting that mammals and wild birds may contribute to viral circulation.
The H3 influenza virus is widely circulated in various species and has been frequently isolated from human, swine, birds, or wild animals. H3 subtypes have established lineages in domestic poultry and have caused mild or severe disease and multiple subtypes such as H3N2, H3N6 and H3N8 viruses have been isolated from domestic poultry globally [23][24][25][26][27][28][29] . Experimental studies showed that avian-origin H3 influenza viruses can replicate in the respiratory tract of mice, implying that H3 isolates from birds can have a potential risk to public health 30,31 .
Co-circulation of AIVs involving H3 and H5 subtypes remains poorly understood. In the winter of 2014, we targeted farms in Wugang city, Hunan Province, China and collected specimens from domestic ducks and geese and from the water of the duck farms. In this study, we report novel reassortants of H3 and H5 influenza viruses originated from a common ancestor in domestic ducks and highlight the need for control of H5N6 viral infection in waterfowl from poultry farms.

Results
Sample collection and virus isolation. Two samplings were conducted from November of 2014 to April of 2015 in our long-term surveillance project. Sample collection sites were along a river in Wugang city, Hunan Province, China. Domestic duck and goose farming in this region has grown substantially in recent years. Most farms were built in high-density areas lacking biosecurity measures. For some farms, healthy and sick ducks or geese were housed together. In all, 86 influenza strains from 160 samples from sick birds (positive rate 53.8%) were successfully isolated (unpublished data). No virus was isolated from fecal samples and 10 strains were selected for phylogenetic analysis and biological experiments (Table 1).

Phylogeny and detection of novel reassortment.
To investigate the genetic relationship between these co-circulated AIVs, we first compared the sequences of both key viral genes. HA genes of the four H3N2, three H3N6 and one H3N8 viruses shared 87.8-100% nucleotide identity. The HA gene of DK/HuN/146/2014 (H3N6) shared 87. .2% identity at the nucleotide level with the four H3N2 viruses. The nucleotide identity of the HA gene of DK/HuN/199/2014 (H3N8) and the four H3N2 viruses was only 88.1-92.3% identical and was the most similar to A/aquatic bird/Korea/CN-1/2004 (H3N6) (94%). The nucleotide identity of HA for the four H3N2 viruses was 88.2-100%. Phylogenetic analysis of the H3 data sets showed that N2, N6, and N8 were commonly reassorted in the local area (Fig. S1). NA genes of the four H3N2 viruses shared 93-99.1% nucleotide identity and clustered into an Eurasian lineage of avian-like H3N2 viruses. The NA gene of A/duck/Hunan/199/2014 (H3N8) had the most similarity with A/chicken/Vietnam/G14/2008 (H3N8) (97%). N2 viruses isolated in our study were of two genetically distinct groups, suggesting multiple introductions and co-infection at local areas. The three N6 isolates, one H3 and two H5, were clustered closely with supported high bootstrap values ( Fig. 1; Fig. S1A), indicating a possible H5N6 origin of the novel reassortant H3N6 virus (Fig. 2). At the time of manuscript preparation, a H3N6 virus-A/duck/Guangxi/175D12/2014-isolated from a native duck in Guangxi Province in 2014, was reported 32 . This sequence was added to our analysis and it was phylogenetically close to H5N6 (Fig. 1 Molecular characteristics. Molecular analysis revealed the presence of the PEKQTR/GLF motif of a single basic amino acid (aa) (Arginine) residues present at the cleavage site of HA protein of the newly isolated H3N6 and H3N2 and H3N8, which was characteristic of low pathogenic AIVs (LPAIV). Such a motif was different from the RERRRKR/GLF of HA of the two H5N6 viruses, representing high pathogenicity in chickens (Table 2). Several amino acid changes in HA, including T160A, Q226L and G228S, was reported to promote an affinity of AIVs for human-type receptors 35,36 . Analysis of the receptor-binding site revealed characteristics of an avian-like receptor binding motif present at the position of 226 and 228 in HA. However, the mutation of 160A was detected

Virulence of H3N2, H3N6, H3N8 and H5N6 viruses in mice. Four influenza viruses tested in mam-
malian cells were evaluated for pathogenicity in mice. After inoculation of six-week-old BALB/c mice with 10 6 EID 50 of each virus, no disease or death occurred during the two-week observation period (Fig. 4A). DK/ HuN/121/2014 (H3N2) and DK/HuN/199/2014(H3N8) replicated to high titers not only in lungs, but also in the nasal turbinates (Fig. 4B,D). Although DK/HuN/146/2014(H3N6) and DK/HuN/144/2014(H5N6) replicated efficiently in lungs, they barely replicated in nasal turbinates (Fig. 4C,E), suggesting that the gene recombination of these two strains might not be suited for efficient replication in mammals prior to forming a stable lineage. No virus was detected in brain, spleen, or kidney. Thus, AIVs originating from ducks or geese caused infection in mice, but no obvious disease or lethality.

Discussion
Hunan Province in China is on the Eastern Asian-Australian Flyway of migratory birds. Duck and goose farms in this region are poorly built and domestic duck and goose farming occurs in high-density settings and in a free-range manner with no biosecurity measures, making this region an excellent ecosystem for AIVs cohabitation from domestic or migratory birds. In addition, ducks or geese from some poultry farms were imported and subsequently sold in live-poultry markets, which offer additional opportunities for virus reassortment and evolution with foreign AIVs circulating outside of Hunan Province. Therefore, this pattern of breeding, farming, and marketing has led to the co-existence of influenza viruses of different sources in farms, and the facilitation of interspecies transmission and viral gene reassortment. To prevent and control epidemic viral distribution, measures including closed feeding, free-transportation ban and active surveillance should be strictly implemented.
Our phylogenetic analyses demonstrated that reassortment frequently occurred in H3 and N6 subtypes in local duck and goose farms. Previous work indicated that H5N6 viruses were novel reassortants that emerged in Southeast Asia, with N6 derived from H6N6 18,20,22,42 . Phylogenetic analyses showed that all NA, PB2, PB1, PA, NP, M, and NS of the novel reassortant H3N6 were derived from the recently emerged H5N6 (Fig. 1 and Fig. S1), while its HA gene was from H3N2 or H3N8 isolated from southern China. Meanwhile, we successfully isolated H3N2, H3N8, and H5N6 viruses from ducks and geese at the same location, implying that co-circulation of multiple subtypes facilitates influenza viral evolution and reassortment in aquatic birds.
Waterfowl are chief natural hosts of H3N2, H3N6, H3N8, and H5N6 viruses. In the literature, we confirmed that most H3N2, H3N8, and H5N6 viruses reported from 2012 to 2014 were isolated from waterfowl, especially mallard and Muscovy ducks, and few were isolated from chickens. These results suggested that waterfowl, but not domestic chickens, contributed to the evolution of H3N2, H3N8, and H5N6 viruses. Human infections with H5N6 virus in China underscore a potential public health risk of the virus. In our study, the two H5N6 isolates had low mammalian pathogenicity, despite efficient replication in mammalian cells. Similarly, the novel H3N6 reassortant also had low virulence in mice. Thus, H3N6 and H5N6 AIVs have not yet fully adapted and may cause severe diseases in mammals if allowed to evolve further.
In summary, our study demonstrates that domestic duck and goose farms in southern China provide an ecosystem for co-circulation of multiple AIVs and facilitate the generation of novel reassortants such as H3N6 that could potentially infect mammals. Thus, large-scale surveillance of related AIVs in waterfowl in farms and mammals in surrounding areas are urgently needed in southern China.    Virus isolation. A total of 160 samples were collected from eight poultry farms along a river from ducks and geese. In addition, 10 fecal samples and 10 water samples were also collected. Each tissue, swab, fecal or water sample was placed in 2 mL of minimal essential medium supplemented with penicillin (2000 U/mL) and streptomycin (2000 U/mL). The samples were immediately frozen with dry ice and kept on dry ice during transfer to the laboratory. All of the individual samples were inoculated into 10-day-old embryonated chicken eggs for 48 h at 37 °C. The allantoic fluid samples were collected and tested for HA activity with 1% chicken red blood cells. All the positive ones were aliquoted and stored at − 80 °C. To avoid cross-contamination during egg inoculation and allantoic fluid collection, strict sterile techniques were implemented. All of the instruments used during the procedure were autoclaved before use and disinfected with 70% ethanol after use with each egg. Each specimen was divided into two aliquots and only one aliquot was used for the virus isolation procedure. Where HA assay was positive, hemagglutinin inhibition (HI) assay was performed to determine the HA subtype of the isolated AIV and Newcastle disease virus (NDV). Hemagglutinin (HA) subtypes were first determined by chicken serum anti-HA of each subtypes, then specific PCR methods was used to further identify HA and NA subtypes.

Methods
Viruses and cells. The H3N2, H3N6, H3N8 and H5N6 viruses used in this study were isolated by using 10-day-old specific pathogen-free embryonated chicken eggs. The viruses were stocked in − 70 °C until used. Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco's modified Eagle's medium (Gibco) supplemented with 5% fetal bovine serum plus with 100 UI/mL penicillin and 100 μ g/mL streptomycin. A549 cells were maintained in F-12K Nutrient Mixture (Gibco) supplemented with 10% fetal bovine serum plus with 100 UI/mL penicillin and 100 μ g/mL streptomycin. All cells were incubated at 37 °C with 5% CO 2 .  and GTR+ Γ for PB2. All sequences were aligned in MUSCLE (version 3.8.31) 46 , guided by the aligned protein sequences.

Viral growth curves in cells.
Viruses were inoculated into MDCK and A549 monolayers with 10 4 50% egg-infectious-dose (EID 50 ). One hour after infection, the cells were replaced with fresh OPTI-MEM (containing 0.25 μ g/mL TPCK-trypsin for H3 subtype viruses) and incubated at 37 °C. Virus-containing culture supernatants were collected at various time points, hours post-infection (h.p.i), and titrated in eggs. The growth data shown was the average of three independent experiments.
Mouse study. To evaluate virulence and replication of AIVs in mammals, five groups of BALB/c mice (6 weeks old female from Vital River Co. Ltd., Beijing, China) were inoculated with viruses at 10 6 EID 50 in a volume of 50 μ L. To determine the replication of the viruses in mice, three mice were euthanized at 3 days post-inoculation (d.p.i), their nasal turbinates, lungs, kidneys, spleens, and brains were collected for virus titration in eggs. The rest of the mice were observed for disease and death for two weeks.