Pathogenicity and transmission of a swine influenza A(H6N6) virus

Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222 (alanine to valine) and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored.


INTRODUCTION
Influenza A virus (IAV) is an enveloped, segmented, single-and negative-stranded RNA virus belonging to the family Orthomyxoviridae. Migratory waterfowl are the natural reservoirs for IAVs, but these viruses also infect humans, domestic poultry, wild birds, pigs, dogs, cats, horses, mink and marine mammals, including seals and whales 1 Human IAVs bind preferentially to N-acetylneuraminic acid-α2, 6-linked galactose (Neu5Acα2,6-Gal) receptors, whereas avian influenza viruses (AIVs) prefer N-acetylneuraminic acid-α2,3-linked galactose (Neu5Acα2,3-Gal) receptors. [2][3][4] Swine are considered a 'mixing vessel' of IAVs because they have both Neu5Acα2, 3-Gal and Neu5Acα2,6-Gal receptors throughout their respiratory tract. With these receptors, swine facilitate the generation of novel influenza reassortants and enable avian-like IAVs to obtain the ability to bind to human receptors, 5 as has been hypothesized to have occurred during the genesis of viruses that caused the 1957 H2N2 and 1968 H3N2 influenza pandemics. 6 H6-subtype IAVs have been detected in various migratory waterfowl and domestic poultry in Eurasia and North America. 7 Most H6 viruses introduced from waterfowl into domestic poultry have gained only limited spread. However, during 2000-2005, subtype H6N2 IAVs caused illness outbreaks among domestic poultry in CA, USA. [8][9][10] In addition, H6 IAVs have been shown to replicate well in mice without pre-adaptation, indicating that these viruses could cause cross-species infection in mammals. 11,12 Laboratory experiments showed that humans can be infected with H6 IAVs through experimental inoculation. 13 Furthermore, findings from serologic surveillance suggested that veterinarians exposed to H6 IAV-infected domestic birds can become infected with the virus, 14 and in 2013, an avianorigin H6N1 IAV was reported to cause human infection, but there has been no evidence of subsequent human-to-human transmission. 15 Since 2002, H6 IAVs have been one of the predominant IAV subtypes circulating in live bird markets in southern China, [16][17][18] and some of these H6 viruses recognized human receptors. 19 In 2010, after an avian-origin H6N6 swine influenza A virus (SIV) was isolated from sick pigs in southern China, it was found that the virus had been transmitted to and was circulating among the swine population; seroprevalence rates ranged from 1.8% to 3.4%. 20,21 The hemagglutinin (HA) protein of the currently circulating H6N6 SIV has amino acids 222V and 228S, compared with amino acids 222A and 228G in its potential AIV precursors. In other IAV subtypes, HA amino acids 222V and 228S have been reported to affect virus replication in mammals. 7,22 The virus that caused the 2009 H1N1 pandemic, influenza A (H1N1)pdm09, was a swine-origin IAV. 23 After its discovery in humans, this virus quickly moved to swine and other animal populations worldwide. [24][25][26][27][28] During the past few years in southern China, A(H1N1)pdm09 virus has become one of the predominant viruses among domestic swine. [29][30][31] Frequent reassortments between influenza A(H1N1)pdm09 and other endemic SIVs have been observed. 32,33 The aims of our study were to understand the impacts of two acquired mutations in HA of H6N6 virus on its receptor-binding properties and to assess the transmission potential of the H6N6 virus. We also aimed to assess the potential risks posed by reassortants of H6N6 virus with A(H1N1)pdm09 virus because such reassortants would be expected to emerge if both viruses continue to circulate in swine.

Virus and RNA extraction
In 2010, an avian-like H6N6 SIV, A/swine/Guangdong/k6/2010 (H6N6), was isolated from swine in southern China. 20 To study the virus, we used an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) to extract RNA from the isolate; extraction was performed in a Biosafety Level 3 (BSL-3) laboratory.

Cells
Madin-Darby canine kidney (MDCK) cells and human embryonic kidney 293 T cells (both from American Type Culture Collection, Manassas, VA, USA) were used for virus propagation; the cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco/ BRL, Grand island, NY, USA). A549 cells (American Type Culture Collection) used in assays were maintained in Advanced DMEM/F-12 (Gibco/BRL). The medium for each of the three cell lines was supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA), penicillin-streptomycin, and amphotericin B (Gibco/BRL), and the cells were held at 37°C in 5% CO 2 .

Molecular cloning, mutagenesis and reverse genetics
The full-length cDNA for eight genes of A/swine/Guangdong/k6/2010 (H6N6) virus were amplified by using the SuperScript One-Step RT-PCR system (Invitrogen, Carlsbad, CA, USA) and then cloned into a pHW2000 vector. 34 The site-directed mutagenesis on residues 222 and 228 of HA was performed using a QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Primers are available upon request.
Eleven recombinant viruses were generated (Table 1) by reverse genetics as previously described. 35 The recombinant viruses were confirmed by Sanger sequencing at the Life Sciences Core Laboratories Center at Cornell University (Ithaca, NY, USA).

Virus glycan receptor-binding assay
The glycan stock solution (1 mg/mL) was prepared in 50% glycerol in 1 × phosphate-buffered saline (PBS) (v/v), according to the manufacturer's instructions. The protein concentration in viruses was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. For the binding analysis, we further diluted the sialic acid receptors (N-linked glycans, 3′SLN and 6′SLN) and the viruses in a PBS solution (pH = 7.4) containing 0.01% bovine serum albumin and 0.002% Tween-20 (1 × Kinetics Buffer 10 × ; FortéBIO Inc., Menlo Park, CA, USA) with 10 μM neuraminidase inhibitor (zanamivir hydrate; Moravek Inc., Brea, CA, USA) and 10 μM oseltamivir phosphate (American Radiolabeled Chemicals Inc., St Louis, MO, USA). The binding assay was performed by using a FortéBIO Octet K2 interferometer equipped with streptavidin biosensor tips (FortéBIO Inc.). In summary, the biotinylated receptors were first coated onto Plaque assays were performed on MDCK cells in six-well tissue culture plates. Serial dilutions were prepared from the virus stock, and 800 μL of each dilution was incubated in MDCK cells at 37°C with 5% CO 2 for 1 h. The inocula were then aspirated, and the cells were overlaid with 2 mL of 1% agarose containing TPCK-treated trypsin (1.5 μg/mL). Cultures were incubated for 3 days at 37°C and then fixed with methanol and stained with 1% crystal violet to reveal plaques.

Glycan microarray and data analyses
The viruses were purified using 25% sucrose as previously described. 37 The virus labeling, glycan microarray hybridization and data analyses were performed as previously described. 38

Animal experiments
To test the transmissibility of the two testing viruses (rgH6N6-222V/ 228S and rgH6N6 × pdm09-222V/228S), we designed six experiment groups for each virus: three aerosol transmission and three direct contact transmission groups. Two 4-month-old female ferrets (Triple F Farms) were included in each of the 12 groups: one as a virusinoculated ferret to be inoculated intranasally with a testing virus (10 6 TCID 50 viral load in a 1-mL volume) and the other as an exposure ferret to be exposed to the virus through indirect (that is, aerosol) or direct contact with the virus-inoculated ferret. Before the experiments were conducted, all 24 ferrets tested negative for antibodies to rgH6N6-222V/228S (wild-type-like), A/California/04/2009(H1N1), A/Perth/16/2009(H3N2), A/Victoria/361/2011(H3N2) and A/Minnesota/307875/2012(H3N2) influenza viruses. In the aerosol transmission groups, the virus-inoculated and exposure ferrets were housed in the same cage on different sides of a 1-cm-thick, double-layered, steel partition with 5-mm perforations (Allentown Inc., Allentown, NJ, USA). The airflow in the cage went from the exposure ferret to the virus-inoculated ferret. In the direct-contact transmission groups, the virus-inoculated and exposure ferrets were housed together in the same cage without a partition. In all cages, the exposure ferret was put into the cage 1 day after the virus-inoculated ferret was inoculated with a testing virus.
Nasal wash fluids were collected from virus-inoculated ferrets at one and two days post inoculation (DPI) and from exposure ferrets at 1 DPI; thereafter, nasal wash fluids were collected every other day until 10 DPI for both groups of ferrets. Body temperature and weight were measured before nasal wash fluids were collected.
Serum samples were collected from all ferrets at 14 DPI, immediately before they were killed. Virus titers in nasal wash fluids were determined by TCID 50 in MDCK cells and confirmed by 50% egg infectious dose (EID 50 ) in 9-day-old embryonated chicken eggs.
To test the replication efficiency of testing viruses in the ferret respiratory track, we killed two of the three virus-inoculated ferrets in each contact transmission group at 3 DPI. The turbinates, trachea, bronchi and lungs were collected, and virus titers were determined by TCID 50 in MDCK cells.
Biosafety and animal handling All laboratory and animal experiments were conducted under BSL-2 conditions, with investigators wearing appropriate protective equipment, and in compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Mississippi State University.

Phylogenetic analyses
We conducted multiple sequence alignments by using the MUS-CLE software package. 39 We used GARLI version 0.96 40 and maximum likelihood criteria to perform phylogenetic analyses, and we conducted bootstrap resampling analyses with 1000 runs by using PAUP* 4.0 Beta 41 with a neighbor-joining method as described elsewhere. 42

H6N6 SIV differs genetically from the H6N1 AIV that infected humans
Phylogenetic analyses showed that the HA genes from H6N6 SIVs and H6 AIVs, including the strain that caused human infection, belong to different sublineages within a Eurasian lineage ( Figure 1A). The PB2, NP and NS genes of H6N6 and H6N1 viruses belong to the same genetic lineages, but the PB1, PA and MP genes belong to different lineages ( Figures 1B-1H). None of these genes was genetically close to those of influenza A(H1N1)pdm09 virus or other circulating H3N2 and H1N1 SIVs in southern China ( Figures 1B-1H). The HA protein in the H6N6 SIV is 73.2% identical to that of the H6N1 AIV that was isolated from a human in southern China. The HA in the H6N6 SIV has amino acids 222V and 228S, whereas the HA in the human H6N1 AIV has amino acids 222A and 228S; the corresponding progenitors of these SIVs (that is, subtype H6 AIVs) have amino acids 222A and 228G.
H6N6 virus binds to α2,3and α2,6-linked sialic acid receptors, and mutations A222V and G228S affect virus receptor affinity The glycan array, which contained a total of 152 α2,3-linked and α2,6linked glycans, was used to determine the receptor-binding profile of H6N6 SIV and the effect of mutations V222A and S228G on the glycan-binding profile of H6N6 IAV. Results showed that rgH6N6 × Figure 1 Contiued.

Transmission of H6N6 wild-type SIV and rgH6N6 virus possible between ferrets
We used a ferret model to determine the transmissibility of rgH6N6-222V/228S virus by direct and indirect (aerosol) contact. In the directcontact transmission experiment, the rgH6N6-222V/228S virusinoculated ferrets did not show obvious clinical signs of illness. At 1 DPI, nasal wash fluids from these ferrets had virus titers ranging from 10 3.67 to 10 3.83 TCID 50 /mL, and at 2 DPI, titers peaked at 10 4.5 TCID 50 /mL; viral shedding continued until 5 DPI in these ferrets ( Figure 4). HI assay results showed that serum collected from these virus-inoculated ferrets at 14 DPI had virus titers ranging from 1:320 to 1:1280, indicating all ferrets seroconverted (Table 3). Ferrets exposed to the virus-inoculated ferrets through direct contact had no detectable viral shedding when MDCK cells were used for detection; however, one of the three direct-contact ferrets showed seroconversion ( Table 3). As in the direct-contact transmission experiment, rgH6N6-222V/ 228S virus-inoculated ferrets in the aerosol transmission study did not exhibit clinical signs of illness. At 1 DPI, nasal wash fluids from these virus-inoculated ferrets had median rgH6N6 virus titers ranging from 10 3.00 to 10 4.00 TCID 50 /mL, and virus titers peaked at 2 DPI at 10 4.50 TCID 50 /mL; these virus-inoculated ferrets continued to shed viruses until 6 DPI ( Figure 5). HI assay results showed that serum collected from these virus-inoculated ferrets at 14 DPI had virus titers ranging from 1:320 to 1:1280, indicating all ferrets had seroconverted   3′SLN and 6′SLN) and four synthetic N-linked glycans (N32, N33, N52 and N53). 3′SLN, N32 and N52 were used to represent as Neu5Acα2,3-Gal; 6′SLN, N33 and N53 were used to represent as Neu5Acα2,6-Gal.  Table S1.

Risk assessment of H6N6 swine influenza A virus H Sun et al
Risk assessment of H6N6 swine influenza A virus H Sun et al ( Table 3). None of the three exposure ferrets in the aerosol transmission study had detectable viral shedding when MDCK cells were used as the detection method, and HI assay results showed that none of the ferrets had seroconverted before being killed at 14 DPE (Table 3). However, when we used embryonated chicken eggs as the detection method, the nasal wash fluids collected from one of the three ferrets at 6 and 8 DPE had an EID 50 of 10 (Table 4).
To validate the aerosol transmissibility of this H6N6 virus, we repeated the experiment with the wild-type H6N6 isolate at an animal BSL-3 facility. The results from this independent experiment showed that the wild-type H6N6 isolate caused seroconversion in only one of the two direct-contact exposure ferrets and one of the two aerosol-exposure ferrets (data not shown), supporting that H6N6 virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets.

Internal genes of influenza A(H1N1)pdm09 virus did not facilitate transmission of H6N6 virus among ferrets
To assess the risks posed by a potential reassortant rgH6N6 × pdm09 strain, which could result from co-circulating H6N6 SIV and influenza A(H1N1)pdm09 virus, we determined transmissibility of the reassortant virus by direct contact and aerosol contact in ferrets. The three rgH6N6 × pdm09-222S/228V virus-inoculated ferrets had weight loss and slightly elevated body temperatures at 3 DPI, but the ferrets showed clinical recovery from 4 DPI onward. Nasal wash fluids collected from the virus-inoculated ferrets at 1 DPI had virus titers of 10 4.67 -10 6.00 TCID 50 /mL; at 2 DPI, virus titers peaked at 10 6.33 TCID 50 /mL and continued to shed until 6 DPI ( Figure 5). All three virus-inoculated ferrets seroconverted, with HI titers ranging from 1:640 to 1:1280, at 14 DPI (Table 3).
In the direct-contact transmission experiment, two of the three exposure ferrets had no overt signs of illness. However, these ferrets had detectable virus loads (range, 10 4.00 -10 4.50 TCID 50 /mL in nasal wash fluids at 4 DPE (Figure 4), and viral shedding was sustained for at least 5 days. All three direct-contact exposure ferrets seroconverted, with HI titers of 1:640, at 14 DPE (Table 3).
In the aerosol transmission experiment, the exposure ferrets had no detectable viral shedding when MDCK cells were used as the detection method. However, the nasal wash fluids collected from one of the three exposure ferrets at 2 DPE had a virus titer of 10 EID 50 /mL when embryonated chicken eggs were used for detection ( Figure 5; Table 4), but none of these exposure ferrets seroconverted by 14 DPE (Table 3).

Internal genes of influenza A(H1N1)pdm09 virus increased replication efficiency of H6N6 virus in ferret lower respiratory tract
To evaluate the effects of the internal genes on the pathogenesis of H6N6 virus, we inoculated ferrets with rgH6N6-222S/228G and rgH6N6 × pdm09-222S/228G viruses and compared the replication efficiencies of the viruses in ferret respiratory track tissues. Results showed rgH6N6-222S/228G replicated without prior adaptation in ferret nasal turbinate, trachea and lung but not in bronchi (Figure 6). At 3 DPI, the turbinate, trachea and lung tissues of the ferrets inoculated with rgH6N6-222S/228G (wild-type) virus had virus titers of 10 5.53 , 10 3.84 and 10 2.87 TCID 50 /g, respectively. In the ferrets inoculated with rgH6N6 × pdm09-222S/228G virus, the turbinate, trachea, bronchi and lung tissues at 3 DPI had virus titers of 10 6.00 , 10 3.67 , 10 4.26 and 10 3.59 TCID 50 /g, respectively. Thus, the virus titers in respiratory tract tissues, especially lower respiratory tract tissues, such   15,43,44 and to H6N6 viruses in swine. 20 Virus mutation and reassortment rates have been key measures in virologic risk assessments of influenza. 45 The presence of genetically diverse H6 IAVs and active evolutionary events increases the possibility for a virus of this subtype to develop pandemic potential and present a risk to public health. Compared with its progenitor AIV, H6N6 SIV has mutations 222V and 228S in the 220-loop of its HA. Mutations in this 220-loop (for example, at residues 222, 225, 226 and 228) have been shown to affect receptor-binding specificity. For example, mutations Q226L and G228S enabled H3 viruses to bind sialic acid α2,3-Gal and sialic acid α2,6-Gal receptors. 46 The D222G substitution (corresponding to residue 225 in H3 viruses) enabled influenza A(H1N1)pdm09 viruses to acquire dual receptor specificity for complex α2,3-linked and α2,6linked sialic acids; the substitution also increased the virulence of this   Figure 6 Mean titers of influenza viruses recovered from respiratory tract tissues of ferrets after nasal inoculation of virus. Ferrets were inoculated with 10 6 50% tissue culture infectious doses (TCID 50 ) of each virus shown: the wild-type-like rgH6N6-222V/228S and rgH6N6xpdm09-222V/228S. Two ferrets were killed three days after inoculation, and virus titers in nasal turbinate, trachea, bronchus and lung of each ferret were determined by using end-point titration in Madin-Darby canine kidney cells. The titers were quantified as the mean titer from at least three sections of each tissue. The limit of virus detection was 10 0.699 TCID 50 /mL. The dashed line indicates the lower limit of detection. Black bars represent error bars.
virus. 47 We recently showed that the W222L substitution in HA could facilitate infection of H3N2 IAV in dogs, possibly by increasing the binding affinity of the virus to canine-specific receptors with Neu5Acα2,3-Galβ1,4-(Fucα-) or Neu5Acα2,3-Galβ1,3-(Fucα-)-like structures. 38 In addition, mutation W222R in HA can increase influenza virus infectivity in mice 48,49 by introducing a hydrogen bond between the virus HA and the host glycan receptor. 50 Previous studies showed that turkey and chicken erythrocytes express α2,3and α2,6-linked sialic acids that horse erythrocytes almost exclusively express α2,3-linked sialic acids, 51 and that guinea pig erythrocytes disproportionately express α2,3and α2,6-linked sialic acids. 52 The receptor-binding profiles we obtained in this study with turkey, chicken, guinea pig, dog and horse erythrocytes suggest that mutations V222A and G228S, which are associated with a change in affinity from avian to swine, especially mutation G228S, changed receptor-binding specificity. This mutation G228S led to a minimum 16-fold increase of receptor-binding affinities for guinea pig and horse erythrocytes and a minimum two-fold decrease in binding affinities to chicken erythrocytes (Table 2). Thus, mutation G228S could affect virus binding to both α2,3-linked and α2,6-linked sialic acids, but the effects on α2,6-linked sialic acids were significantly higher than that on α2,3-linked sialic acids. These results were confirmed by the virus glycan-binding assay results, which showed that wild-type H6N6 virus can bind to α2,3and α2,6-linked sialic acids and that substitution G228S (avian to swine) significantly increased the binding affinities of H6N6 IAV to three α2,6-linked sialic acids that we tested (Figure 2). In addition, glycan microarray data analysis confirmed that mutations A222V and G228S (avian to swine) can increase HA-binding affinities to glycans, including α2,6-linked sialic acid (Figure 3). The results from virus glycan binding were consistent with those using recombinant HA proteins of H6N1 human influenza virus, which has amino acid 228S. 53 In summary, these findings indicated that mutations A222V and especially G228S, could facilitate the binding ability of H6N6 virus to α2,6-linked sialic acids. The conservation of S228 between the H6N1 human isolate and H6N6 SIV suggests that the G228S mutation likely facilitated transmission of H6 IAV to swine or humans.
In H6N6 SIV-infected and rgH6N6 virus-infected ferrets, virus was shed at high titers until 6 DPI, similar to viral shedding by other H6 viruses. 11,12 Previous studies in ferrets showed that H6N2 AIVs replication was more efficiently in lungs than nasal turbinates at 5 DPI. 11 However, our findings show that the minimum H6N6 SIV load in nasal turbinates was 10-fold higher than that in lungs at 3 DPI ( Figure 6). In our experiments, it is likely that H6N6 SIV replicated better in the upper than lower respiratory tract of ferrets; such a situation would lead to constant viral shedding and to transmissibility of this virus among ferrets. Additional experiments are needed to understand whether mutations A222V and G228S (avian to swine) alter viral tissue tropisms in ferrets.
A number of studies have shown that AIVs, such as highly pathogenic H5N1 and low pathogenicity H9N2 viruses, have increased aerosol transmissibility after acquiring the internal genes of influenza A(H1N1)pdm09 virus. [54][55][56] However, the results of our transmission studies showed that the internal genes of A(H1N1)pdm09 virus did not increase transmissibility of H6N6 SIVs through aerosol or direct contact. Nonetheless, it is possible that additional mutations acquired through adaptation or genetic reassortments between H6N6 SIV and influenza A(H1N1)pdm09 virus would increase the transmissibility of H6N6 SIV.
In the pathogenesis studies, virus replicated in lungs of rgH6N6 virus-infected ferrets, but the ferrets exhibited no obvious signs of illness. Virus titers in lungs of rgH6N6 × pdm09 virus-infected ferrets were ≥ 5-fold higher than those in rgH6N6 virus-infected ferrets. In addition, ferrets infected with rgH6N6 × pdm09 virus showed slightly elevated temperatures and weight loss at 1 and 2 DPI.
We showed that the method used for detecting virus loads in nasal wash fluids or tissues affects the accuracy of the results. For example, embryonated chicken eggs were more sensitive than MDCK cells in detecting H6N6 SIVs in the virus transmission group: viruses in aerosol-exposed ferrets were detectable in embryonated chicken eggs but not in MDCK cells (Table 4).
In summary, our findings suggest that subtype H6N6 virus can bind to α2,6-linked sialic acids, indicating H6N6 virus as a virus with zoonotic potential. Although H6N6 SIV has limited transmissibility between ferrets and probably cannot yet be transmitted between ferrets through infectious aerosolized droplets, the virus could evolve into a more transmissible H6 virus through additional adaptation and reassortment. Thus, evolution of this H6N6 virus and other H6 AIVs should be closely monitored.