Characterization of an H9N2 avian influenza virus from a Fringilla montifringilla brambling in northern China

Virology 476 (2015) 289–297

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Characterization of an H9N2 avian influenza virus from a Fringilla montifringilla brambling in northern China$ Jing Yuan a,b,c, Lili Xu a,b,c, Linlin Bao a,b,c, Yanfeng Yao a,b,c, Wei Deng a,b,c, Fengdi Li a,b,c, Qi Lv a,b,c, Songzhi Gu a,b,c, Qiang Wei a,b,c,nn, Chuan Qin a,b,c,n a

Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Comparative Medicine Center, Peking Union Medical College, Beijing, China Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Beijing, China c Key Laboratory of Animal Models of Human Diseases, State Administration of Traditional Chinese Medicine, Beijing, China b

art ic l e i nf o

a b s t r a c t

Article history: Received 22 October 2014 Returned to author for revisions 4 December 2014 Accepted 10 December 2014

Avian H9N2 influenza viruses circulating in domestic poultry populations are occasionally transmitted to humans. We report the genomic characterization of an H9N2 avian influenza virus (A/Brambling/Beijing/ 16/2012) first isolated from a healthy Fringilla montifringilla brambling in northern China in 2012. Phylogenetic analyses revealed that this H9N2 virus belongs to the BJ/94-like sublineage. This virus had a low pathogenicity for chickens and was able to replicate at a low level in mouse lung tissue. Transmission studies in ferrets showed that this H9N2 strain shed high levels of the virus in nasal and throat swabs. In vitro receptor binding assays, the virus bound only to α-2,6 linkage receptors and not to the avian-type α-2,3 linkage receptors, suggesting that H9N2 influenza viruses present potential public health risks. Therefore, attention should be paid to H9N2 influenza viruses and the close surveillance of H9N2 viruses in poultry. & 2014 Elsevier Inc. All rights reserved.

Keywords: H9N2 avian influenza virus Pathogenesis Ferret Transmission Droplet spread Receptor-binding properties

Introduction Avian influenza viruses are influenza A viruses belonging to the family of Orthomyxoviridae which are categorized into 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes according to the antigenicity of the surface glycoproteins HA and NA (Tong et al., 2012; Webster et al., 1992). Among these subtypes, the H9N2 subtype is of great concern because it is endemic in the poultry populations across Asia and the Middle East and has occasionally been transmitted from poultry to mammalian species (Butt et al., 2005; Lin et al., 2000; Peiris et al., 2001). Furthermore, phylogenetic analyses revealed that H9N2 viruses were the donors

☆ GenBank accession numbers: These newly identified sequences were deposited into GenBank under the accession numbers KC464595 to KC464602. n Correspondence to: Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Comparative Medicine Center, Peking Union Medical College, Pan Jia Yuan Nan Li No. 5, Chao Yang District, Beijing 100021, China. Tel.: þ 86 10 67761942; fax: þ 86 10 67761943. nn Correspondence to: Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Comparative Medicine Center, Peking Union Medical College, Pan Jia Yuan Nan Li No. 5, Chao Yang District, Beijing 100021, China. Tel.: +86 10 67761942; fax: +86 10 6776194. E-mail addresses: [email protected] (Q. Wei), [email protected] (C. Qin).

http://dx.doi.org/10.1016/j.virol.2014.12.021 0042-6822/& 2014 Elsevier Inc. All rights reserved.

of the internal genes of the H5N1 viruses in Hong Kong in 1997 (Guan et al., 1999) and the novel H7N9 viruses in mainland China in 2013 (Gao et al., 2013; Kageyama et al., 2013). Recent research also demonstrated that H9N2 viruses replicate efficiently in experimental mice without adaptation (Choi et al., 2004) and can be transmitted via respiratory droplets in ferrets after obtaining the internal genes of the 2009H1N1 pandemic (Kimble et al., 2011). However, to date, limited data are available to describe the relative infectivity and transmissibility of H9N2 viruses. Therefore, surveillance of the H9N2 virus in poultry is required for us to better understand the ecology and epidemiology of AIV and the potential risk that these viruses pose to human health. In November 2012, we first isolated an H9N2 virus (A/Brambling/ Beijing/16/2012) from a wild Fringilla montifringilla brambling in northern China, and phylogenetic analyses revealed that this H9N2 virus was the donor of the internal genes of the novel H7N9 viruses in mainland China in 2013 (Liu et al., 2013). F. montifringilla are very common wild birds in China. Huge numbers of them inhabit the plains, hills, mountains, and forests. The brambling is a migratory bird that flies south for wintering. They have many opportunities for close contact with human beings. These characteristics suggest that the viruses that infect these birds could have many opportunities to infect humans.

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The ferret model has been widely used to study the transmission of the H5, H7, and H9 subtypes of avian influenza viruses (AIV), as well as the 1918H1N1 and 1957H2N2 viruses (Belser et al., 2007; Herlocher et al., 2001; Maines et al., 2006, 2005; Salomon et al., 2006; Shinya et al., 2005; Yen et al., 2007). Ferrets are susceptible to infection with human influenza viruses and develop some symptoms of influenza that closely resemble those observed in humans. Importantly, the respiratory tract of ferrets expresses predominantly human-type receptors and is thus very similar to human respiratory epithelia (van Riel et al., 2007; Xu et al., 2010). However, no airborne transmission of wild H9N2 AIV has been reported in mammals (Wan et al., 2008). In this study, we report the genomic information of an H9N2 avian influenza virus first isolated from a brambling. Phylogenetic analyses reveal that it belong to the BJ/94-like sublineage. Because this H9N2 virus was the donor of the internal genes of the novel H7N9 viruses in mainland China in 2013 (Liu et al., 2013), we systematically examined the pathogenicity, transmissibility, and receptor binding specificity of this new ancestor virus. This comprehensive characterization of emerging host viruses is necessary to understand the evolution of H9N2 viruses and provides information that is important for preventing and controlling potential pandemics in the future.

Results Phylogenetic and sequence analysis The H9N2 viruses that have recently circulated in China are primarily descendants of the BJ/94-like and G1-like genotypes (Bi et al., 2010; Sun et al., 2010; Xu et al., 2007; Zhang et al., 2011). Phylogenetic analyses based on the HA protein showed that the A/Brambling/Beijing/16/2012 virus belongs to the BJ/94-like (A/chicken/Beijing/1/1994) sublineage (Fig. 1), which demonstrates that BJ/94-like viruses may be the primary epidemic H9N2 strains currently circulating in northern China. The amino acid sequence of the HA cleavage site is RSSR/GLF. This sequence contains two basic amino acids but is still characteristic of a low-pathogenic AIV (Callan et al., 1997; Guo et al., 2000). Eight potential glycosylation sites were observed in the HA protein. A potential glycosylation site at amino acid residue 313 was observed in the new isolate, suggesting this mutation may affect virus-induced cell fusion and its receptor binding ability (Kaverin et al., 2004). The receptor-binding pocket of HA1 has the key amino acid L226 (H3 numbering), thereby providing H9N2 AIV with the ability to bind to the α2,6-linked sialic acid receptors, which enables it to infect and replicate within mammalian hosts

Fig. 1. The phylogenetic analyses for the HA and NA genes of the A/Brambling/Beijing/16/2012 virus in comparison with other H9N2 strains. (A) An analysis based on the HA gene. (B) An analysis based on the NA gene.

J. Yuan et al. / Virology 476 (2015) 289–297

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Pathogenicity of the H9N2 isolates in white leghorn chickens

(Matrosovich et al., 2001; Wan et al., 2008). The new isolate contains the HA and PA genes with HA-K363 (H9 numbering) and PA-L672. A recent mutational analysis showed that the transmissibility phenotype predominantly mapped to the HA and PA genes and that HA-K363 and PA-L672 are important for airborne transmissibility among chickens. This analysis also showed that the recent H9N2 viruses with 672L in PA and 363K in HA are predominant in China (Zhong et al., 2014), suggesting the potential for the airborne transmission of H9N2 strains. However, the new isolate does not contain the E627K or D701N mutations (Li et al., 2005b) in basic polymerase 2 (PB2), so it retains avian virus characteristics. Furthermore, there are amino acid substitutions in 31S in M2, indicating that the new isolate might be resistant to amantadine (Suzuki et al., 2003). It is worth noting that there is an N383D substitution at residue 383 in acidic polymerase (PA) that may increase the virulence of the virus (Song et al., 2011). In the present study, a substitution of V149A was found in the NS1 sequence of the new isolate; this substitution may affect the ability of the virus to antagonize interferon induction in chicken embryonic fibroblasts (Li et al., 2004). However, all of these viral characteristics that have been predicted based on the amino acid sequences must be verified experimentally (Table 1).

H9N2 viruses are typically associated with egg production drops and occasionally with 10–20% mortality in the field (Aamir et al., 2007; Capua and Alexander, 2004; Choi et al., 2004). To understand the pathogenicity of this H9N2 virus in chickens, 6-week-old white leghorn SPF chickens were inoculated i.n. with 0.1 ml 107 EID50 of virus and observed for 21 days for clinical signs and death. This virus did not induce clinical signs or deaths in chickens during the observation period of 3 weeks, except for inactivity and a slight decrease in appetite. On day 1 p.i., virus was isolated from the oropharyngeal cavity of ten chickens and from the cloaca of five chickens. At 5 days post-infection, shedding viruses were detected in the oropharyngeal cavity of ten chickens, and eight chickens continued to shed virus from the cloaca. At 7 days post-infection, nine and six chickens were shedding virus from the oropharyngeal cavity and cloaca, respectively. The virus titers in the oropharyngeal and cloacal swabs peaked at three days post-infection. Moreover, the virus replicated better in the trachea compared with the intestine and to relatively higher titers, indicating that the H9N2 influenza virus primarily spreads via the respiratory route. All of the chickens seroconverted by day 21 post-inoculation (Table 2). Pathogenicity of the H9N2 isolates in mice Avian influenza viruses were considered to be restricted to replication in avian species, but H9N2 avian influenza viruses have been previously isolated from pigs and humans (Guo et al., 1999; Peiris et al., 1999; Xu et al., 2004). We used BALB/c mice as a model to evaluate the ability of the H9N2 virus to replicate in mammalian hosts. Five-week-old BALB/c mice were inoculated i.n. with 107 EID50 of the H9N2 virus, organs from 4 mice were collected on days 3, 5, and 7 p.i. for virus titration, and 10 mice were observed for 2 weeks. This virus could only be detected in the mouse lungs with a peak titer of 1.5 log10EID50/100 ml on day 3 post-inoculation, and the inoculated mice stayed healthy and continue to gain weight during

Table 1 Molecular characterization at selected sites of HA, PA, NS1, and M2 of the A/ Brambling/Beijing/16/2012 virus based on the mammalian adaptation of molecular markers. Genotypes

Cleavage site

Amino acid position HA

BJ/94-like

RSSR/GLF

226 L

PA 363 K

PB2

672 L

383 D

627 E

701 D

M

NS1

31 S

149 A

Table 2 Pathotyping and replication of the H9N2 virus in chickensa. Days postinfection

Virus isolated from swabs Oropharyngeal

1 3 5 7

No. of survivors/ no. tested

No. of seroconverted chickens/no. tested

HI titer (log10)c

10/10

10/10

3.78 7 0.21

Cloacal

No. of chickens shedding virus/no. tested

Titerb (log10 EID50/100 ml)

No. of chickens shedding virus/no. tested

Titerb (log10 EID50/100 ml)

10/10 10/10 10/10 9/10

3.197 0.05 4.677 0.07 2.69 7 0.12 0.54 7 0.07

5/10 10/10 8/10 6/10

0.58 7 0.07 3.217 1.12 2.32 7 0.26 0.65 7 0.17

a One group of ten 6-week-old specific-pathogen-free white leghorn chickens was inoculated intranasally with 107 EID50 of the virus in a 0.1 ml volume and observed for 3 weeks after infection. b The mean titer in log10EID50/100 ml of swab media of the positive chickens. c Sera were harvested 3 weeks after infection, and seroconversion was confirmed by the HI test.

Table 3 The viral distribution of avian H9N2 influenza virus in different tissues in micea. Days post-infection

Virus titer (log10 EID50/g, mean7 SD) Heart

3 5 7

– – –

b

Liver

Spleen

Lung

BALF

Kidney

Brain

Intestine

– – –

– – –

1.5 70.00 1.5 70.00 –

1.43 7 0.12 1.21 70.51 –

– – –

– – –

– – –

a SPF mice were intranasally inoculated with the A/Brambling/Beijing/16/2012 virus at 107 EID50. On days 3, 5, and 7 after infection, four mice were sacrificed for virus titration. The results are expressed as the means 7 SD. b –, Not detectable.

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the observation period. No viruses were recovered from the other organs, and no deaths were observed (Table 3). Sera were harvested from the ten observed mice at 14 days p.i., and seroconversion was confirmed by the HI test. Seroconversion was found in all of the mice with HI titers of 640–1280.

Pathogenicity and transmissibility in ferrets Two groups of three ferrets each were inoculated with 107 EID50 of A/Brambling/Beijing/16/2012 or 106 EID50 of A/California/ 07/2009 (H1N1). At 24 h post-inoculation, each inoculated animal was housed individually with a naïve ferret in a transmission cage. The ferrets were monitored as described in the Materials and methods section. Sneezing was not observed in any of the ferrets inoculated with the H9N2 viruses. However, lethargy and anorexia were noted in two of the inoculated ferrets, and these conditions usually lasted 2 to 3 days. The inoculated ferrets experienced a slight body weight loss (average o 7.5%) over the course of 9 days (Table 3 and Fig. 2A (left)). A transient elevation in body temperature was detected in all three H9N2-infected ferrets. The temperatures were highest between 2 to 4 days p.i. when a majority of the ferrets showed peak viral shedding in the nasal and throat swabs (Fig. 2A (right)).

As shown in Fig. 2B, virus shedding from the upper respiratory tract began at 1 d.p.i. and continued until 7 d.p.i. By day 14 p.i, the inoculated ferrets developed high titers of anti-H9 antibodies (5120, 5120, and 10240 for the inoculated ferrets). However, the virus was not transmitted from the inoculated ferrets to the naïve ferrets via respiratory droplets. No virus could be detected in either the nasal or throat swabs from the ferrets in the airborne contact group (Fig. 2B and Table 4), and the convalescent sera (21 d.p.e.) of the contact ferrets was negative for virus-specific antibodies according to the HI assay, indicating a lack of aerosol transmission of this virus. To validate our system of detecting aerosol transmission, we undertook transmission studies performed using the prototypic human virus, A/California/07/2009 (H1N1). High titers of virus were detected in all of the ferrets inoculated with this virus (Fig. 2B). The aerosol contacts began shedding virus by day 1 p.e. and continued to shed virus for up to 5 days. All of the ferrets showed clinical signs, including sneezing, and developed high antibody titers against A/California/07/2009 (H1N1) (Fig. 2B and Table 4). Receptor-binding properties HA receptor specificity plays an important role in the transmission of influenza viruses (Belser et al., 2007; Maines et al., 2009; Sun

Fig. 2. The pathogenicity and transmissibility of the A/Brambling/Beijing/16/2012 virus in ferrets. (A) Left: body weight changes of the pre-inoculated ferrets. The data are expressed as each ferret relative to the value (100%) before inoculation. Right: temperature changes of the pre-inoculated ferrets. (B) Viral shedding by the inoculated or airborne contact ferrets as assessed by the nasal and throat swabs. The data are presented as the mean7 SD of the log10 EID50/ml. The dashed red line in these panels indicates the lower limit of detection. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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293

Table 4 The transmission of A/Brambling/Beijing/16/2012 or A/California/07/2009 (H1N1) influenza viruses between ferrets. Virus

A (H9N2) A (H1N1) a b c

Seroconversionc Airborne contacts

Inoculated ferrets Sneezing

Virus shedding

Onset of shedding (d.p.i.)a

Peak titer (Log10 EID50/ml) b

0/3

3/3

1

5.833

3/3

3/3

1

5.833

Seroconversionc

Sneezing

Virus shedding

Onset of shedding (d.p.e.)

0/3

0/3

–

–

0/3

3/3

3/3

1

4.833

3/3(1280)

3/3(5120, 10,240) 3/3(1280)

Peak titer (Log10 EID50/ml)

Virus titers in the nasal and throat swabs. Calculated as log10 EID50/ml of the nasal or throat swabs. Homologous virus was used in the HI assays to detect anti-H9 and anti-H1 antibodies.

Fig. 3. The receptor-binding assays for the A/Brambling/Beijing/16/2012 virus. (A) The hemagglutination assays for the H9N2 influenza viruses using cRBCs with different treatments. This figure shows the HA titers of the test viruses with 0.5% cRBCs treated as follows: cRBCs, untreated; desial cRBCs, treated with VCNA; α-2,3 cRBC, VCNA treated and resialylated with α-2,3 glycans; α-2,6 cRBC, VCNA treated and resialylated with α-2,6 glycans. (B) Direct binding assay with synthetic sialylglycopolymers. Left: affinity to synthetic α-2,6-linked sialic acids. Right: affinity to synthetic α-2,3-linked sialic acids.

et al., 2010; Wan et al., 2008). Affinity of the viral HA protein for α2,6-glycan is required for the transmission of human influenza virus among ferrets (Belser et al., 2007; Maines et al., 2006). We examined the receptor-binding specificity of this virus in hemagglutination assays using resialyated cRBCs. The A/Brambling/Beijing/16/2012 virus only bound to cRBCs resialylated with α-2,6-glycans. The two control viruses, CA/04(H1N1) and AH/05(H5N1), selectively bound to the α-2,6 and α-2,3 substrates, respectively, confirming the specificity and validity of the hemagglutination assays using resialyated cRBCs (Fig. 3A). Chandrasekaran et al. (2008) reported that the sialylated glycans in the α-2,3 and α-2,6 linkages have different topologies and that the HAs of H1N1 and H3N2 influenza viruses that have adapted to humans specifically bind to the long α-2,6 glycan topology, whereas the HA of the H5N1 viruses (e.g., A/Vienam/ 1203/04 and A/Hong Kong/486/97) does not. To further confirm the receptor-binding properties of this H9N2 virus, we tested the binding of the HAs of the H9N2 virus with different glycans using a solid-phase binding assay. As shown in Fig. 3B, the A/Brambling/Beijing/16/2012 virus bound to only the α-2,6 glycan but not the α-2,3, consistent with the result of the

receptor-binding analysis that was obtained using hemagglutination assays.

Discussion The H9N2 AIV has resulted in enormous economic losses since its discovery in China in 1994(Guo et al., 2000; Xu et al., 2007). Although the H9N2 viruses have been detected in chickens and ducks in many provinces in China since 1993(Li et al., 2005a; Zhang et al., 2009), their low pathogenic nature to poultry has made them a low priority for animal disease control. More seriously, the H9N2 viruses caused human infections in China in 1999, 2003, and 2013 (Butt et al., 2005; Peiris et al., 1999; WHO, 2014), and some poultry workers in China, India, Cambodia, Romania, America, Nigeria, and Vietnam were reportedly serologically positive for H9N2 viruses (Blair et al., 2013; Coman et al., 2013; Gray et al., 2011; Okoye et al., 2013; Pawar et al., 2012; Uyeki et al., 2012; Wang et al., 2009; Wang et al., 2014), implying a substantial threat to public health. Recent studies indicated that the H9N2 viruses contributed the six internal genes to the newly emerged H7N9 virus in southern China and to

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Fig. 4. Schematic presentation of paired transmission cages. The transmission cages were specifically designed to allow transmission experiments to be conducted in negatively pressurized isolator cages (0.8 m  0.8 m  0.8 m) in an animal biosafety level 3 facility. Three ferrets were inoculated with 107 EID50 of A/Brambling/Beijing/16/ 2012 or 106 EID50 of A/California/07/2009 (H1N1). The ferrets were housed in cages in which each inoculated animal was housed individually next to a naive ferret. Each ferret cage was 40 cm wide  40 cm high  45 cm long, and the 2 cages were separated by 2 stainless steel grids (1), with a grid size of 0.5 cm2, 8 cm apart. Negative pressure within the isolator cage was used to direct a modest (o 0.1 m/s) flow of high-efficiency particulate air (HEPA)–filtered air (2) from the inoculated ferret to the naive ferret. The outlet airflow (3) was HEPA filtered to prevent the continuous circulation of infectious influenza A virus particles and to prevent cross-contamination. Arrows indicate the airflow direction.

the H10N8 virus that caused three human infections in Jiangxi province, China (Chen et al., 2014; Gao et al. , 2013; Zhang et al., 2013). These facts prompted us to assess the biologic properties and pandemic potential of H9N2 influenza viruses circulating in poultry. Limited human-to-human transmission of viruses with the H9N2 subtypes has occurred (Butt et al., 2005; Guo et al., 1999; Peiris et al., 1999). This avian influenza virus does not spread readily from person to person. Therefore, avian influenza viruses must overcome a host range restriction to become established in the human population. The HA glycoprotein is most likely a major determinant of host switching, primarily because of its role in host cell receptor recognition (Glaser et al., 2005; Matrosovich et al., 2000; Rogers and Paulson, 1983). Human influenza virus strains preferentially bind to oligosaccharides that terminate with sialic acid linked to galactose by α-2,6-linkages (Sia-α2,6-Gal), whereas the HAs of avian influenza virus strains prefer oligosaccharides that terminate with a sialic acid linked to galactose by α-2,3-linkages (Sia-α-2,3Gal) (Connor et al., 1994; Rogers and Paulson, 1983; Stevens et al., 2006). A shift from Sia-α-2,3-Gal binding to Sia-α-2,6-Gal-binding specificity is most likely a critical step in the adaptation of avian influenza viruses to human hosts (Herfst et al., 2012). To evaluate whether the H9N2 virus has the potential to be transmitted among humans through the upper respiratory tract, we examined the receptor-binding properties of this virus using hemagglutination assays and the solid-phase binding assay. Remarkably, this H9N2 avian influenza virus, first isolated from F. montifringilla, binds only to α-2,6 linkage receptors and not to the aviantype α-2,3 linkage receptors. These viruses displayed a human, but not an avian, virus-like receptor specificity. This effect clearly correlated with the presence of Leu-226 in the RBS of these viruses. Studies of the earliest virus isolates from human pandemics and swine epidemics demonstrated that these viruses possess a human viruslike receptor specificity and suggested that a switch in receptor specificity is an essential element (in addition to changes in other viral genes) in the initiation of a pandemic (Connor et al., 1994; Matrosovich et al., 2000). Because of these findings and the human virus-like receptor specificity of the poultry H9N2 viruses, this virus may be considered a particularly plausible candidate for the generation of new pandemic strains. Human-to-human transmission of influenza viruses can occur through direct contact, indirect contact via fomites (i.e., contaminated environmental surfaces), and/or airborne transmission via small aerosols or large respiratory droplets. The pandemic and epidemic influenza viruses that have circulated in humans over the past century were all transmitted via the airborne route, in contrast to many other respiratory viruses that are transmitted exclusively via contact (Herfst et al., 2012). In this study, we observed that this virus could not be transmitted between ferrets via respiratory droplets.

This study shows that although the efficient transmission of avian influenza viruses in a ferret model requires the adaptation of HA from the avian-type to human-type receptor specificity, other viral factors are also important determinants of virus transmission. For example, amino acid changes in the PB2 protein are associated with mammalian adaptation, efficient transmission via respiratory droplets between ferrets, and replication in human cells (Bussey et al., 2010; Hatta et al., 2001; Li et al., 2005b; Mehle and Doudna, 2009; Yamada et al., 2010). The NA protein is also likely to contribute to viral transmissibility, as shown by Chen et al. (2012). During the budding process, the NA protein cleaves sialic acids from cellular receptors to facilitate the release of viral particles from the infected cell surface. An optimal interplay between the activities of HA and NA is required for efficient virus replication (Wagner et al., 2002). In addition, a recent study reported that the balance between HA and NA activities is critical for the efficient respiratory droplet transmission of a pandemic 2009H1N1 virus in ferrets (Yen et al., 2011). In addition to the receptor-binding preference conferred by HA, internal gene combinations also play a determinative role in virus transmissibility in mammals (Zhang et al., 2013). Similar to other avian influenza viruses circulating in poultry in Southern China (Chen et al., 2004; Deng et al., 2013; Li et al., 2010; Wang et al., 2014), the H9N2 viruses formed multiple genotypes. The DK/ZJ/C1036/09-like internal gene combination was detected in the H9N2 viruses with different groups of HA and NA genes that were isolated between 2009 and 2013 in nine of the 12 provinces investigated, and was also detected in the H7N9 and H10N8 viruses that have infected humans (Chen et al., 2014; Zhang et al., 2013), suggesting that this predominant internal gene combination is more stable and compatible with different surface genes. Therefore, the H9N2 viruses pose a threat to human health not only because they will likely cause new influenza pandemic, but also because they can transfer different subtypes of influenza viruses from avian species to humans. Despite this restriction in aerosol transmission, four key factors in avian H9N2 viruses should be noted. First, a number of studies have demonstrated that H9N2 viruses are undergoing extensive evolution and reassortment (Kimble et al., 2011; Li et al., 2005a; Sorrell et al., 2009; Xu et al., 2007), fueling their pandemic potential, including the novel H7N9 viruses that caused the 2013 outbreak in China (Liu et al., 2013). It is worth noting that the most closely related genes, PB2, PB1, and PA, were all from this A/brambling/Beijing/16/2012(H9N2) virus. Second, there have been several lines of evidence that H9N2 viruses have transmitted to pigs (Peiris et al., 2001; Xu et al., 2004). Numerous recent H9N2 isolates contain a human-like amino acid residue at position 226 (i.e., 226L) in their HAs and show preferential binding to a-2,6-glycan receptors (Matrosovich et al., 2001). Furthermore, 226L-containing H9N2 viruses have been shown to replicate

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efficiently in differentiated human airway epithelial cells (Wan and Perez, 2007). Third, serological data from separate studies suggest that there may be more human cases of H9N2 infection than previously predicted (Guo et al., 1999; Peiris et al., 1999) and that the possibility of a limited level of human-to-human transmission cannot be absolutely excluded (Butt et al., 2005). Fourth, the fact that H9N2 influenza viruses are not highly pathogenic for poultry makes them more, rather than less, likely to contribute to a pandemic (Webster et al., 1992). In fact, viruses that are less pathogenic for poultry have a greater opportunity to become widespread because they do not raise concern but instead permit their hosts to survive unhindered. Thus, they are free to continue to reassort and are more likely to have an opportunity to find the best genetic combination (Webster et al., 1992) that permits the infection of humans and facilitates further person-to-person transmission. Two previous pandemic strains (H2N2 in 1957 and H3N2 in 1968) were also derived from viruses with low pathogenicities (Horimoto and Kawaoka, 2001). Therefore, avian H9N2 viruses are in an ideal position to undergo further adaptation for more efficient transmission among mammals and humans. In summary, we report the genetic characterization of an H9N2 avian influenza virus first isolated from a clinically normal F. montifringilla brambling in northern China in 2012. This H9N2 virus had a low pathogenicity for chickens and was able to replicate in the lung tissues of mice, though at a low level, and the virus was able to replicate in the respiratory tract of ferrets. However, efficient aerosol transmission was not observed between ferrets. This virus had overcome host range restriction to bind to human receptors. However, considering the widespread prevalence of H9N2 viruses in poultry, the human virus-like receptor specificity of some avian and swine H9N2 isolates, co-circulation of H9N2 with H3N2 viruses in Asian swine, and the repeated direct transmission to humans, the public health threat of H9N2 viruses cannot be overemphasized. Further studies should aim to dissect the molecular constraints that limit aerosol transmission of H9N2 viruses and the natural glycan profiles of the mammalian respiratory tract.

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poultry market in Zhejiang Province in April 2005. This virus was also propagated in the allantoic sacs and amniotic cavities of 9day-old embryonated chicken eggs and then passaged once in MDCK cells. Cells MDCK cells were obtained from the ATCC and were tested for mycoplasma contamination. The cells were maintained in Eagle’s minimal essential medium (MEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml of streptomycin and were cultured at 37 1C with 5% CO2. All viruses were propagated in Madin–Darby canine kidney (MDCK) cells for the solid-phase binding assay. Sequencing and phylogenetic analysis Viral RNA was extracted directly from the allantoic fluid using an RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany). The RNA (11 μl) was reverse transcribed into cDNA using the Uni12-primer (AGCAAAAGCAGG) and a SuperScript III reverse transcriptase (200 U) reaction mixture (20 μl) (Invitrogen). PCR was performed using specific primers for eight genes (primer sequences are available upon request) to sequence the full-length genome (Hoffmann et al., 2001). The PCR products were purified and sequenced (Invitrogen, Shanghai, China). We performed sequencing using an ABI 3730  l automatic DNA analyzer (Life Technologies, USA) and the ABI BigDye Terminator v3.1 cycle sequencing kit (Life Technologies, USA) according to the manufacturer’s recommendations. A neighbor-joining phylogenetic tree based on the amino acid sequence of the HA or NA protein was constructed in MEGA5 using the Jones–Taylor–Thornton amino acid replacement model with 1000 bootstrap replicates. Pathogenic analysis

Materials and methods The experimental protocol was evaluated and approved by the Institute of Animal Use and Care Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (ILAS-PC2013-010). For all experiments involving live viruses, the animals were housed in negative-pressure isolators with HEPA filters in a biosafety level 3 (BSL3) animal facility in accordance with the institutional biosafety manual. Virus Pooled lung, trachea, liver, cecal tonsil, and gizzard tissue from a F. montifringilla brambling in northern China in 2012 were injected into embryonated chicken eggs, and the avian influenza virus A/Brambling/Beijing/16/2012(Br/BJ/12(H9N2)) was isolated from lung samples according to a previously reported method (Lv et al., 2011). The viruses were purified by three rounds of limiting dilution in 9-day-old SPF embryonated chicken eggs that were incubated at 37 1C for 48 h. Aliquots of the virus were stored at  80 1C. The median egg infectious dose (EID50) of each virus was determined in SPF embryonated chicken eggs, and the titers were calculated using the Reed– Muench method (Reed and Muench, 1938). A/California/04/2009 (CA/04(H1N1)) and A/California/07/2009 (H1N1) were the prototypic strains of the 2009 pandemic A (H1N1) influenza viruses that were collected in the United States in California during April 2009. The AH/05(H5N1) avian influenza virus was isolated from fecal samples taken from a duck in a

Studies with H9N2 were conducted in a biosafety level 3 (BSL-3) laboratory that was approved by the China National Accreditation Service for Conformity Assessment. All of the animals were treated strictly according to the guidelines of Laboratory Animal Management from the National Council for Science and Technology. Ten 6-week-old white leghorn chickens (Beijing MERIAL) were inoculated intranasally (i.n.) with 107 EID50 of virus, and the clinical symptoms and lethality were observed for 21 days. Oropharyngeal and cloacal swabs were collected from the inoculated chickens at 1, 3, 5, and 7 d.p.i. and transferred to 1 ml of PBS. The viral titers of the oropharyngeal and cloacal swabs were determined by end-point titration in SPF embryonated eggs (Matsuoka et al., 2009). The sera were harvested from the inoculated chickens at 21 d.p.i. to confirm seroconversion via hemagglutination inhibition (HI) analysis. In the mammalian studies, female 5-week-old BALB/c mice (n ¼25) (Institute of Laboratory Animal Sciences, Beijing) were anesthetized and inoculated intranasally with 50 μl (107 EID50) of virus. Ten mice were selected randomly for monitoring of the clinical signs, weight loss, and mortality daily for up to 14 days post-inoculation (d.p.i), and their sera were collected at 14 d.p.i and treated with receptor-destroying enzyme from Vibrio cholerae. Seroconversion was confirmed by the HI assay. Mice with HI titers lower than 10 were considered influenza-free. The remaining 15 mice were euthanized at 3, 5, and 7 d.p.i, their lung tissues were transferred to 1 ml of PBS, and the clarified homogenates were titrated for virus infectivity in 9-day-old SPF embryonated eggs from initial dilutions of 1:10. The viral titers were expressed as the mean log10 EID50/100 ml.

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Airborne transmission experiments in ferrets Castrated adult ferrets that were 6–10 months of age and seronegative for the currently circulating influenza viruses according to an HI assay were used in this study. Prior to infection, the ferrets were housed in an ABSL3 facility and monitored for 5 to 7 days to measure their body temperatures. A subcutaneous implantable temperature transponder (BioMedic Data Systems; http://www. bmds.com) was placed in each ferret to obtain identification and temperature readings. The temperatures were recorded daily. The transmission experiments were conducted in cages that were designed to prevent direct contact between animals but still allowed airflow between an inoculated ferret and a neighboring naïve ferret (Fig. 4). Three ferrets each in 2 groups were inoculated with 107 EID50 of A/Brambling/Beijing/16/2012 or 106 EID50 of A/California/07/2009 (H1N1). At 24 h p.i., each inoculated animal was housed individually with a naïve ferret in a transmission cage to test the transmissibility of the virus. All of the animals were observed for clinical signs, and body temperatures were measured daily as an indicator of disease. Nasal and throat swabs were collected from the inoculated and naïve animals at 1, 3, 5, 7, and 9 d.p.i. or days post-exposure, respectively, and transferred to 1 ml of PBS. The viral titers were determined by end-point titration in SPF embryonated eggs. The post-exposure sera were collected from the inoculated or contact ferrets at 14 d.p.e. to test the seroconversion by HI analysis. All of the ferrets were allocated randomly to the experimental groups and processed. HI assay Standard HI assays were performed on sera using 0.5% turkey erythrocytes in accordance with the WHO guidelines for established procedures (World Health Organization Global Influenza Surveillance Network, 2011). Sera were collected from mice, chickens, and ferrets and tested for H9N2 virus-specific antibodies. Receptor-binding analysis using hemagglutination assays The hemagglutination assays using resialylated chicken red blood cells (cRBCs) were performed as described previously (Tong et al., 2012; Zhong et al., 2014), with minor modifications. The cRBCs were enzymatically desialylated with V. cholerae neuraminidase (VCNA; Roche, www.roche.com) and were then resialylated using either α26-(N)-sialyltransferase or α2-3-(N)-sialyltransferase (Calbiochem, www.calbiochem.com) or CMP-sialic acid (Sigma, www.sigmaal drich.com). Solid-phase binding assay An analysis of the receptor specificity of the influenza virus was performed using a direct solid-phase assay (Chandrasekaran et al., 2008). In brief, microtiter plates (Corning) were incubated with the sodium salts of sialylglycopolymers (poly-l-glutamic acid backbones containing N-acetylneuraminic acid linked to galactose through either an α-2,3 bond (Neu5Acα2,3Galβ1,4GlcNAcβ1-pAP) or an α-2,6 bond (Neu5Acα2,6Galβ1,4GlcNAcβ1-pAP) bond) in PBS at 4 1C overnight. After removing the glycopolymer solution, the plates were blocked with 0.1 ml of PBS containing 2% bovine serum albumin (Invitrogen) at room temperature for 1 h. The plates were washed with PBS five times and then incubated in a solution containing influenza virus (128 hemagglutination units in PBS) at 4 1C for 12 h. After three washes with PBS, an antibody (1:1000 dilution, IRR Ltd., Catalog no. FR-51) against the virus was added to the plates and the plates were incubated at 4 1C for two additional hours. The plates were then washed three times with ice-cold PBS and incubated with the horseradish peroxidase (HRP)-conjugated protein A at 4 1C. After four washes with ice-cold PBS, the plates were incubated with

O-phenylenediamine in PBS containing 0.01% H2O2 for 10 min at room temperature, and the reaction was stopped with 0.05 ml of 1 M HCl. The absorbance was determined at 490 nm. The AH/05 (H5N1) and CA/04 (H1N1) viruses were used as positive controls for α-2,3 and α-2,6 binding, respectively. Statistical analysis The data are expressed as the mean 7SD. The differences among the different groups were analyzed by one-way ANOVA followed by a post-hoc Bonferroni correction, and the differences between the two groups were analyzed using Student’s t-test. All of the statistical analyses was performed using SPSS 11.5 software.

Acknowledgments This work was supported by grants from the National Science and Technology Major Projects of Infectious Disease (2012ZX10004501004-003, 2012ZX10004501-004-004), the National Natural Science Foundation of China (31370203), the Natural Science Foundation of Beijing (7142106), and the Fundamental Research Funds for the Central Universities (2012Y02). References Aamir, U.B., Wernery, U., Ilyushina, N., Webster, R.G., 2007. Characterization of avian H9N2 influenza viruses from United Arab Emirates 2000 to 2003. Virology 361, 45–55. Belser, J.A., Lu, X., Maines, T.R., Smith, C., Li, Y., Donis, R.O., Katz, J.M., Tumpey, T.M., 2007. Pathogenesis of avian influenza (H7) virus infection in mice and ferrets: enhanced virulence of Eurasian H7N7 viruses isolated from humans. J. Virol. 81, 11139–11147. Bi, J., Deng, G., Dong, J., Kong, F., Li, X., Xu, Q., Zhang, M., Zhao, L., Qiao, J., 2010. Phylogenetic and molecular characterization of H9N2 influenza isolates from chickens in Northern China from 2007-2009. PLoS One 5, e13063. Bussey, K.A., Bousse, T.L., Desmet, E.A., Kim, B., Takimoto, T., 2010. PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J. Virol. 84, 4395–4406. Butt, K.M., Smith, G.J., Chen, H., Zhang, L.J., Leung, Y.H., Xu, K.M., Lim, W., Webster, R.G., Yuen, K.Y., Peiris, J.S., Guan, Y., 2005. Human infection with an avian H9N2 influenza A virus in Hong Kong in 2003. J. Clin. Microbiol. 43, 5760–5767. Callan, R.J., Hartmann, F.A., West, S.E., Hinshaw, V.S., 1997. Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases. J. Virol. 71, 7579–7585. Capua, I., Alexander, D.J., 2004. Avian influenza: recent developments. Avian Pathol. 33, 393–404. Chandrasekaran, A., Srinivasan, A., Raman, R., Viswanathan, K., Raguram, S., Tumpey, T.M., Sasisekharan, V., Sasisekharan, R., 2008. Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat. Biotechnol. 26, 107–113. Chen, L., Blixt, O., Stevens, J., Lipatov, A., Davis, C., Collins, B., Cox, N., Paulson, J., Donis, R., 2012. In vitro evolution of H5N1 avian influenza virus toward humantype receptor specificity. Virology 422, 105–113. Choi, Y.K., Ozaki, H., Webby, R.J., Webster, R.G., Peiris, J.S., Poon, L., Butt, C., Leung, Y. H., Guan, Y., 2004. Continuing evolution of H9N2 influenza viruses in southeastern China. J. Virol. 78, 8609–8614. Connor, R.J., Kawaoka, Y., Webster, R.G., Paulson, J.C., 1994. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 17–23. Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., Chen, J., Jie, Z., Qiu, H., Xu, K., Xu, X., Lu, H., Zhu, W., Gao, Z., Xiang, N., Shen, Y., He, Z., Gu, Y., Zhang, Z., Yang, Y., Zhao, X., Zhou, L., Li, X., Zou, S., Zhang, Y., Yang, L., Guo, J., Dong, J., Li, Q., Dong, L., Zhu, Y., Bai, T., Wang, S., Hao, P., Yang, W., Han, J., Yu, H., Li, D., Gao, G.F., Wu, G., Wang, Y., Yuan, Z., Shu, Y., 2013. Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368, 1888–1897. Glaser, L., Stevens, J., Zamarin, D., Wilson, I.A., Garcia-Sastre, A., Tumpey, T.M., Basler, C.F., Taubenberger, J.K., Palese, P., 2005. A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity. J. Virol. 79, 11533–11536. Guan, Y., Shortridge, K., Krauss, S., Webster, R., 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proc. Natl. Acad. Sci. U.S.A 96, 9363–9367. Guo, Y., Krauss, S., Senne, D., Mo, I., Lo, K., Xiong, X., Norwood, M., Shortridge, K., Webster, R., Guan, Y., 2000. Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 267 (279-228).

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