Review

Pandemic potential of avian influenza A (H7N9) viruses Tokiko Watanabe1,2,3, Shinji Watanabe2,4, Eileen A. Maher1, Gabriele Neumann1, and Yoshihiro Kawaoka1,2,3,5 1

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 575 Science Drive, Madison, WI 53711, USA 2 ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama 332-0012, Japan 3 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 1088639, Japan 4 Laboratory of Veterinary Microbiology, Department of Veterinary Sciences, University of Miyazaki, Miyazaki, 889-2192, Japan 5 Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

Avian influenza viruses rarely infect humans, but the recently emerged avian H7N9 influenza viruses have caused sporadic infections in humans in China, resulting in 440 confirmed cases with 122 fatalities as of 16 May 2014. In addition, epidemiologic surveys suggest that there have been asymptomatic or mild human infections with H7N9 viruses. These viruses replicate efficiently in mammals, show limited transmissibility in ferrets and guinea pigs, and possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. In this review, we summarize the characteristic features of the novel H7N9 viruses and assess their pandemic potential. Influenza A virus as a zoonotic pathogen Influenza A viruses are maintained in wild waterfowl, poultry, humans, pigs, and horses; in addition, infection of dogs, marine mammals, and several other mammalian species has been reported (reviewed in [1]). Influenza A viruses are divided into subtypes according to the antigenicity of their two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA); to date, 18 HA and 11 NA subtypes have been identified [1–3]. In humans, only viruses of three HA subtypes (H1, H2, and H3) and two NA subtypes (N1 and N2) have caused annual epidemics and sporadically occurring pandemics in the last and current centuries. Influenza A viruses of all subtypes (except for viruses of the H17N10 and H18N11 subtypes, whose genomic material has been identified in bats [2,3]) have been detected in waterfowl, which is considered the natural host of influenza A viruses [1]. In waterfowl, most influenza A viruses replicate in the intestinal tract and spread to other birds via the fecal–oral route, whereas the respiratory tract is the major site of influenza A virus replication in mammals [1]. Corresponding author: Kawaoka, Y. ([email protected], [email protected]). Keywords: avian influenza H7N9 viruses; transmission; pandemic potential. 0966-842X/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.08.008

Despite the wide host range of influenza A viruses, their transmission from avian to mammalian species, or vice versa, is rare due to host range restrictions. Influenza A viruses circulating in avian species (so-called ‘avian influenza viruses’) rarely infect humans, and influenza A viruses circulating in humans (‘human influenza viruses’) rarely infect avian species [4–7]. Recently, however, avian influenza A viruses of the H5N1 and H7N9 subtypes have caused hundreds of cases of human infections. So far, sustained human-to-human transmission of these viruses has not been reported. Nonetheless, additional adaptive mutations and/or reassortment with circulating human viruses may enable H5N1 or H7N9 viruses to efficiently infect humans and transmit among them. Because humans lack protective antibodies against these viruses, humantransmitting H5N1 or H7N9 viruses could spread worldwide, resulting in an influenza pandemic. In this review, we focus on the avian H7N9 influenza viruses that recently infected humans in China (Figure 1), describing their biological features and pandemic potential. Human infections with avian H7N9 influenza viruses in China in 2013–2014 To date, two sizable waves of human infection with H7N9 viruses have been documented (Figure 2A). The first wave started with a human case of H7N9 influenza virus infection in Shanghai on 19 February 2013 (this case was officially reported on 31 March 2013) [8]. In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China by the end of April. Most cases were reported from the provinces of Jiangsu, Zhejiang, and Shanghai, which are all located in the eastern part of China (Figure 1 and Figure 2B). The detection of H7N9 viruses in live poultry markets [9–13], and epidemiological data suggesting that contact with poultry or contaminated environments in live bird markets was the likely source of many (although not all) human cases (see section ‘Epidemiology of human H7N9 virus infections’ below), prompted the Chinese government to close live poultry markets in several provinces in mid-April, 2013 [14,15]. This measure most likely led to Trends in Microbiology, November 2014, Vol. 22, No. 11

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Figure 1. The geographic distribution of confirmed human cases of avian influenza A (H7N9) virus infection, as of 16 May 2014 (440 total cases). The number of cases in each province is based on data reported by the Centre for Health Protection, Hong Kong PRC SAR (http://www.chp.gov.hk/en/index.html; map revised from a version available at http://www.cidrap.umn.edu/infectious-disease-topics/h7n9avian-influenza#literature). The darker the green color, the higher the number of cases.

the rapid decline in new human H7N9 cases during the following 2 weeks [16,17]. The second wave of human infections with H7N9 viruses started in the fall of 2013 [18] (Figure 2A), perhaps spurred by the lower fall temperatures and/or the reopening of poultry markets. The number of confirmed human H7N9 virus infections spiked in January and February of 2014, with more than 30 new cases over several consecutive weeks (http://www.who.int/ influenza/human_animal_interface/influenza_h7n9/17_ ReportWebH7N9Number_20140408.pdf?ua=1). Since February 2014, the number of new human cases has declined, although new infections continue to be reported (Figure 2A). The second, ongoing wave is characterized by a larger number of human H7N9 virus infections, and by more extensive geographic spread. While most human cases during the first wave were reported in Eastern China, the majority of human H7N9 virus infections during the second wave occurred in the southern province of Guangdong (Figure 2B). As of 16 May 2014, a total of 440 human infections with H7N9 viruses have been confirmed with 122 associated deaths (unofficial statement; http://www.cidrap.umn.edu/sites/default/files/public/ downloads/topics/cidrap_h7n9_update_051614_0.pdf); 425 of the cases occurred in China (http://www.info.gov.hk/ gia/general/201405/17/P201405170325.htm), whereas the remaining 15 were exported cases (http://www.chp.gov.hk/ files/pdf/2014_avian_influenza_report_vol10_wk19.pdf). Epidemiology of human H7N9 virus infections Epidemiological studies have shown that H7N9 virus infections have affected mainly middle-aged or older individuals (Figure 2C; i.e., the median age at infection 624

is 63 years) [19–29]. Interestingly, two-thirds of the infected individuals have been male [19,20,22–24,27–29] (Figure 2C). The high number of cases among elderly men may reflect socio-economical differences among age groups and genders, since elderly men may have frequent workrelated or non-job-related contact with poultry. Most H7N9 influenza patients exhibit general influenza-like symptoms, including fever and cough, and more than half of the infections typically progress to severe pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure [8,11,19,21,23,25,27,30–38]. Most H7N9 virus-infected patients possessed at least one underlying medical condition, such as chronic obstructive pulmonary disease (COPD), diabetes, hypertension, obesity, and/or chronic lung and heart disease [19,21–23,25,26,28,31, 39,40], suggesting that these comorbidities may increase the risk of severe H7N9 virus infection. The following lines of evidence suggest that contact with live poultry is the source of human H7N9 virus infection: (i) most H7N9 patients had been exposed to live poultry prior to the onset of illness [10,11,19–21,27–29,31–33,37–43]; (ii) surveillance in live bird markets resulted in the isolation of H7N9 viruses with close homology to the H7N9 viruses isolated from humans in the same area [9–13,41]; (iii) a worker who had culled H7N9 virus-infected poultry developed a mild illness that was traced to a confirmed H7N9 virus infection [44]; and (iv) serological surveillance of poultry workers in areas where human cases had been reported revealed antibodies to H7N9 viruses in >6% of the individuals tested, whereas no antibodies to H7N9 viruses were detected in the general population [45]. Collectively, these findings suggest an association between human H7N9 infections and poultry, likely in live bird markets, as underscored by the rapid decline in human H7N9 infections upon closure of these markets in mid-April, 2013 [15–17]. The finding of H7N9 seropositive poultry workers [45] indicates that subclinical human H7N9 infections occurred. Several sentinel surveillance studies identified H7N9 virus-positive individuals who exhibited only mild-to-moderate influenza virus symptoms and recovered quickly [27,46,47]. In fact, several studies suggested that a significant number of unidentified, mild cases of human H7N9 infections may have occurred [48,49]. Although sustained H7N9 virus transmission among humans has not been reported, the potential for humanto-human transmission cannot be ruled out in several family clusters [13,27,50,51]. In some of these clusters, H7N9 infections occurred in blood-related family members, implying that close contacts in household settings, and perhaps also genetic factors, may be risk factors for infection with H7N9 viruses. Genesis of H7N9 influenza viruses Phylogenetic analyses have revealed that the novel H7N9 viruses likely emerged via reassortment of at least four avian influenza A virus strains (Figure 3). The HA gene of the H7N9 viruses belongs to the Eurasian lineage of avian influenza viruses and is closely related to that of avian H7N3 viruses isolated from ducks in Eastern China in 2010–2011 [8,9,11,33,52–55]. The NA gene of the novel

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Figure 2. The number of confirmed human cases of avian influenza A (H7N9) virus infection in 2013–2014. The number of laboratory-confirmed cases of human infection with H7N9 influenza virus by week of onset of illness (A) and by Chinese provinces by wave (B). Blue and red bars indicate the number of human cases of H7N9 virus infection detected in the first and second waves, respectively. Provinces are categorized into two groups: northern and southern regions of China. (C) Human cases of H7N9 influenza virus infection by age- and gender-groups. Data for graphs (A) and (C) are based on FluTrackers 2013/14 Human Case List of Provincial/Ministry of Health/ Government Confirmed Influenza A (H7N9) Cases with Links (http://www.flutrackers.com/forum/showthread.php?t=202713). The number of cases per province in (B) is based on the data shown at the CIDRAP website (http://www.cidrap.umn.edu/sites/default/files/public/downloads/topics/cidrap_h7n9_update_051614_0.pdf).

H7N9 viruses is closely related to that of avian H2N9 and/ or H11N9 influenza viruses isolated from wild migratory birds along the East Asian flyway [8,9,11,33,52–55]. The remaining six viral genes likely originated from two distinct subgroups of an H9N2 sub-lineage (formed by the reassortment of a major H9N2 lineage in China with a Eurasian wild bird virus) circulating in Eastern China [8,9,11,33,52–55]. This genetic heterogeneity suggests that several reassortment events occurred during the generation and ongoing evolution of H7N9 viruses [38,52,53,55–62]. Viral determinants of influenza virus host range A key question stemming from the H7N9 incident is: how was this avian influenza virus able to overcome the host restriction barrier so easily and infect humans? Two viral proteins are known to play a major role in the host range of influenza viruses: the surface glycoprotein, HA, and the polymerase subunit, PB2, which determine

host-specific receptor-binding and replicative ability, respectively. HA determines viral receptor-binding specificity Influenza virus infections are initiated by the binding of HA to receptors on host cells. Human influenza viruses preferentially bind to sialic acid-a2,6-galactose (SAa2,6 Gal), which is the predominant sialyloligosaccharide species expressed on epithelial cells in the upper respiratory tract of humans (reviewed in [63]). By contrast, avian influenza viruses preferentially recognize sialic acida2,3-galactose (SAa2,3 Gal), which is the major sialyloligosaccharide species expressed in the intestinal tract of waterfowl [63], where avian viruses replicate efficiently. Typically, avian influenza viruses exhibit low affinity for SAa2,6 Gal (i.e., ‘human-type’ receptors), and therefore, a shift of HA receptor-binding specificity from SAa2,3 Gal to SAa2,6 Gal is thought to be critical for avian influenza viruses to replicate and transmit efficiently in humans. 625

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Duck H7N3 Possible H7N9 precursor

Eurasian lineage

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Wild bird H2N9/ H11N9 Eurasian lineage

Chicken H9N2 sublineage 1

PB2-627K PB2-701N PB2-591K/R HA-226L/I

Eurasian lineage Poultry H9N2 lineage in China

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Figure 3. The genesis of avian influenza A (H7N9) influenza virus. The novel H7N9 viruses likely resulted from the reassortment of at least four avian influenza A virus strains. The hemagglutinin (HA) gene of the H7N9 viruses is closely related to the Eurasian lineage of avian influenza viruses and to that of the avian H7N3 viruses recently isolated from ducks in Eastern China. The neuraminidase (NA) gene of the novel H7N9 viruses is closely related to that of avian H2N9 and/or H11N9 influenza viruses isolated from wild migratory birds along the East Asian flyway. The remaining six viral genes likely originated from two distinct subgroups of an H9N2 sub-lineage circulating in poultry in Eastern China. The virus encircled by the broken line represents a possible precursor of H7N9 avian influenza viruses. Although the H7N9 viruses currently circulating in birds do not encode determinants of mammalian adaptation (i.e., PB2-627K, PB2-701N, or PB2-591K/R and HA-226L/I), such mutations can arise during H7N9 virus replication in humans. This figure was created by modification of a figure in [53].

Previous studies revealed that the amino acid at position 226 (HA numbers in this article refer to the amino acid position in H3 HA after the removal of the signal peptide) of H3 HAs affects binding to human-type receptors [64]. Although sequence analyses of most H7N9 viruses revealed leucine or isoleucine residues at HA position 226 (i.e., the human virus-type residue) (Table 1) [9,33,52,54], purified H7N9 HAs bearing these human virus-type residues at position 226 still preferentially bound to SAa2,3 Gal in studies using soluble recombinant trimeric HAs expressed in mammalian cells [65]. Therefore, the H7N9 viruses may require additional adaptive mutations in HA, such as a Gly-to-Ser mutation at position 228 of HA [66], to efficiently bind to the human-type receptors. Nonetheless, several groups have demonstrated that whole H7N9 viruses possessing leucine or isoleucine at position 226 of HA bind to both SAa2,3 Gal and SAa2,6 Gal [65–74], whereas a H7N9 virus encoding glutamine (i.e., the avian virus-type residue) preferentially bound to SAa2,3 Gal [75], suggesting that the binding of H7N9 virions to human-type receptors might be affected by other viral components (i.e., the neuraminidase). 626

In addition, although the leucine residue at position 226 of HA is characteristic of human influenza viruses, it is encoded by most avian H7N9 virus isolates, indicating that it did not arise during H7N9 virus replication in humans. Therefore, the leucine or isoleucine residue at position 226 of HA in H7N9 viruses likely emerged during virus replication in poultry, and may now facilitate the infection of mammalian cells. PB2 determines viral replicative ability The PB2 protein is one of the three subunits of the viral polymerase complex, which catalyzes viral replication and transcription in the nucleus of infected cells. A lysine residue at position 627 of PB2 (as found in most human influenza viruses) confers efficient replication to avian influenza viruses in mammals [76]. By contrast, glutamic acid at this position, as is found in most avian influenza viruses, significantly restricts avian influenza virus replication in mammals. Currently, the mechanism through which the amino acid at position 627 of PB2 directs viral replicative ability is thought to involve interactions with other viral and/or host proteins [77–81], most likely in a

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Table 1. Selected amino acids of human-infecting H7N9 viruses Viral protein PB2 HA

NA

Amino acid position 627 128 (138) a 151 (160) a 177 (186) a 217 (226) a 69-73 c 289 (294) d

Human virus K A K G I No deletion R

Avian virus E Ab Ab Gb Qb No deletion R

A/Anhui/1/2013 K A A V L Deletion R

A/Shanghai/1/2013 K S A G Q Deletion K

A/Chicken/Shanghai/S1053/2013 E A A V L Deletion R

a

Amino acid positions of HA are based on H7 HA numbering (H3 HA numbering is shown in parentheses).

b

Amino acids characteristic of H7 avian influenza virus.

c

Amino acid positions of NA are based on N9 NA numbering.

d

Amino acid positions of NA are based on NA numbering of human-infecting H7N9 viruses (avian N9 NA numbering is shown in parentheses).

temperature-sensitive manner. Avian body temperature is 418C [82], whereas the temperatures in the human lung and upper respiratory tract are generally considered to be 378C and 338C, respectively. Consequently, most avian influenza viruses replicate more efficiently at 418C than at 338C [83]. Having a lysine at position 627 of PB2 confers efficient replication to avian influenza viruses at 338C and 378C [83,84], enabling them to establish robust infections in mammals. Additionally, the PB2-627K mutation has recently been shown to increase the transmissibility of avian influenza viruses [85–87]. Sequence analyses have shown that H7N9 viruses isolated from avian hosts possess glutamic acid at position 627 of PB2, whereas many human H7N9 viruses encode lysine at this position (Table 1); this finding suggests that the PB2-627K mutation likely emerges during virus replication in humans, as occurred in the human cases of infection with H7N7 avian influenza viruses in the Netherlands in 2003 [88]. Interestingly, some human H7N9 virus isolates that lack PB2-627K possess other potentially mammalianadapting amino acid changes in PB2 that may compensate for the lack of the mammalian-adapting lysine residue at position 627. Some of the human H7N9 virus isolates that encode PB2-627E possess an aspartic acid-to-asparagine mutation at position 701 of PB2 [11], a mutation known to improve avian virus replication in mammalian cells [89]. Moreover, a PB2-D701N mutation has been shown to increase the replicative ability and virulence in mice of an H7N9 virus encoding PB2-627E [90]. Another human H7N9 isolate lacking PB2-627K acquired a glutamine-tolysine mutation at position 591 of PB2. A basic amino acid at this position was shown to compensate for the lack of PB2-627K in pandemic 2009 H1N1 viruses [91,92]. A comparison of viruses encoding PB2-627E or PB2-627E/591K showed higher virus titers and virulence in mice for the latter virus [90]. Together, these findings demonstrate that the H7N9 viruses currently circulating in birds do not encode strong determinants of mammalian adaptation in PB2 (namely, PB2-627K, PB2-701N, or PB2-591K/R), but that such mutations arise easily during H7N9 virus replication in humans. Contribution of amino acids in PA to H7N9 virulence Viral proteins other than HA and PB2 also contribute to H7N9 virulence, albeit to a lesser extent than these major

virulence determinants. Previous studies have suggested a potential role for PA in the adaptation and pathogenicity of avian influenza viruses in a mammalian host [93–95]. Several computational and phylogenetic analyses have identified amino acid mutations in the H7N9 PA protein (another subunit of the viral polymerase complex) that are typically found in human, but not in avian, influenza viruses [58,96–98]. Indeed, experimental testing revealed that some of these amino acids (i.e., A100 V, R356K, and N409S) affect H7N9 replicative ability and virulence [96]. Interestingly, however, replacement of human-type amino acids in PA with the amino acids commonly found in avian influenza viruses slightly increased their replicative ability in human cells and their virulence in mice, contrary to the expected attenuating effect [96]. Although the exact reason for this unexpected finding is unknown, it may be that these mutations are introduced to optimize virus replication in different environments. Risk assessment of human-to-human transmission of H7N9 viruses in animal models The primary concern with H7N9 viruses is that they may gain efficient human-to-human transmissibility and cause a pandemic. Influenza viruses transmit from human to human through direct and indirect contact via aerosols, respiratory droplets, and fomites. Several research groups have evaluated the transmissibility of H7N9 viruses in two animal models, namely ferrets and guinea pigs [68,72,73,99–103]. These studies were carried out with patient-derived H7N9 viruses encoding HA-226L/PB2627K (i.e., possessing the mammalian-adapting markers in both proteins) or HA-226Q/PB2-627K (i.e., possessing the mammalian-adapting marker in PB2, but not in HA). Efficient transmission of both human H7N9 viruses through direct contact (i.e., pair-housing an infected and a naı¨ve animal) was observed in the ferret and guinea pig models (i.e., viruses were recovered from all contact animals – naı¨ve animals that were housed in a cage adjacent to each of the infected animals) [72,99,101]. By contrast, transmission via respiratory droplets was limited in ferrets and guinea pigs (i.e., viruses were recovered only from a few contact animals) [68,72,99,100,102,103], except in one study in which efficient respiratory droplet transmission was reported for one of two human H7N9 isolates tested in ferrets [73], whereas no transmission was detected for avian H7N9 isolates tested under the same conditions [68,73]. 627

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Table 2. Amino acid changes found in H7N9 viruses isolated from contact ferrets in transmission studiesa Viral protein PB2 PB1 PA HA b

NA NP

Amino acid position 678 523 678 349 531 61 (71) 121 (131) 123 (133) 125 (135) 130 (140) 149 (158-159) d 210 (219) 10 27 109 225

Reference virus D M S E R T R N A R N A T A I I

Xu et al. [103] Y

Watanabe et al. [68]

Richard et al. [100]

Belser et al. [72]

I N G K I K D T Kc

M D E I

I T T

L

a

Viruses isolated from the nasal washes of contact ferrets in the human-infecting H7N9 virus (A/Anhui/1/2013) group were sequenced. Zhang et al. found no amino acid substitutions in viruses recovered from the contact ferrets [73]. No sequence data were presented in Zhu et al. [99].

b

Amino acid positions of HA are based on H7 HA numbering. H3 HA numbering is shown in parentheses.

c

The HA R140K mutation (H3 numbering) corresponds to the HA R148K mutation (H7 numbering) described by Xu et al. [103] because they counted the signal peptides of HA.

d

The H7 HA possesses amino acid insertions corresponding to the position between residues 158 and 159 in the H3 HA.

Sequence analyses of viruses obtained from infected and contact animals revealed several mutations in the latter group (Table 2), except for one study that did not detect amino acid changes in viruses recovered from contact animals [73]. Three non-synonymous mutations, T71I, R131K, and A135T, in HA (H3 numbering), and one non-synonymous mutation, A27T, in NA (N9 numbering) were identified in viruses collected from contact ferrets 68. Positions 131 and 135 are located near the receptorbinding pocket (Figure 4). At position 71, both threonine and isoleucine are commonly found among H7 HAs, and the location of this position ‘underneath’ the receptorbinding pocket suggests a possible effect on HA stability Modeled humantype receptor

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G186V A219E R131K

Q226L/I

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Figure 4. Amino acid changes in the hemagglutinin (HA) of viruses recovered from contact ferrets in the human-infecting H7N9 virus groups. The 3D structure of A/ Anhui/1/2003 (H7N9) HA (PDB ID: 4BSE) is shown in complex with human receptor analogues and a close-up view of the globular head. Mutations shown in cyan (i.e., A138S, G186 V, and Q226L/I) are known to increase the binding of avian H5 and H7 viruses to human-type receptors. Mutations that emerged in HA of humaninfecting H7N9 viruses during replication and/or transmission in ferrets are shown in green (see also Table 2). The human receptor analogue is shown in orange. Images were created with MacPymol [http://www.pymol.org/].

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(note that a stabilizing mutation played a critical role in the selection of a ferret-transmissible H5 virus [104]). Another study detected the double substitution of N133D/N158-159D in HA (H3 numbering) [note that these mutations correspond to N124D/N149D (H7 numbering) described in [100]; however, since H7 HA possesses amino acid insertions relative to H3 HA, there is no amino acid in the H3 HA corresponding to that at position 149 of the H7 HA] together with an M523I mutation in the PB1 polymerase protein [100]. Interestingly, the N133D/N158159D double mutation reduced the thermostability of HA and increased HA binding to avian-type – but not humantype – receptors, contrary to what may have been expected for a ferret-transmissible virus [100]. In this context, it is worth noting that the N133D/N158-159D double mutation was also detected after replication of a human H7N9 virus isolate in avian species [105]. The R140 M or R140K mutation detected in viruses isolated from contact ferrets in two independent studies [72,103] localize to the rim of the HA receptor-binding pocket, and it is thus conceivable that they affect HA receptor-binding properties. In addition to the R140K mutation in HA, this transmissible virus also possessed a D678Y mutation in PB2, an I109T mutation in the nucleoprotein NP, and a T10I mutation in NA [103]. Collectively, these data demonstrate the inherent capability of H7N9 viruses to transmit among mammals, although the efficiency of transmission is lower than that of human influenza viruses. In patients treated with NA inhibitors, resistant H7N9 variants have been detected encoding the R292K mutation in NA that confers resistance to oseltamivir [106– 110]. Oseltamivir-resistance mutations frequently reduce viral fitness (reviewed in [111]), although compensatory amino acid changes can restore it [112]. The NA-R292K mutation was found to reduce H7N9 viral fitness in cultured cells in one study [106]; however, another study did not detect an effect of the NA-R292K mutation in an H7N9

Review virus on virulence in mice or transmissibility in guinea pigs [102], suggesting that oseltamivir-resistant H7N9 viruses may be competitive in nature. Natural isolates of highly pathogenic avian H5N1 viruses do not transmit among ferrets (reviewed in [113]), whereas novel H7N9 have limited transmissibility in mammalian models. Therefore, the pandemic potential of novel H7N9 viruses appears to be greater than that of highly pathogenic H5N1 viruses. Combined with the emergence of already partially adapted phenotypes and the relatively high fitness of oseltamivir-resistant H7N9 viruses, these novel viruses pose a significant pandemic threat. Concluding remarks To date, the novel H7N9 influenza viruses have not caused a pandemic in humans due to their inability to support sustained human-to-human transmission. However, these viruses exhibit high replicative ability and limited transmissibility in mammals, have acquired mammalian-adapting amino acid changes, may reassort with circulating human viruses (based on the reported coinfection of a patient with H7N9 and human H3N2 viruses) [114], and readily acquire resistance to the NA inhibitor oseltamivir [68,72,73,99,100]. Moreover, humans lack protective immunity to H7N9 infection [68] and frequently show relatively weak antibody responses when infected with these viruses [45,115]. Therefore, a better understanding of the molecular mechanisms of pathogenicity, transmissibility, and immunogenicity of the novel H7N9 influenza viruses, combined with continued surveillance in avian and human populations, will be vital to develop countermeasures against H7N9 infections in humans. Acknowledgments We thank Dr Susan Watson for editing the manuscript. This work was supported by National Institute of Allergy and Infectious Diseases Public Health Service research grants, by RO1 AI080598 and R56 AI099275, by ERATO (Japan Science and Technology Agency), by the Strategic Basic Research Programs of the Japan Science and Technology Agency, Japan, and by J-GRID (Japan Initiative for Global Research Network on Infectious Diseases).

References 1 Wright, P.F. et al., eds (2013) Orthomyxoviruses, Lippincott Williams & Wilkins 2 Tong, S. et al. (2012) A distinct lineage of influenza A virus from bats. Proc. Natl. Acad. Sci. U.S.A. 109, 4269–4274 3 Tong, S. et al. (2013) New world bats harbor diverse influenza a viruses. PLoS Pathog. 9, e1003657 4 Beare, A.S. and Webster, R.G. (1991) Replication of avian influenza viruses in humans. Arch. Virol. 119, 37–42 5 Hinshaw, V.S. et al. (1984) Are seals frequently infected with avian influenza viruses? J. Virol. 51, 863–865 6 Murphy, B.R. et al. (1984) Avian-human reassortant influenza A viruses derived by mating avian and human influenza A viruses. J. Infect. Dis. 150, 841–850 7 Webster, R.G. et al. (1978) Intestinal influenza: replication and characterization of influenza viruses in ducks. Virology 84, 268–278 8 Centers for Disease Control and Prevention (2013) Emergence of avian influenza A (H7N9) virus causing severe human illness – China, February–April 2013. MMWR Morb. Mortal. Wkly. Rep. 62, 366–371 9 Shi, J.Z. et al. (2013) Isolation and characterization of H7N9 viruses from live poultry markets – implication of the source of current H7N9 infection in humans. Chin. Sci. Bull. 58, 1857–1863

Trends in Microbiology November 2014, Vol. 22, No. 11

10 Han, J. et al. (2013) Epidemiological link between exposure to poultry and all influenza A (H7N9) confirmed cases in Huzhou city, China, March to May 2013. Euro Surveill. 18, pii=20481 11 Chen, Y. et al. (2013) Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 381, 1916–1925 12 Wang, C. et al. (2014) Relationship between domestic and wild birds in live poultry market and a novel human H7N9 virus in China. J. Infect. Dis. 209, 34–37 13 Liu, T. et al. (2014) One family cluster of avian influenza A (H7N9) virus infection in Shandong, China. BMC Infect. Dis. 14, 98 14 Murhekar, M. et al. (2013) Avian influenza A (H7N9) and the closure of live bird markets. Western Pac. Surveill. Response J. 4, 4–7 15 Xu, J. et al. (2013) Reducing exposure to avian influenza H7N9. Lancet 381, 1815–1816 16 Yu, H. et al. (2014) Effect of closure of live poultry markets on poultryto-person transmission of avian influenza A H7N9 virus: an ecological study. Lancet 383, 541–548 17 Chowell, G. et al. (2013) Transmission potential of influenza A/H7N9, February to May 2013, China. BMC Med. 11, 214 18 Chen, E. et al. (2013) Human infection with avian influenza A (H7N9) virus re-emerges in China in winter 2013. Euro Surveill. 18, pii: 20616 19 Lu, S. et al. (2014) Prognosis of 18 H7N9 avian influenza patients in Shanghai. PLoS ONE 9, e88728 20 Ding, H. et al. (2014) Epidemiologic characterization of 30 confirmed cases of human infection with avian influenza A (H7N9) virus in Hangzhou, China. BMC Infect. Dis. 14, 175 21 Wang, X.F. et al. (2013) Clinical features of three avian influenza H7N9 virus-infected patients in Shanghai. Clin. Respir. J. http:// dx.doi.org/10.1111/crj.12087 22 Wang, C. et al. (2014) Comparison of patients hospitalized with influenza A subtypes H7N9, H5N1, and 2009 pandemic H1N1. Clin. Infect. Dis. 58, 1095–1103 23 Gao, H.N. et al. (2013) Clinical findings in 111 cases of influenza A (H7N9) virus infection. N. Engl. J. Med. 368, 2277–2285 24 Dudley, J.P. and Mackay, I.M. (2013) Age-specific and sex-specific morbidity and mortality from avian influenza A (H7N9). J. Clin. Virol. 58, 568–570 25 Liu, S. et al. (2013) Epidemiological, clinical and viral characteristics of fatal cases of human avian influenza A (H7N9) virus in Zhejiang Province, China. J. Infect. 67, 595–605 26 Ji, H. et al. (2014) Epidemiological and clinical characteristics and risk factors for death of patients with avian influenza A H7N9 virus infection from Jiangsu Province, Eastern China. PLoS ONE 9, e89581 27 Li, Q. et al. (2014) Epidemiology of human infections with avian influenza A (H7N9) virus in China. N. Engl. J. Med. 370, 520–532 28 Zhang, W. et al. (2013) Epidemiologic characteristics of cases for influenza A (H7N9) virus infections in China. Clin. Infect. Dis. 57, 619–620 29 Cowling, B.J. et al. (2013) Comparative epidemiology of human infections with avian influenza A H7N9 and H5N1 viruses in China: a population-based study of laboratory-confirmed cases. Lancet 382, 129–137 30 Tang, X. et al. (2014) ARDS associated with pneumonia caused by avian influenza A H7N9 virus treated with extracorporeal membrane oxygenation. Clin. Respir. J. http://dx.doi.org/10.1111/crj.12140 31 Chen, E. et al. (2013) The first avian influenza A (H7N9) viral infection in humans in Zhejiang Province, China: a death report. Front. Med. 7, 333–344 32 Xie, L. et al. (2013) Clinical and epidemiological survey and analysis of the first case of human infection with avian influenza A (H7N9) virus in Hangzhou, China. Eur. J. Clin. Microbiol. Infect. Dis. 32, 1617–1620 33 Gao, R. et al. (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368, 1888–1897 34 Wang, Q. et al. (2013) Emerging H7N9 influenza A (novel reassortant avian-origin) pneumonia: radiologic findings. Radiology 268, 882–889 35 Yu, L. et al. (2013) Clinical, virological, and histopathological manifestations of fatal human infections by avian influenza A (H7N9) virus. Clin. Infect. Dis. 57, 1449–1457 36 Li, J. et al. (2013) Environmental connections of novel avian-origin H7N9 influenza virus infection and virus adaptation to the human. Sci. China Life Sci. 56, 485–492 629

Review 37 Mei, Z. et al. (2013) Avian influenza A (H7N9) virus infections, Shanghai, China. Emerg. Infect. Dis. 19, 1179–1181 38 To, K.K. et al. (2014) Unique reassortant of influenza A (H7N9) virus associated with severe disease emerging in Hong Kong. J. Infect. 69, 60–68 39 Lu, S. et al. (2013) Clinical findings for early human cases of influenza A (H7N9) virus infection, Shanghai, China. Emerg. Infect. Dis. 19, 1142–1146 40 Ai, J. et al. (2013) Case–control study of risk factors for human infection with influenza A (H7N9) virus in Jiangsu Province, China, 2013. Euro Surveill. 18, 20510 41 Bao, C.J. et al. (2013) Live-animal markets and influenza A (H7N9) virus infection. N. Engl. J. Med. 368, 2337–2339 42 Sun, J. et al. (2014) Comparison of characteristics between patients with H7N9 living in rural and urban areas of Zhejiang Province, China: a preliminary report. PLoS ONE 9, e93775 43 Yang, P. et al. (2013) A case of avian influenza A (H7N9) virus occurring in the summer season, China. J. Infect. 67, 624–625 44 Lv, H. et al. (2013) Mild illness in avian influenza A (H7N9) virusinfected poultry worker, Huzhou, China, April 2013. Emerg. Infect. Dis. 19, 1885–1888 45 Yang, S. et al. (2014) Avian-origin influenza A (H7N9) infection in influenza A (H7N9)-affected areas of China: a serological study. J. Infect. Dis. 209, 265–269 46 Ip, D.K. et al. (2013) Detection of mild to moderate influenza A/H7N9 infection by China’s national sentinel surveillance system for influenza-like illness: case series. BMJ 346, f3693 47 Yang, P. et al. (2013) Surveillance for avian influenza A (H7N9), Beijing, China, 2013. Emerg. Infect. Dis. 19, 2041–2043 48 Yu, H. et al. (2013) Human infection with avian influenza A H7N9 virus: an assessment of clinical severity. Lancet 382, 138–145 49 Cowling, B.J. et al. (2013) Preliminary inferences on the age-specific seriousness of human disease caused by avian influenza A (H7N9) infections in China, March to April 2013. Euro Surveill. 18, 20475 50 Jie, Z. et al. (2013) Family outbreak of severe pneumonia induced by H7N9 infection. Am. J. Respir. Crit. Care Med. 188, 114–115 51 Qi, X. et al. (2013) Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation. BMJ 347, f4752 52 Liu, D. et al. (2013) Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 381, 1926–1932 53 Lam, T.T. et al. (2013) The genesis and source of the H7N9 influenza viruses causing human infections in China. Nature 502, 241–244 54 Kageyama, T. et al. (2013) Genetic analysis of novel avian A (H7N9) influenza viruses isolated from patients in China, February to April 2013. Euro Surveill. 18, 20453 55 Wu, A. et al. (2013) Sequential reassortments underlie diverse influenza H7N9 genotypes in China. Cell Host Microbe 14, 446–452 56 Cui, L. et al. (2014) Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus. Nat. Commun. 5, 3142 57 Zhang, L. et al. (2013) Rapid reassortment of internal genes in avian influenza A (H7N9) virus. Clin. Infect. Dis. 57, 1059–1061 58 Neumann, G. et al. (2014) Identification of amino acid changes that may have been critical for the genesis of A (H7N9) influenza viruses. J. Virol. 88, 4877–4896 59 Meng, Z. et al. (2014) Possible pandemic threat from new reassortment of influenza A (H7N9) virus in China. Euro Surveill. 19, pii:20699 60 Yu, X. et al. (2014) Influenza H7N9 and H9N2 viruses: coexistence in poultry linked to human H7N9 infection and genome characteristics. J. Virol. 88, 3423–3431 61 Han, J. et al. (2014) Cocirculation of three hemagglutinin and two neuraminidase subtypes of avian influenza viruses in Huzhou, China, April 2013: implication for the origin of the novel H7N9 virus. J. Virol. 88, 6506–6511 62 Liu, J. et al. (2014) Complex reassortment of polymerase genes in Asian influenza A virus H7 and H9 subtypes. Infect. Genet. Evol. 23, 203–208 63 de Graaf, M. and Fouchier, R.A. (2014) Role of receptor binding specificity in influenza A virus transmission and pathogenesis. EMBO J. 33, 823–841 630

Trends in Microbiology November 2014, Vol. 22, No. 11

64 Rogers, G.N. et al. (1983) Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304, 76– 78 65 Xu, R. et al. (2013) Preferential recognition of avian-like receptors in human influenza A H7N9 viruses. Science 342, 1230–1235 66 Tharakaraman, K. et al. (2013) Glycan receptor binding of the influenza A virus H7N9 hemagglutinin. Cell 153, 1486–1493 67 Zhou, J. et al. (2013) Biological features of novel avian influenza A (H7N9) virus. Nature 499, 500–503 68 Watanabe, T. et al. (2013) Characterization of H7N9 influenza A viruses isolated from humans. Nature 501, 551–555 69 Dortmans, J.C. et al. (2013) Adaptation of novel H7N9 influenza A virus to human receptors. Sci. Rep. 3, 3058 70 Yang, H. et al. (2013) Structural analysis of the hemagglutinin from the recent 2013 H7N9 influenza virus. J. Virol. 87, 12433–12446 71 Ramos, I. et al. (2013) H7N9 influenza viruses interact preferentially with alpha2,3-linked sialic acids and bind weakly to alpha2,6-linked sialic acids. J. Gen. Virol. 94, 2417–2423 72 Belser, J.A. et al. (2013) Pathogenesis and transmission of avian influenza A (H7N9) virus in ferrets and mice. Nature 501, 556–559 73 Zhang, Q. et al. (2013) H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science 341, 410–414 74 Xiong, X. et al. (2013) Receptor binding by an H7N9 influenza virus from humans. Nature 499, 496–499 75 Shi, Y. et al. (2013) Structures and receptor binding of hemagglutinins from human-infecting H7N9 influenza viruses. Science 342, 243–247 76 Hatta, M. et al. (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842 77 Labadie, K. et al. (2007) Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. Virology 362, 271–282 78 Rameix-Welti, M.A. et al. (2009) Avian influenza A virus polymerase association with nucleoprotein, but not polymerase assembly, is impaired in human cells during the course of infection. J. Virol. 83, 1320–1331 79 Moncorge, O. et al. (2010) Evidence for avian and human host cell factors that affect the activity of influenza virus polymerase. J. Virol. 84, 9978–9986 80 Mehle, A. and Doudna, J.A. (2008) An inhibitory activity in human cells restricts the function of an avian-like influenza virus polymerase. Cell Host Microbe 4, 111–122 81 Foeglein, A. et al. (2011) Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase. J. Gen. Virol. 92, 1650–1661 82 Prinzinger, R. et al. (1991) Body-temperature in birds. Comp. Biochem. Phys. A 99, 499–506 83 Hatta, M. et al. (2007) Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog. 3, 1374–1379 84 Massin, P. et al. (2001) Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses. J. Virol. 75, 5398–5404 85 Van Hoeven, N. et al. (2009) Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air. Proc. Natl. Acad. Sci. U.S.A. 106, 3366–3371 86 Steel, J. et al. (2009) Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog. 5, e1000252 87 Watanabe, T. et al. (2014) Circulating avian influenza viruses closely related to the 1918 virus have pandemic potential. Cell Host Microbe 15, 692–705 88 Jonges, M. et al. (2014) Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A (H7N7) infection as determined by Illumina ultradeep sequencing. J. Virol. 88, 1694–1702 89 Li, Z. et al. (2005) Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J. Virol. 79, 12058–12064 90 Mok, C.K. et al. (2014) Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts. J. Virol. 88, 3568–3576 91 Yamada, S. et al. (2010) Biological and structural characterization of a host-adapting amino acid in influenza virus. PLoS Pathog. 6, e1001034

Review 92 Mehle, A. and Doudna, J.A. (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proc. Natl. Acad. Sci. U.S.A. 106, 21312–21316 93 Kim, J.H. et al. (2010) Role of host-specific amino acids in the pathogenicity of avian H5N1 influenza viruses in mice. J. Gen. Virol. 91, 1284–1289 94 Bussey, K.A. et al. (2011) PA residues in the 2009 H1N1 pandemic influenza virus enhance avian influenza virus polymerase activity in mammalian cells. J. Virol. 85, 7020–7028 95 Mehle, A. et al. (2012) Reassortment and mutation of the avian influenza virus polymerase PA subunit overcome species barriers. J. Virol. 86, 1750–1757 96 Yamayoshi, S. et al. (2014) Virulence-affecting amino acid changes in the PA protein of H7N9 influenza A viruses. J. Virol. 88, 3127–3134 97 Liu, Q. et al. (2013) Genomic signature and protein sequence analysis of a novel influenza A (H7N9) virus that causes an outbreak in humans in China. Microbes Infect. 15, 432–439 98 Xu, W. et al. (2013) PA-356R is a unique signature of the avian influenza A (H7N9) viruses with bird-to-human transmissibility: potential implication for animal surveillances. J. Infect. 67, 490–494 99 Zhu, H. et al. (2013) Infectivity, transmission, and pathology of human-isolated H7N9 influenza virus in ferrets and pigs. Science 341, 183–186 100 Richard, M. et al. (2013) Limited airborne transmission of H7N9 influenza A virus between ferrets. Nature 501, 560–563 101 Gabbard, J.D. et al. (2014) Novel H7N9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model. J. Virol. 88, 1502–1512 102 Hai, R. et al. (2013) Influenza A (H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat. Commun. 4, 2854 103 Xu, L. et al. (2014) Novel avian-origin human influenza A (H7N9) can be transmitted between ferrets via respiratory droplets. J. Infect. Dis. 209, 551–556

Trends in Microbiology November 2014, Vol. 22, No. 11

104 Imai, M. et al. (2012) Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/ H1N1 virus in ferrets. Nature 486, 420–428 105 Pantin-Jackwood, M.J. et al. (2014) Role of poultry in the spread of novel H7N9 influenza virus in China. J. Virol. 88, 5381–5390 106 Wu, Y. et al. (2013) Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses. Cell Res. 23, 1347–1355 107 Hu, Y. et al. (2013) Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance. Lancet 381, 2273–2279 108 Yen, H.L. et al. (2013) Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population. MBio 4, e00396–e413 109 Lin, P.H. et al. (2014) Virological, serological, and antiviral studies in an imported human case of avian influenza A (H7N9) virus in Taiwan. Clin. Infect. Dis. 58, 242–246 110 Shen, Z. et al. (2013) Host immunological response and factors associated with clinical outcome in patients with the novel influenza A H7N9 infection. Clin. Microbiol. Infect. 20, O493–O500 111 Govorkova, E.A. (2013) Consequences of resistance: in vitro fitness, in vivo infectivity, and transmissibility of oseltamivir-resistant influenza A viruses. Influenza Other Respir. Viruses 7 (Suppl 1), 50–57 112 Bloom, J.D. et al. (2010) Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science 328, 1272–1275 113 Imai, M. et al. (2013) Transmission of influenza A/H5N1 viruses in mammals. Virus Res. 178, 15–20 114 Zhu, Y. et al. (2013) Human co-infection with novel avian influenza A H7N9 and influenza A H3N2 viruses in Jiangsu province, China. Lancet 381, 2134 115 Guo, L. et al. (2014) Human antibody responses to avian influenza A (H7N9) virus, 2013. Emerg. Infect. Dis. 20, 192–200

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