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neumann 2/7/07 13:21 Page 617 Antiviral Therapy 12:617–626 Review Molecular pathogenesis of H5N1 influenza virus infections Gabriele Neumann1*, Kyoko Shinya2 and Yoshihiro Kawaoka1,3 1 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA The Avian Zoonosis Research Centre, Tottori University, Tottori, Japan 3 Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2 *Corresponding author: Tel: +1 608 263 7114; Fax: +1 608 262 9641; E-mail: [email protected] Highly pathogenic H5N1 influenza viruses have become endemic in poultry populations throughout Southeast Asia and continue to infect humans with a greater than 50% case fatality rate. So far, human-to-human transmission of these viruses has been limited. Here, we discuss the molecular features of H5N1 influenza viruses that might affect their pathogenicity, and explain the current lack of efficient human-to-human transmission. Such knowledge is critical in evaluating the pandemic risk these viruses pose. Introduction Influenza viruses belong to the family Orthomyxoviridae, which is comprised of five genera. Only one genus – Influenzavirus A – causes pandemic disease and will, therefore, be the focus of this article. Influenza A viruses are further classified into subtypes based on the antigenicity of their haemagglutinin (HA) and neuraminidase (NA) molecules (reviewed in [1]). To date, 16 HA subtypes (H1–H16) and nine NA subtypes (N1–N9) have been described. Despite the large number of potential combinations, viruses of only a limited number of subtypes (H1N1, H1N2, H2N2, H3N2) have circulated in humans. The genome of influenza A viruses is composed of eight segments of single-stranded, negative-sense RNA that each encode one or two proteins (reviewed in [2]). The enveloped viruses possess two surface glycoproteins (HA and NA). These proteins are critical for virus binding and internalization, as well as for the release of newly assembled virions from infected cells. The M1 matrix protein is thought to form a matrix underneath the lipid bilayer. The virions contain eight viral ribonucleoprotein (vRNP) complexes, each composed of a viral RNA (vRNA) segment and its attached polymerase proteins (PB2, PB1 and PA). The vRNAs are wrapped around the nucleoprotein NP. Other viral proteins include the M2 ion channel protein, the interferon antagonist NS1, the nuclear © 2007 International Medical Press 1359-6535 export protein NEP and the PB1-F2 protein, which is encoded by a second reading frame in segment 2 and might induce apoptosis [3]. Influenza pandemics Influenza viruses cause a highly contagious respiratory disease that manifests in local epidemics or global pandemics. Last century, there were three pandemics that killed an estimated 20 million–50 million people worldwide in 1918/1919 (‘Spanish influenza’), about 70,000 people in the US in 1957 (‘Asian influenza’) and about 33,800 people in the US in 1968 (‘Hong Kong influenza’). How new pandemic viruses emerge is poorly understood, although the introduction of a new HA subtype into immunologically naive populations is a key factor. Viruses containing a new HA subtype can be introduced into human populations via the transmission of a wholly avian influenza virus (which probably occurred in the 1918 influenza pandemic) or from human/avian reassortant viruses (which caused the 1957 and 1968 pandemics). Highly pathogenic H5N1 influenza viruses Most avian influenza viruses do not replicate efficiently in humans [4] and their direct transmission to humans 617 neumann 2/7/07 13:21 Page 618 G Neumann et al. was therefore considered a rare event that posed little threat. This assumption proved wrong in Hong Kong in 1997, when 18 individuals, six of whom died, were infected with highly pathogenic avian influenza viruses of the H5N1 subtype [5–7]. In parallel, H5N1 virus outbreaks occurred in live poultry markets in Hong Kong. From 1997 to 2003, these virus outbreaks were confined to Southeast Asia and involved few human infections. In 2003, however, an outbreak of H5N1 avian infections started that has since spread throughout Southeast and South Asia and has been accompanied by an increasing number of human infections. The viruses responsible for this outbreak have become endemic in poultry populations in Southeast Asia. The next remarkable event in H5N1 virus evolution occurred in 2005 at Qinghai Lake, China, when thousands of waterfowl succumbed to H5N1 virus infection [8–10]. Waterfowl are the natural reservoir of influenza viruses and, as such, are typically asymptomatic to infection with highly pathogenic influenza viruses. The Qinghai Lake H5N1 virus sublineage is now dominant in northern China and has spread to Europe and Africa [9]. Since 2003, the number of human infections with highly pathogenic H5N1 viruses has increased, with a fatality rate in humans that exceeds 50%. Moreover, several family clusters of H5N1 virus infections have now been reported [11–13]. Equally alarming is the increased pathogenicity of recent H5N1 viruses in mice and ferrets [14,15], and the ability of some recent H5N1 viruses to cause systemic infections in humans [16]. Both of these findings suggest that highly pathogenic H5N1 viruses are adapting to mammalian species. Here, we review the molecular features that might determine the pathogenicity and transmissibility of highly pathogenic H5N1 influenza viruses. Pathogenicity is defined as the ability to cause disease, while transmissibility refers to the ability of the infectious agent to spread among host organisms. Currently, the relationship between these two properties is not understood. For example, high pathogenicity might not be required for transmissibility, but can result in high virus loads and/or systemic infections which might facilitate virus transmission. Roles of the HA protein and virus receptor distribution in virus pathogenicity Efficient virus binding to host cells is critical to virus dissemination during epidemics and pandemics. Both the binding to host cell receptors and the subsequent fusion of the viral and cellular membranes are mediated by the HA protein, which is, for these reasons, an important determinant of virulence and host restriction. 618 Receptor specificity of the HA protein and receptor distribution on host cells Historically, host range restriction of influenza viruses was explained as a mismatch between the HA receptor binding specificity and the receptor distribution on host cells: Epithelial cells in the human trachea contain on their surface sialic acid (SA) that is predominantly linked to galactose by an α2,6-linkage (SA-α2,6-Gal) [17,18], which is preferentially recognized by human influenza viruses [19–22]. By contrast, human influenza viruses do not efficiently bind to SA-α2,3-Gal sialyloligosaccharides, which are predominantly expressed by epithelial cells in the intestinal tract of waterfowl (the main replication site of avian influenza viruses). SA-α2,3-Gal sialyloligosaccharides are efficiently recognized by avian influenza viruses [19–22], resulting in the infection of avian, but not human, cells. This explanation, however, proved too simplistic, when a wholly avian H5N1 influenza virus isolated from an infected individual in 1997 in Hong Kong was shown to bind to SA-α2,3-Gal, but not to SA-α2,6-Gal, sialyloligosaccharides [23], suggesting that viruses with ‘avian-type’ receptor binding specificity can infect human cells. This prompted researchers to reexamine the distribution of influenza virus receptors in human respiratory organs, which led to the finding of a more complex pattern than was previously thought. Studies with in vitro-differentiated human epithelial cells from tracheal and bronchial tissues suggested that non-ciliated epithelial cells (that is, most epithelial cells) contain SAα2,6-Gal sialyloligosaccharides on their surfaces, whereas ciliated cells (a minor epithelial cell population) express SA-α2,3-Gal sialyloligosaccharides [24]. This distribution of sialyloligosaccharides corresponds to the preferential infection of non-ciliated cells by human influenza viruses [24]. The finding that human cells contain ‘avian-type’ influenza virus receptors (that is, those expressing SAα2,3-Gal sialyloligosaccharides) raised two fundamental questions: If avian influenza viruses can infect humans, why are these infections rare, and why do these viruses not spread among humans? Possible explanations come from a study that examined receptor distribution on human respiratory tissue and found appreciable differences between tissues of the upper and lower respiratory tract [25]. Epithelial cells in nasal mucosa, paranasal sinuses, pharynx, trachea and bronchi express primarily SA-α2,6-Gal sialyloligosaccharides [25], whereas the cells that line the alveolar walls express SA-α2,3-Gal sialyloligosaccharides [25]. This finding, and a similar observation by others [26], confirmed the presence of ‘avian-type’ influenza virus receptors on human cells. In addition, it might explain the low frequency of human infection by avian influenza viruses and the current inability of these © 2007 International Medical Press neumann 2/7/07 13:21 Page 619 Factors for influenza H5N1 virus pathogenicity viruses to efficiently transmit among humans: SA-α2,3Gal sialyloligosaccharides are confined to the lower respiratory tract of humans, which greatly reduces the odds of avian influenza virus infection. Moreover, the lack of ‘avian-type’ receptors in the upper airways probably prevents efficient virus replication in this setting and thereby transmission, which occurs via droplets generated by coughing and sneezing. Recently, Nicholls et al. [27] challenged this concept by demonstrating infection of upper respiratory organs with an H5N1 avian virus. Also, de Jong et al. [28] reported that individuals infected with recent H5N1 viruses had higher vRNA levels in the pharynx than individuals infected with human H3N2 or H1N1 viruses. Overall, further quantitative analyses should be carried out to assess avian influenza virus binding to and infection of cells in the upper respiratory tract of humans. Influenza virus receptor binding specificity is primarily determined by the amino acids that line the receptor binding pocket. Several studies have identified residues that are crucial for binding to SA-α2,3-Gal or SA-α2,6Gal sialyloligosaccharides: For H2 and H3 influenza viruses, Glu226 and Gly228 confer specificity for ‘aviantype’ receptors, whereas Leu226 and Ser228 mediate efficient binding to SA-α2,6-Gal sialyloligosaccharides [19,22,29,30]. Consequently, the HA proteins of avian influenza viruses encode Glu226 and Gly228, whereas human influenza virus HA encodes Leu226 and Ser228. For H1 influenza viruses, the amino acid at position 190 of HA is crucial: Glu190 in avian influenza viruses mediates efficient binding of SA-α2,3Gal sialyloligosaccharides, whereas Asp190 in human and swine influenza viruses confers specificity to human-type receptors [29,31–33]. Recently, Tumpey et al. [34] reported that conversion of Asp to Glu at position 190 and Asp to Gly at position 225 converts the receptor specificity of the pandemic 1918 virus from the human to avian type. This conversion does not significantly reduce virus titres in nasal washes and is still associated with severe disease in inoculated animals, but abolishes transmissibility of the virus among ferrets, indicating the importance of receptor specificity for efficient transmissibility. Most avian H5N1 influenza viruses possess ‘aviantype’ amino acids in their HA and, therefore, bind to SAα2,3-Gal but not to SA-α2,6-Gal sialyloligosaccharides [23,35]. All 1997 human H5N1 isolates tested showed ‘avian-type’ receptor binding specificities [23,35]. In 2003, however, two H5N1 viruses, isolated from infected individuals in Hong Kong, showed decreased levels of binding to SA-α2,3-Gal sialyloligosaccharides and low but detectable levels of binding to SAα2,6-Gal sialyloligosaccharides [35]. Both viruses contained a unique Ser-to-Asn substitution at position 227 of the HA1 protein, confirming that a single Antiviral Therapy 12:4 Pt B amino acid change is sufficient to affect HA receptor binding specificity. Stevens et al. [36] solved the threedimensional structure of the HA of an H5N1 virus and showed that replacement of two amino acids at positions 226 and 228 (H3 numbering) can convert avian-like to human-like receptor binding specificity. Another study demonstrated that three of 21 recent human H5N1 isolates recognize both sialyloligosaccharides [37]; by contrast, none of the avian H5N1 viruses tested recognized SA-α2,6-Gal sialyloligosaccharides. Further analysis has identified two amino acid changes, Asn182 to Lys and Gln192 to Arg (equivalent to positions 186 or 196 in H3 numbering, respectively) that independently converted ‘avian-type’ to ‘humantype’ receptor specificity [37]. The HA crystal structure of the respective HA protein [37] provides an explanation for how these amino acid substitutions alter receptor specificity. These amino acid substitutions were also found in recent human H5N1 viruses isolated from two individuals in Azerbaijan and one individual in Iraq. During replication in humans, highly pathogenic H5N1 viruses can acquire amino acid changes that support their replication in humans and potentially increase their likelihood of becoming pandemic (Figure 1). However, these viruses did not transmit efficiently among humans, indicating that additional factors are necessary. HA cleavage The HA protein is synthesized as a precursor protein (HA0) that is post-translationally cleaved into two disulphide-linked subunits, HA1 and HA2. The hydrophobic N-terminus of HA2 (the so-called ‘fusion peptide’) inserts into the endosomal membrane, initiating fusion between the viral and endosomal membranes. Therefore, without HA cleavage, influenza virus is not infectious. HA cleavability is a critical determinant of virulence and pathogenicity [38–40]. The cleavability of the HA protein is determined by the amino acid sequence at the cleavage site. Single Arg residues at the HA cleavage site of avirulent avian and non-avian influenza A viruses [41,42] (with the notable exception of H7N7 equine influenza viruses), are cleaved by a limited number of proteases in the respiratory tract and/or intestinal organs, resulting in localized infections with typically mild or no disease symptoms. By contrast, highly pathogenic H5 and H7 viruses cause systemic infections because the multiple basic amino acids at the HA cleavage site of these viruses are recognized by ubiquitous proteases, such as furin and PC6. The proposed consensus motif for furin recognition is QR/K-X-R/K-R (where X is a non-basic amino acid) in the absence of a nearby carbohydrate chain. The presence of such a carbohydrate chain requires 619 neumann 2/7/07 13:21 Page 620 G Neumann et al. Figure 1. Hypothetical scenario for the emergence of pandemic influenza viruses HA, PB2, etc Cells in the lower respiratory tract of humans contain ‘avian-type‘ influenza virus receptors, in contrast to cells in the upper respiratory tract. This may explain the ability of avian viruses (pale grey) to infect humans and cause disease. The inability of these viruses to replicate in the upper respiratory tract might prevent efficient human-to-human transmission. If the avian viruses acquire mutations in HA, PB2 and other proteins that allow efficient replication in the upper respiratory tract (dark grey), efficient human-to-human transmission may occur, resulting in a pandemic. extended motifs of the following kind: B(X)-X(B)-R/KX-R/K-R or Q-X-X-R-X-R/K-R (where X represents non-basic amino acids and B, basic amino acids). Avian influenza viruses of low pathogenicity can become highly pathogenic through the acquisition of multibasic HA cleavage sites, a finding that further demonstrates the importance of HA cleavability for the pathogenicity of avian influenza viruses. Examples include outbreaks of highly pathogenic H5N2 viruses in Pennsylvania in 1983 [43] or in Mexico in 1994 [44], of highly pathogenic H7N1 viruses in Italy in 1999 [45], or of highly pathogenic H7N3 viruses in Chile in 2002 [46] or in Canada in 2004 [47]. The role of terrestrial poultry in the acquisition of HA mutations Influenza viruses from terrestrial poultry display reduced affinity for SA-α2,3-Gal sialyloligosaccharides relative to viruses isolated from waterfowl [23]. Like human virus isolates, land-based poultry viruses of different subtypes (including H7N1 and H9N2) have an extra glycosylation site in their HA and a deletion in their NA stalk. These features were also found in the HA and NA proteins of highly pathogenic H5N1 poultry viruses [23,48–50]. Terrestrial poultry might, therefore, serve as an intermediary host for avian influenza viruses to acquire mutations that support their transmission to humans [23,51]. Avian H5N1 influenza viruses can also be transmitted directly from aquatic birds to humans, as was demonstrated by the isolation from infected individuals of two H5N1 influenza viruses isolated in 2003 that lacked the typical features of poultry adaptation [52]. 620 Role of the replication complex in virus pathogenicity Given that the ability of an influenza virus to cause disease probably depends on its ability to replicate efficiently to produce high virus loads and outpace cellular antiviral responses, it is not surprising that the components of the viral replication complex are involved in viral pathogenicity. Influenza virus replication is mediated by the three polymerase proteins (PB2, PB1 and PA) and the nucleoprotein NP. PB2, which binds to type 1 cap structures of cellular mRNAs, and NP, which encapsidates influenza vRNAs, have been implicated in host range restriction [53–55]. A human virus containing an avian virus PB2 gene required a ‘human-like’ amino acid at position 627 of the PB2 protein to form plaques in Madin–Darby canine kidney cells [55]. This finding was not fully appreciated until 2001, when the pathogenicity of H5N1 influenza viruses in mice was linked to the nature of the amino acid at position 627 of PB2 [56]. Most human influenza viruses contain Lys at this position (and a few possess Arg). Lys is also found at PB2-627 in the 1997 H5N1 viruses that were highly pathogenic in mice. By contrast, the 1997 H5N1 viruses that were of low pathogenicity in mice contain Glu at this position, which is also found in all avian viruses (with the exception of the Qinghai Lake H5N1 viruses and their descendants). Reverse genetics experiments demonstrated that replacement of PB2-627Glu with Lys renders viruses highly pathogenic, whereas the reciprocal experiment generates a variant of low pathogenicity in a mouse model [56]. PB2-627Lys © 2007 International Medical Press neumann 2/7/07 13:21 Page 621 Factors for influenza H5N1 virus pathogenicity continues to be found in a substantial number (albeit not all) of H5N1 viruses isolated from infected humans [28,57–59], and has been found in H5N1 viruses isolated from tigers in Thailand in 2004 and 2006 [60]. Interestingly, PB2-627Lys was also found in an H7N7 virus isolated from a fatal case of pneumonia in the Netherlands in 2003 [61]. By contrast, virus isolates from nonfatal cases and from chickens in this outbreak contained Glu at this position. PB2-627Lys thus appears to be selected during replication in mammals. Proof for this concept comes from the finding that chicken H5N1 viruses isolated from the brains of infected mice all contained this substitution [62]. However, during the Qinghai Lake outbreak in China from May to July 2005 [9,63], avian H5N1 viruses were isolated that encoded PB2-627Lys. Descendants of the Qinghai Lake viruses have maintained this mutation and continue to circulate in northern China and to cause outbreaks in Europe and Africa, attesting to their biological fitness. For H5N1 viruses isolated from infected individuals, both Lys and Glu have been found at PB2-627. This might be explained by differences in sampling time points. How does PB2-627 affect viral pathogenicity? Several studies have shown that the amino acid at PB2627 determines the replicative ability of the virus and affects host range [64–67]. Viruses with PB2-627Lys grow more efficiently in mouse, but not avian, cells compared with those containing Glu at this position [64]. Moreover, an artificial mini-replicon system showed higher replication levels for PB2 proteins encoding 627Lys than for those encoding 627Glu [67]. The amino acid at position 627 of PB2 does not, however, affect the tissue tropism of the virus in mice [64]. Collectively, these findings indicate that PB2627Lys supports virus replication in mammalian cells and provide an explanation for its selection in mammalian species. Viruses containing PB2-627Lys probably replicate to high titres in mammals resulting in efficient virus dissemination and the ability to overwhelm the host defences. The amino acid at position 627 of PB2 is now widely recognized as a crucial determinant of H5N1 virus pathogenicity. However, other amino acids in the viral replication complex also affect viral replication and pathogenicity. Two studies suggest an important role for amino acid PB2-701 in viral pathogenicity [68,69]. An Asn-to-Asp replacement at this position attenuated a duck H5N1 virus in mice, whereas the reciprocal replacement enabled an otherwise non-pathogenic duck H5N1 virus to replicate in mice [68]. Similar findings were made with an H7N7 virus, in which PB2-701Asn conferred superior replicative abilities relative to PB2701Asp [69]. The underlying molecular mechanism for this effect is unknown. Antiviral Therapy 12:4 Pt B In another study [70], exchanging the HA and NA genes of H5N1 viruses of high and low pathogenicity did not alter their pathogenicity, yet pathogenicity in mice and ferrets was determined by the origin of the three polymerase genes [70]. In vitro assays revealed higher replicative abilities for the replication complex of the highly pathogenic variant compared with those of the less pathogenic variant [70]. Thus, as observed with viruses containing PB2-627Lys or PB2-627Glu, increases in replicative ability can translate to increased pathogenicity. Collectively, these findings suggest that human-type receptor binding specificity and efficient replication in mammals (the latter mediated by mutations in the replication complex) probably facilitate efficient viral replication in humans. It was feared that the combination of these two features would produce highly pathogenic and potentially pandemic viruses. However a human H5N1 virus isolated in 2006 in Turkey that contained the PB2-627Lys mutation and a mutation in HA that facilitated its binding to human-type receptors [71] did not cause a pandemic, suggesting that yet other mutations are needed for efficient human-to-human transmission. Role of NS gene and NS1 protein in virus pathogenicity A marked cytokine imbalance occurs in individuals infected with H5N1 viruses – particularly in those with fatal outcome [28,72,73]. This imbalance is characterized by high levels of interferon-induced protein 10, monokine induced by interferon (IFN)-γ, monocyte chemotactic protein 1, interleukin (IL)-8, IL-10, IL-6, IFN-γ and tumor necrosis factor (TNF)-α. Cytokine and chemokine imbalance was also found in in vitro in H5N1-virus-infected primary macrophages [74–76] and human primary alveolar and bronchial epithelial cells [77]. In addition, mRNA upregulation of death receptor ligands such as TNF-related apoptosisinducing ligand (TRAIL), but not Fas ligand, has been documented in human monocyte-derived macrophages infected with a highly pathogenic H5N1 virus [78]. This upregulation was accompanied by an increased sensitivity of virus-infected cells to TRAIL-induced apoptosis, which suggests a role in the cytotoxicity of these viruses and thus their pathogenicity. Recent studies also indicated that H5N1 viruses activate the mitogen-activated protein kinase pathway [79]; interestingly, however, no difference was found in the ability of H5N1 and H1N1 viruses to activate the transcription factor NF-κB. Thus, a picture emerges in which highly pathogenic H5N1 viruses are more potent activators of signal transduction pathways than are other influenza viruses, resulting in an upregulation of 621 neumann 2/7/07 13:21 Page 622 G Neumann et al. cytokines and chemokines and a possible explanation for the unusual pathogenicity of these viruses. A number of studies suggest a role for the influenza viral NS gene in viral pathogenicity [76,80,81]. In pigs, recombinant viruses containing the 1997 H5N1 NS gene were more pathogenic than control viruses containing a non-H5N1 NS gene [80,81]. The 1997 H5N1 NS gene induced high levels of chemokines and conferred resistance to the antiviral effects of IFN. Hence, highly pathogenic H5N1 viruses can resist IFN, allowing them to continue to replicate and sustain the upregulation of cytokines and chemokines. The NS gene encodes two proteins, the interferon antagonist NS1 and the nuclear export protein NEP (also called NS2). Depending on the strain, NS1 encompasses 202–238 amino acids with an N-terminal RNA-binding domain and a C-terminal effector domain. The RNA-binding domain resides in the Nterminal 73 amino acids. The effector domain contains binding sites for the cleavage and polyadenylation specificity factor [82,83] and the polyA-binding protein II (PABII) [84], both of which are crucial for the functions of NS1. The NS1 protein is an IFN antagonist that ensures efficient virus replication in IFN-competent hosts. In interferon-deficient systems, such as Vero cells or STAT1–/– mice, influenza virus lacking NS1 replicates efficiently [85–89]. To counteract the host cell defence system, NS1 interferes with two major pathways: IFNβ production and the activation of IFN-induced antiviral genes. To inhibit IFN-β production, NS1 blocks the activation of transcription factors, such as NF-κB, IFN regulatory factor 3 or activation protein 1 [90–92]. The mechanism by which NS1 achieves this is not fully understood; however, several studies indicate that the RNA-binding activity of NS1 is crucial for this function [93,94], probably because it sequesters the doublestranded RNA that is essential for the activation of the IFN response. In addition to blocking transcription factor activation, NS1 also directly interferes with the activation of the IFN-β promoter. As stated above, NS1 also interferes with IFN-βstimulated genes, such as protein kinase R (PKR) [95–99]. PKR expression is stimulated by IFN-β and the NS1-mediated inhibition of IFN-β also affects PKR expression in infected cells. Moreover, NS1 has a direct effect on PKR levels by sequestering dsRNA, a known PKR activator, and, possibly, by directly interacting with PKR [98]. In influenza-virus-infected cells the cellular PKR inhibitor, P58IPK, is activated, suggesting another means by which influenza virus interferes with PKR expression. It is not known, however, whether NS1 is directly involved in this mechanism. Recently, a new concept has emerged in which dsRNA-binding by 622 NS1 prevents the dsRNA-dependent activation of 2′-5′ oligosynthetase [100], which in turns activates RNase L, a key player in the innate immune response. NS1 has also been shown to inhibit adaptive immunity by suppressing dendritic cell maturation, migration and T-cell-stimulatory activity [101]. The NS1 proteins of H5N1 viruses isolated from infected individuals in 1997 in Hong Kong confer resistance to the antiviral effects of IFN and induce high levels of proinflammatory cytokines [76,80,81]. The resultant cytokine imbalance, which has also been observed in victims of H5N1 virus infections [73,102], might be crucial to the pathogenicity of these viruses. Likewise, the 2003 H5N1 viruses similarly induce high levels of certain cytokines in patients and cell culture [72,75], although the NS genes of these two groups of viruses differ. The NS1 proteins of highly pathogenic H5N1 viruses isolated in the 1997 outbreak differ from other NS1 proteins by an aspartic-acid-to-glutamic-acid change at amino acid 92, a substitution that is critical for the pathogenicity and/or cytokine resistance of these viruses in pig and mouse models [80,81,103]. In addition, a study of reassortant and mutant H5N1 geese viruses in chickens found a crucial role for amino acid 149 of NS1 [104]. How these mutations affect the biological properties of NS1 is unknown; however, the recently solved crystal structure of the NS1 effector domain [105] might provide clues as to the molecular mechanisms by which NS1 modulates host cell responses. Large-scale genome analyses to identify amino acid changes that affect pathogenicity and/or transmissibility Since the first human infections with highly pathogenic H5N1 viruses in Hong Kong in 1997, researchers have searched for key amino acids that predict the pathogenicity and/or transmissibility of these viruses, in order to predict the outcome of human infections. Bioinformatics approaches were initially hampered by the low number of available H5N1 virus genomic sequences; in fact, until recently, even the number of complete genomic sequences for non-H5N1 influenza viruses was surprisingly small. However, over the last few years, tremendous efforts have been made to sequence complete influenza viral genomes. Data from a large-scale sequence analysis of avian influenza viruses were published recently [106] and revealed a previously unrecognized PDZ domain ligand (PL) at the C-terminus of NS1. PDZ domains are modular protein interaction domains that are found in proteins involved in signalling pathways. Human and avian influenza virus NS1 proteins differ © 2007 International Medical Press neumann 2/7/07 13:21 Page 623 Factors for influenza H5N1 virus pathogenicity in their PL motifs, resulting in different binding patterns to human PDZ domains [106]. Human H5N1 viruses isolated during the outbreaks in 1997 and 2003–2004 are characterized by ‘avian-type’ PL domains [106], which might interfere more severely with signal transduction in human cells than do their human counterparts. Although hypothetical at this point, this scenario demonstrates the potential of largescale genome analyses for the identification of amino acids that affect pathogenicity. Comparisons of avian and human influenza virus sequences have identified ‘avian-like’ and ‘human-like’ signature amino acids (reviewed in [107,108]). The conservation of these amino acids in avian or human virus isolates, respectively, suggests a biological function; however, direct experimental evidence for an involvement in host range restriction, virulence and/or pathogenicity is lacking for most of these signature amino acids. An alternative approach to identifying H5N1 virus amino acids critical for viral pathogenicity involved comparing genomic sequences of viruses isolated from fatal and non-fatal human cases [28]. This approach, however, yielded no changes that correlated with outcome, suggesting that other factors, such as the immune status and/or genetic factors of the infected individual, have a crucial role in the disease course. Summary and outlook Molecular studies of H5N1 influenza viruses have identified amino acids that contribute crucially to pathogenicity. However, none of the viruses containing these key amino acids has acquired the ability to spread efficiently among humans; in fact, high pathogenicity might not be a requirement for a pandemic virus. Efficient human-to-human transmission will probably require a combination of several identified and as yet unidentified key changes in the viral genome that facilitate efficient binding to and replication in epithelial cells of the upper respiratory tract, as well as efficient suppression of host immune responses. With the advent of bioinformatics approaches and the growing number of genomic influenza virus sequences available, additional determinants of pathogenicity will probably be identified and tested for their biological significance. The combination of experimental and bioinformatics approaches should generate valuable data on influenza virus pathogenicity, virulence and transmissibility. It should be borne in mind, however, that a defined set of mutations that determines pathogenicity and transmissibility might not exist; rather, different combinations of mutations may render a virus pathogenic and/or pandemic, depending on the viral background. Antiviral Therapy 12:4 Pt B Acknowledgements We thank Susan Watson for editing the manuscript. We also thank those in our laboratories who contributed to the data cited in this review. Our original research was supported by National Institute of Allergy and Infectious Diseases Public Health Service research grants; by CREST (Japan Science and Technology Agency), and by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Wright PF, Neumann G, Kawaoka Y. Orthomyxoviruses. In Fields Virology, 2007; pp. 1691–1740. Edited by DM Knipe, PM Howley, DE Griffin, RA Lamb, MA Martin, B Roizman & SE Straus. 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