key: cord-0007889-wu7i548j
authors: Sriwilaijaroen, Nongluk; Suzuki, Yasuo
title: Molecular Basis of a Pandemic of Avian-Type Influenza Virus
date: 2014-05-27
journal: Lectins
DOI: 10.1007/978-1-4939-1292-6_38
sha: b9e22577a56e0c6bd8db76e3d9f49effe27beb0f
doc_id: 7889
cord_uid: wu7i548j

Despite heroic efforts to prevent the emergence of an influenza pandemic, avian influenza A virus has prevailed by crossing the species barriers to infect humans worldwide, occasionally with morbidity and mortality at unprecedented levels, and the virus later usually continues circulation in humans as a seasonal influenza virus, resulting in health-social-economic problems each year. Here, we review current knowledge of influenza viruses, their life cycle, interspecies transmission, and past pandemics and discuss the molecular basis of pandemic acquisition, notably of hemagglutinin (lectin) acting as a key contributor to change in host specificity in viral infection.

An infl uenza pandemic is grim as it is unpredictable, rapidly spreads throughout the world, and is mostly associated with severe clinical disease and death in humans, leading to the serious socioeconomic problems. Infl uenza is an infectious respiratory illness epidemically caused by human infl uenza A, B, and C viruses (classifi ed on the basis of serologic responses to matrix proteins and nucleoproteins), which are single-stranded, negative-sense RNA viruses of the family Orthomyxoviridae [ 1 ] . Type A viruses have the greatest genetic diversity, harboring numerous antigenically distinct subtypes of the two main viral surface glycoproteins; so far, 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes have been identifi ed. All possible combined 16 × 9 subtypes, except for H17-18 and N10-11 subtypes, which are detected only in bats (mammals) and exhibit functions completely different from those of the other subtypes [ 2 -4 ] , are maintained in waterfowls, mainly in wild ducks. These avian viruses are occasionally transmitted to other species that are immunologically naïve, in which they may only cause outbreaks or may acquire mutations so as to be effi ciently transmitted between new hosts, and they can lead to a pandemic in human populations. An infl uenza pandemic has continued to circulate as an epidemic with an antigenic variant each year, and two human infl uenza A subtypes, H3N2 Hong Kong/68 and H1N1 2009 variants, have been seasonally found among humans.

The unexpectedly rapid emergence of A/H1N1 2009 swine pandemic recently, ongoing outbreaks of avian infl uenza viruses in humans, circulating avian/human/swine infl uenza viruses in pigs, and the emergence of infl uenza A viruses that are resistant to currently available anti-infl uenza virus drugs have raised a great concern that a pandemic could spread rapidly without time to prepare a public health response to stop the illness spread and could threaten human health and life throughout the world, becoming a major impediment to socioeconomic development. We have collected available information in the infl uenza A virus fi eld, especially factors playing important roles in determining viral transmission, in order to know how best to perform surveillance, prevent, slow, or limit a future pandemic.

Infl uenza A virus contains eight (−) ssRNA genomic segments that encode at least ten proteins; nine are structural proteins and 1-4 depending on the virus strain and host species are nonstructural proteins ( see Fig. 1 ; also see Table 1 ). Once inside the host, the virus is able to escape the host's innate immune responses in two ways: mainly by viral nonstructural protein 1 (NS1) attacking multiple steps of the type I IFN system, resulting in evasion of type I IFN responses [ 20 ] , and by viral NA removing decoy receptors on mucins ( see Figs. 2 and 3 ), cilia, and cellular glycocalyx and preventing self-aggregation of virus particles [ 22 ] . Also, the virus is capable of evasion of adaptive immune responses: evasion of the preexisting humoral or neutralizing antibodies and seasonal vaccines by antigenic variation in HA and NA antigens [ 23 ] , and evasion of cellular immune response by amino acid substitutions in cytotoxic T-lymphocyte (CTL) epitopes of viral proteins, resulting in a decrease in CTL response [ 24 ] . Furthermore, although the precise functions of PB1-F2 remain unclear and are virus strain-specifi c and host-specifi c, it has been thought that PB1-F2 plays roles in both innate and adaptive immune responses in order to support infl uenza virus infection [ 25 ] .

The replication cycle of an infl uenza A virus ( see Fig. 2 ) starts from attachment of viral HAs to sialic acid (Sia, 5-amino-3,5dideoxy-D -glycero -D -galacto -2-nonulosonic acid or neuraminic acid (Neu)) receptors on the host cell surface ( see Subheading 5.3 ) . This attachment mediates internalization of the virus into the cell by receptor-mediated endocytosis. While an early endosome gradually matures, the acidity in the endosome gradually increases. The low pH activates the integral membrane protein M2 of infl uenza virus, which is a pH-gated proton channel in the viral lipid envelope, conducting protons into the virion interior. Acidifi cation of the virus interior causes weakening of electrostatic interaction, leading to dissociation of M1 proteins from the viral RNP complexes (unpacking of the viral genome). The low pH in late endosomes also triggers a conformational change in HAs, resulting in exposure of their fusion peptides that immediately bind hydrophobically to the endosomal membrane (fusion), followed by release of vRNPs into the cytoplasm. During the course of the endocytic pathway, sialidase of NAs has been shown to be active [ 26 ] , possibly in order to promote HA-mediated fusion [ 27 ] ; however, further studies are needed to determine the exact mechanisms of this NA function. It should be noted that HA fusion will not occur if the HA protein (HA0) is not cleaved to form HA1 and HA2 by a membrane-bound host protease either before or during the release of progeny virions or with incoming viruses prior to endocytosis at the cell surface ( see Subheading 5.2 ) . Infl uenza A virus structure. Infl uenza A virus particles are roughly spherical in shape with sizes ranging from 80 to 120 nm in diameter, and each particle is enveloped by a lipid bilayer derived from the host cell membrane. Inside the envelope, each of eight (−) ssRNA genomic segments are wrapped with multiple nucleoproteins (NPs) and bound to RNA polymerases (PB1, PB2, PA) forming the vRNPs. The inner layer of the lipid envelope is attached to M1 molecules bound to vRNPs and to NS2. The outer layer of the lipid envelope is spiked with HA, NA, and M2 molecules with a ratio of about 5/2/1. See color fi gure in the online version The number of nucleotides and that of amino acids may vary depending on the host and strain of infl uenza A virus b NS3 was experimentally found during the adaptation of a human virus within a mouse host [ 19 ] The vRNPs in the cytoplasm are immediately imported into the nucleus most probably by nuclear localization signals in proteins composed of vRNPs, and the viral RNA polymerase transcribes the (−) vRNAs primed with 5′-capped RNA fragments, which are derived from cellular mRNAs by a cap-snatching mechanism, to viral mRNAs and replicates the unprimed (−) vRNAs to complementary RNAs, (+) cRNAs, used as templates to generate (−) vRNAs (transcription and replication). The viral mRNAs are subsequently exported to the cytoplasm for translation into viral proteins by the cellular protein-synthesizing machinery. Viral proteins needed for viral replication and transcription are transported back to the nucleus. The newly synthesized vRNPs are exported from the nucleus to the plasma membrane, mediated by M1 and NS2 

Sia-Gal Sia-Gal-Glc Ganglio-series (-Galβ1-3GalNAc-) Sia-Gal-GalNAc-Sia-penultimate Gal-** Sia-internal Gal-*** Lacto-series Type-I (-Galβ1-3GlcNAc-) Neolacto-series Type-II (-Galβ1-4GlcNAc-) 

Wild waterfowls are the main reservoir of H1-H16 and N1-N9 infl uenza A viruses [ 32 ] ( see Fig. 4 ). Infl uenza A viruses replicate in the gut of wild waterfowls, which are usually asymptomatic. Infected wild waterfowls excrete viruses in feces and spread viruses mainly via virus-contaminated water and fomites (fecal-contaminated-water-oral route 

Having (1) numerous wild waterfowl species as natural reservoirs, (2) various animal hosts, (3) RNA polymerase without proofreading, and (4) segmented genome (3 and 4 causing a high mutation rate), infl uenza A viruses have been diffi cult to control and/or eradicate. Efforts for prevention of the next pandemic, either by minimization of crossinfection between species or rapid identifi cation of novel strains, constitute an essential and primary step for preventing infl uenza infection in human beings. Interspecies transmission of infl uenza A viruses between animal hosts including pigs, horses, and birds, as well as humans, has occasionally been detected, but successful propagation and transmission in their new host have been restricted. In the past 95 years, only four infl uenza A virus strains led to sustained outbreaks in human populations and started pandemics ( see Table 2 ; also see Fig. 5 ). The pandemic virus induced humans to develop immunity that provides selective pressure and drives the virus to evolve their antigenicity. The resulting 1918-derived H1N1 virus, which had antigenic change annually, caused an epidemic with lower death rates and triggered human immunity to the virus over time. Somehow the 1918-derived H1N1 virus underwent dramatic genetic change with acquisition of three novel gene segments, avian-like H2, N2, and PB1 gene segments, resulting in emergence of the H2N2 pandemic in 1957 and disappearance of the 1918-derived H1N1 virus from circulation. However, the 1918-derived H1N1 virus from the pre-1957 period reappeared in 1977, its reemergence believed to be from a laboratory in Russia or Northern China, and caused a (lowgrade) pandemic mostly affecting young people less than 20 years of age due to immunological memory to the virus in most elderly people. After that, H1N1 Russian/77 variant became epidemic yearly until the 2009 H1N1 pandemic emerged, and the virus disappeared from humans. The disappearance of the preexisting H1N1 seasonal virus was suggested to be due in part to stalk-specifi c antibodies boosted from infection with the 2009 H1N1 pandemic virus [ 47 ].

In early 1956, an infl uenza outbreak of a new H2N2 strain occurred in China and spread worldwide, resulting in a pandemic in 1957. It has been believed that the pandemic H2N2 virus evolved via reassortant between avian H2N2 strain and the preexisting circulating human 1918 H1N1 strain; it consisted of three gene segments coding HA (H2), NA (N2), and PB1 derived from an avian virus, with the other fi ve gene segments derived from a previously circulating human virus. New HA and NA surface antigens to human immunity for protection resulted in the Asian infl uenza pandemic virus infecting an estimated 1-3 million people worldwide with approximately two million deaths. The virus became seasonally endemic and sporadic and it disappeared from the human population after the next pandemic appeared in 1968 [ 48 ].

In July 1968, a new infl uenza A virus was detected in Hong Kong. It was identifi ed as H3N2, which was believed to be a result of reassortment between avian and human infl uenza A viruses: the HA and PB1 gene segments were derived from avian infl uenza virus and the other six gene segments, including the NA N2 gene segment, were derived from the 1957 H2N2 virus. The virus killed up to one million humans varying widely depending on the source, less deaths than those in previous pandemics. The relatively small number of death is thought to be due to exposure of people to the 1957 virus, who apparently retained anti-N2 antibodies, which did

Infl uenza Pandemic (1957) (1958) 

Infl uenza Pandemic (1968) (1969) not prevent 1968 infection but limited virus replication and reduced the duration and severity of illness. Although the next pandemic occurred in 2009, the virus is still in circulation globally (as of 2013) as a seasonal infl uenza strain.

In April 2009, a widespread outbreak of a new strain of infl uenza A/H1N1 subtype referred to as swine fl u was reported in Mexico. By June 2009, the virus had spread worldwide, starting the fi rst infl uenza pandemic of the twenty-fi rst century. In August 2010, the infl uenza activity returned to a normal level as seen for seasonal infl uenza; at least 18,000 laboratory-confi rmed deaths from the pandemic 2009 were reported, but infection with the virus resulted in mild disease with no requirement of hospitalization [ 49 ] . To date, the infl uenza A viruses identifi ed among people are 1968 H3N2 and 2009 H1N1 viruses.

Genetic composition of the pandemic 2009 viruses isolated from initial cases indicated that the viruses are composed of PB2 and PA gene segments of North American avian virus origin, PB1 segment of human H3N2 virus origin, HA (H1), NP and NS segments of classical swine virus origin (their genes having been found to circulate in pigs since 1997/1998 known as avian/human/swine triple reassortant H1N2 swine viruses), and NA (N1) and M segments of Eurasian avian-like swine H1N1 virus origin (which emerged in European pigs in 1979); hence, their original description was "quadruple" reassortants ( see Fig. 5 ). Similarity between the HA sequence patterns of the 1918 pandemic and 2009 pandemic viruses and their cross-antibody neutralization [ 50 -53 ] suggested that H1 HA of the 2009 pandemic virus may have originated from or derived from the same origin of the 1918 pandemic virus; probably, the 1918-like pandemic H1N1 virus was transmitted and established in domestic pigs between 1918 and 1920 referred to as the classical swine lineage that circulated continuously in pigs in the USA. Domestic pigs having a short lifespan (4-to 6-monthold pigs are killed for their fl esh, and female breeding pigs (sows) remain in the farm until the age of 4-5 years and then taken to be slaughtered for sausages and bacon) are a frozen-like source for the infl uenza virus due to a lack of selection pressure in pigs. Consequently, the HA sequence of the 2009 swine origin derived from the classical swine lineage is not much different from its origin.

The past pandemics and ongoing direct transmission of avian infl uenza A viruses into humans suggest two plausible mechanisms that would permit infl uenza A viruses to overcome selective pressure and subsequently become established in human populations ( see Fig. 5 ; also see Subheading 5.3.3 ). As shown in Fig. 6 , one mechanism is an adaptation mechanism, in which a nonhuman virus acquires a mutation(s) (a mutation(s) in the HA gene to recognize the Neu5Acα2-6Gal receptor considered to be an essential prerequisite for the beginning of a pandemic) during adaptation to 4 It is not known when and how a future pandemic will emerge, by an adaptation or reassortment mechanism, and it is also impossible to predict which virus subtype will be the next pandemic. In the post-pandemic period, most people develop immunity to the pandemic strain and thus the pandemic virus can continue to cause seasonal outbreaks (epidemics) if it can change its surface antigens (antigenic variation) to evade host immunity.

HA was so named due to its ability to agglutinate red blood cells via binding to Sia on red blood cells. Phylogenetically, 18 HA subtypes are classifi ed into group 1 and group 2 ( see Fig. 7a ). Notable differences in structure between the two groups of HAs are in the region involved in HA conformational change required for membrane fusion; group 1 HAs contain an additional turn of the helix at residues 56-58, blocking accessibility of tert -butyl hydroquinone, which is accessible to group 2 [ 54 ] . HA is encoded by the fourth segment of the infl uenza A viral genome and is assembled as a homotrimeric precursor (HA0) ( see Fig. 7b ). HA is a major target of neutralizing antibodies, plays a pivotal role in avian infl uenza virus pathogenicity, and is a major determinant of host range restriction. It is a lectin that contains one or more carbohydrate recognition domains that determine host specifi city [ 55 ] and plays a crucial role in fusion of the viral envelope and cellular endosomal membrane for release of the viral genome into host cells ( see Fig. 7c-f ).

Change in HA antigen is responsible for epidemics and pandemics. Change in HA antigen either by accumulation of mutations in HA1 of fi ve proposed antigenic sites (based on amino acid sequence comparison among viruses isolated from different years or among variants grown in the presence of mouse monoclonal antibodies) as shown in Fig. 7c [ 56 -59 ] or by intrasubtypic reassortment between distinct clades of co-circulating infl uenza A viruses [ 35 ] is responsible for evasion of recognition by the host antibodies and thus Cleavage site 324P-X-X/R/K-X-X-R/K↓GLF X = nonbasic residue 324P-X-R/K-X-R/K-R↓GLF (due to insertion/substitution) Host proteases Extracellular tissue-restricted trypsin-like proteases Ubiquitously expressed intracellular proteases (e.g., furin and PC6) continuous circulation of the virus in host populations. Amino acid sequencing studies of HAs of avian and animal viruses isolated from different periods of time have shown that the HAs of avians and animals that have shorter lifespans have higher conservation of amino acid sequences than that of human virus isolates, suggesting that avian/animal viruses are subjected to little immune pressure, resulting in less antigenic variation than that of human virus strains [ 60 , 61 ] .

Introduction of a novel HA antigen, resulting from genetic reassortment during mixed infection, from direct introduction of a nonhuman infl uenza virus, or from reintroduction of human infl uenza viruses that had disappeared from circulation, into immunologically naïve human populations is a key factor of an infl uenza virus with pandemic potential.

To enable HA conformational changes that lead to membrane fusion, which is critical for viral infectivity and dissemination, HA0 must be cleaved by a host cell protease into subunits HA1 and HA2; thereby, the host protease is a determinant of tissue tropism of the virus. Infl uenza virus HA0 usually contains a monobasic cleavage site ( see Fig. 7d ), which is recognized by extracellular trypsin-like proteases, such as tryptase Clara from rat bronchiolar 

f. HA is conformationally changed at acidic pH leading to fusion. Negative D136 in H17& H18 has been proposed to electrostatically repulse Sia (4).

H17&H18 membrane fusion has been thought to occur at pH 8.0 on the host cell (3) (4) . 

epithelial Clara cells [ 62 ] and mast cell tryptase from the porcine lung, found only in a few organs, and thus virus infection is localized in a limited number of organs, such as the respiratory and intestinal tracts, resulting in mild or asymptomatic symptoms (including ruffl ed feathers and decreased egg production); hence, the causative viruses are called LPAI viruses [ 63 ] . Multiplication of H5 or H7 LPAI viruses in chickens and turkeys generates HPAI viruses having multiple basic amino acids ( see Fig. 7d ) at the HA cleavage site recognized by intracellular ubiquitous subtilisin-like proteases, such as furin and proprotein convertase 6 (PC6), which are present in a broad range of organs, allowing the virus to infect multiple internal organs with a mortality rate as high as 100 % within 48 h (lethal systemic infection or fowl plaque typically being characterized by cyanosis of combs and wattles, edema of the head and face, and nervous disorders [ 64 -67 ] ). Sequence analysis of the HA cleavage site showed that some LPAI H5 and H7 subtypes contain a purine-rich sequence, and thus a direct duplication (unique insertion) in this region could lead to lysine (K) and/or arginine (R)-rich codons (codon AAA or AAG specifying lysine and codon AGA or AGG specifying arginine); this is a reason why HPAI viruses have been derived only from subtypes H5 and H7 [ 68 ] . Not only the basic cleavage site sequence but also a carbohydrate side chain near the cleavage site contributes to determination of pathogenicity (virulence) if it interferes with the host protease accessibility.

Since the fi rst report of a HPAI H5N1 progenitor strain in 1996 from a farmed goose in Guangdong Province, China (A/gs/ Guangdong/1/96 designated as Clade 0), the world has intermittently experienced HPAI virus outbreaks, both recurrence and new HPAI virus outbreaks, in domestic birds classifi ed into groups or clades (20 clades having been recognized at present) and subdivided into subclades and lineages based on their phylogenetic divergence as the virus continues to evolve rapidly [ 69 , 70 ] . Although they are generally restricted to domestic poultry on farms with high mortality rates and substantial economic losses, HPAI H5N1 viruses have occasionally been isolated from some species of wild waterfowls, including wood ducks and laughing gulls, with varying degrees of severity [ 71 -75 ] ; thus, migration of susceptible waterfowls could spread HPAI H5N1 viruses over long distances, leading to diffi culties for avian infl uenza control. The signifi cant species-related variation in susceptibility to and clinical disease caused by H5N1 virus infection has been determined not only in wild birds but also in other animals. For example, pigs can be infected with HPAI H5N1 viruses, but they have almost no or very weak disease symptoms or only slight respiratory illness. Without infl uenza-like symptoms, the virus may adapt to mammalian hosts in the respiratory tract of this potential intermediate host [ 76 ] , which contains gradual increases in Neu5Acα2-6Gal, a human receptor, over Neu5Acα2-3Gal, an avian receptor, from upper and lower parts of the porcine trachea towards the porcine lung, a primary target organ for swine-adapted virus replication [ 77 ] . Humans can be infected with HPAI H5N1 virus (fi rst report in 1997) with severe disease and high death rate. The ecological success of this virus in crossing the species barrier from poultry to infect diverse species including wild migratory birds and other mammals including pigs, cats, and dogs with sporadic infections in humans often with fatal outcomes [ 78 , 79 ] highlighted the possibility of HPAI H5N1 development to a pandemic strain either by gradual modifi cation of existing structures or rapid modifi cation by reassortment with a human epidemic strain. Although the world has been at phase 3 in WHO's six phases of pandemic alert since 2006 [ 80 ], HPAI H5N1 viruses should not be neglected in efforts to perform surveillance and health management planning.

In addition to HPAI H5N1 viruses, other avian infl uenza viruses including LPAI H5N1, HPAI and LPAI H7N7, HPAI H7N3, and LPAI H9N2 viruses have occasionally crossed the species barrier to infect other mammals including humans ( see Fig. 4 ) and have caused generally mild disease (with conjunctivitis or mild respiratory symptoms) in humans. Recently, LPAI H7N9 virus (a novel avian-avian reassortant virus: HA from wild-duck H7N3 virus, NA from wild-bird H7N9 virus, PA, PB1, PB2, NP, and M from chicken H9N2 virus, and NS from another chicken H9N2 virus) identifi ed in humans in February 2013 in China has killed 45 (due to severe pneumonia) of the 139 laboratory-confi rmed cases (case-fatality ratio of about 32 %) according to WHO data in November 2013 [ 42 , 81 ] . This evidence indicated that HPAI viruses primarily infect poultry and cause severe illness and high death rates in poultry and that they occasionally infect other nonpoultry species with variation in severity depending on the virus strain and host. LPAI viruses spread silently in poultry and occasionally spread to other non-poultry species and often cause mild illness but are capable of causing severe disease, such as disease caused by LPAI H7N9 virus infection in humans. Thus, more studies are needed to understand differences in pathogeneses of these viral infections.

Infl uenza viruses enter the body and search for cells among the host cells in which they can replicate and grow. Infl uenza virus homing is triggered by interactions between viral HA spikes and sialylglycoconjugates on host cell surface [ 82 ] , which play roles in a wide variety of host biological processes, including cell proliferation, apoptosis, and differentiation [ 83 ] . More than 50 types of sialic acids are found in nature, with N -acetylneuraminic acid (Neu5Ac) and N -glycolylneuraminic acid (Neu5Gc) being the most prevalent forms. Not only sialic acid type but also glycosidic linkage type (the most common terminal linkages being α2-3 and

α2-6 linkages), substructure (such as GalNAc or GlcNAc), and other modifi cations (such as fucosylation and sulfation) cause diversity in sialylglycoconjugates ( see Figs. 2 , left panel and 3 ) [ 84 , 85 ] . The sialic acid type and the glycosidic linkage type on the host cell surface are the principal determinants of host range restriction of infl uenza viruses, although other glycan modifi cations may be involved in the virus-receptor binding preference. Therefore, the distribution of sialylglycoconjugates among animal species and tissues, a crucial factor for infl uenza A infection and transmission, has been extensively investigated either by lectin histochemical analysis with Maackia amurensis agglutinin (MAA-I specifi c for Siaα2-3Galβ1-4GlcNAc-, MAA-II for Siaα2-3Galβ1-3GalNAc) and Sambucus nigra agglutinin (SNA specifi c for Siaα2-6Galβ1-4GlcNAc-) or by structural characterization using sequential glycosidase digestion in combination with HPLC and mass spectrometry. Figure 4 shows sialic acid-containing receptors in main target organs in important host species of infl uenza A viruses.

So far (2013), all H1-H16 and N1-N9 avian infl uenza viruses have been reported in 12 bird orders, most having been isolated from the order Anseriformes, especially in the family Anatidae (ducks, swans, and geese), and the order Charadriiformes (shore birds) in the family Laridae (gulls, terns, and relatives). It should be noted that the newest H17N10 and H18N11 viruses recognized in 2012 and 2013, respectively, were found only in bats, the little yellow-shouldered bat Sturnira lilium for H17N10 and the fl at-faced fruit bat Artibeus planirostris for H18N11, in the family Phyllostomidae, a family of frugivorous bats that are abundant in Central and South America [ 2 , 3 ] . Ducks in the Anatinae subfamily belonging to the family Anatidae are the most common source of infl uenza A virus isolation and risk for virus transmission [ 86 ] . Almost all ducks are naturally attracted to aquatic areas including wetlands, lakes, and ponds for resting, feeding, and breeding in their course of migration, allowing infl uenza viruses to be transmitted to and from domestic duck populations. Infected domestic ducks spread the virus to other avian species in a local area [ 73 ] . The duck tracheal and intestinal epithelium was shown to predominantly express Siaα2-3Gal oligosaccharides (the ratio of Siaα2-6Gal to Siaα2-3Gal in the duck trachea being approximately 1:20) [ 87 , 88 ] . Not only Neu5Acα2-3Gal but also Neu5Gcα2-3Gal (not found in chickens) glycans are present in the epithelium of the duck jejunum, cecum, and colon [ 89 ] . Correlated with the duck hosts, the duck-isolated infl uenza viruses preferentially bind to Neu5Ac/Neu5Gcα2-3 receptors (avian receptors) [ 82 , 89 -91 ] . This also agrees with the fi nding that avian infl uenza virus isolates replicate effi ciently in chorioallantoic cells of 10-day-old chicken embryonated eggs that contain N -glycans, which are essential for entry into host cells of infl uenza virus infection [ 92 ] , with molar

percents of α2-3 linkage and α2-6 linkage of 27.2 and 8.3, respectively [ 93 ] .

Studies on sialic acid substructure binding specifi city of infl uenza viruses revealed that although most avian viruses share their preferential binding to terminal Neu5Acα2-3Gal, duck-isolated infl uenza viruses prefer the β1-3 linkage between Neu5Acα2-3Gal and the next sugar residue such as 3′SiaLe c and 3′SiaTF, whereas gull-isolated infl uenza viruses show high affi nity for the β1-4 linkage such as 3′SLN, for fucosylated receptors such as 3′SiaLe x , and for sulfated receptors such as Neu5Acα2-3Galβ1-4(6-O -HSO 3 )GlcNAc (6-O -Su-3′SLN) and 6-O -Su-3′SiaLe X ( see Fig. 3 ) [ 90 ] . These receptor-binding specifi city data of infl uenza viruses are correlated well with intestinal epithelial staining with SNA and MAA lectins showing that the duck intestinal epithelium expressed a high level of Siaα2-3Galβ1-3GalNAc-moieties (preferential to MAA-II), whereas the gull intestinal epithelium dominantly expressed Siaα2-3Galβ1-4GlcNAc-moieties (preferential to MAA-I).

Screening using a virus-receptor binding assay together with molecular modeling revealed that gull-viral HAs with 193R/K displayed increased affi nity for 6-O -Su-3′SLN and 6-O -Su-3′SiaLe X due to favorable electrostatic interactions of the sulfate group of the receptor and positively charged side chain of 193R/K [ 94 , 95 ] . The gull-viral HAs with 222Q exhibited binding affi nity for the fucosylated receptor 3′SiaLe x similar to binding affi nity for the nonfucosylated counterpart 3′SLN, while duck infl uenza viruses showed ineffi cient binding to the fucosylated receptor due to steric interference between its bulky 222K on the HA and the fucose moiety of the receptor [ 94 , 95 ] . Only some gull-isolated infl uenza viruses have potential to infect ducks, indicating that there is a host-range restriction between avian species [ 96 ] .

Several infl uenza A viruses including H1-H13 and N1-N9 subtypes have been isolated from domesticated poultry in the family Phasianidae of the order Galliformes, including turkeys, chickens, quails, and guinea fowls [ 97 , 98 ] . Adapted avian infl uenza viruses in domestic poultry can be divided into two main forms according to their capacity to cause low or high virulence in the infected poultry ( see Subheading 5.2 ). Both forms of avian isolates from poultry before 2002 mainly bind to α2-3 sialyl linkages using either synthetic sialyloligosaccharides or erythrocytes as molecular probes for infl uenza virus binding specifi city [ 99 -102 ] , but since 2002, some of the isolates have shown an increase in binding to α2-6 sialyl linkages ( see Subheading 5.3.3 ). Tissue staining with avian and human infl uenza viruses and with MAA and SNA lectins has shown the presence of Siaα2-3Gal-and Siaα2-6Gal-terminated sialyloligosaccharides in respiratory and intestinal epithelia of gallinaceous poultry, including chickens and quails [ 87 , 103 -108 ] . However, the proportion of α2-3 and α2-6 sialyl linkages in

respiratory and intestinal epithelia of the poultry is still controversial [ 87 , 107 , 109 ] . Thus, more investigations of the structure and distribution of receptors in the replication sites of infl uenza A viruses are needed to understand the basis of viral infection and transmission.

Human-adapted infl uenza A viruses that possess effi cient humanto-human transmission ability mainly target the human upper respiratory tract, where they can be readily spread with a sneeze or cough. Lectin histochemistry of human respiratory tissues demonstrated that epithelial cells in the upper respiratory tract (noselarynx) and in the upper part of the lower respiratory tract (trachea and bronchi) are enriched in α2-6 sialylated glycan receptors with a small proportion of α2-3 sialylated glycans [ 110 ] ; using human airway epithelium (HAE) cells, lectin staining indicated that α2-6linked sialylated receptors are dominantly present on the surface of nonciliated cells, while α2-3-linked sialylated receptors are present on ciliated cells [ 111 , 112 ] . In the lower part of the lower respiratory tract (lung), α2-6-sialylated glycans can be found on epithelial cells of the bronchioles and alveolar type-I cells; α2-3-sialylated glycans can be found on nonciliated cuboidal bronchiolar cells and alveolar type-II cells [ 110 , 113 ] . Recent mass spectromic analysis of glycan structures of human respiratory tract tissues showed that both Sia α2-3 and α2-6 glycans are present in the lung and bronchus [ 114 ] . The pattern of lectin localization correlated with the pattern of virus binding and infection: human-adapted viruses bound extensively to bronchial epithelial cells but intensively to alveolar cells, and the opposite results were found for avian viruses [ 110 ] ; human-adapted viruses and avian viruses preferentially infected nonciliated cells and ciliated cells in the HAE, respectively [ 111 , 112 ] . Clinically, seasonal infl uenza viruses mainly infect the upper respiratory tract [ 115 ] ; however, pulmonary complications of infl uenza virus infection related to secondary bacterial pneumonia (such as by Staphylococcus aureus infection) rather than primary infl uenza pneumonia can occur, especially in children less than 2 years of age, adults more than 65 years of age, pregnant women, and people with comorbid illnesses/poor nutrition [ 115 , 116 ] . The 2009 H1N1pdm viruses mostly attack the upper respiratory tract, resulting in subclinical infections or mild upper airway illness, but some are able to replicate in the lower respiratory tract as seen from diffuse alveolar damage in autopsy tissue samples from patients who died from the 2009 H1N1pdm virus. The viruses probably acquire D222G in HAs, leading to dual receptor specifi city for α2-3and α2-6-linked sialic acids [ 117 ] , and more than 25 % of samples were co-infected with bacteria [ 118 , 119 ] . Either HPAI H5 or H7 infection in terrestrial poultry spreads rapidly and causes damage throughout the avian body [ 120 ] , but HPAI H5N1 infection in humans seems to be restricted to the respiratory tract

and intestine, and H5N1 virus mainly replicates in pneumocytes, frequently causing death with acute respiratory distress syndrome (ARDS) (fatality rate of about 60 %) [ 121 ] . Either HPAI or LPAI H7 infection or LPAI H9, H10, and H6 infection in humans can result in disease in both ocular tissues that predominantly express α2-3-linked Sia receptors [ 122 ] and the respiratory tract with uncomplicated infl uenza-like illness [ 40 , 41 , 123 , 124 ] [ 95 , 126 ] . It should be noted that after the fi rst outbreak of HPAI H5N1 virus in Egypt in 2006 [ 127 ] , the virus has continued to undergo mutations, resulting in sublineages A-D; at present (2013), sublineages B and D are dominant in Egypt, while sublineage A has not been detected. Hemagglutination of an H9 human isolate, A/Hong Kong/1073/99 (H9N2), with guinea pig erythrocytes was shown to be inhibited by both α2-3 and α2-6-linked sialic acid containing polymers [ 128 ] . This characteristic of avian H5, H7, and H9 viruses highlights the possibility of the potential of these avian infl uenza viruses for development to infect and spread among humans in the future. Similar to H2 and H3 HAs of pandemic H2N2 in 1957 and H3N2 in 1968 ( see Fig. 7e ), Q226L and G228S/N224K mutations in H5 HA [ 129 , 130 ] , Q226L and G228S mutations in H7 HA [ 131 ] , and Q226L mutation in H9 HA [ 132 ] have been experimentally shown to be associated with preferential binding to the α2-6 human-type receptor. Notably, the H5 virus harboring either Q226L-G228S [ 129 ] or Q226L-N224K [ 130 ] mutation in combination with loss of the 158-161 glycosylation site (N158D or T160A) near the receptor binding pocket and T318I or H107Y substitution in the stalk region (believed to increase the stability of the HA variant) has been shown to have preferential binding to Siaα2-6Gal, effi cient respiratory droplet transmission in ferrets, and viral attachment to human tracheal epithelia. Nonetheless, another viral factor(s) has been believed to be involved for avian viruses to gain effi cient human-to-human transmission ( see Subheadings 6 and 7 ).

Pigs serve as intermediate hosts for pandemic generation due to being mixing reservoirs of infl uenza A viruses, allowing genetic reassortment [ 133 ] . Indeed, interspecies transmission of avian and human viruses to pigs and vice versa has been documented in nature [ 134 -136 ] , and the recent pandemic H1N1 2009 has been confi rmed to be of swine origin [ 137 ] . Lectin staining demonstrated high levels of α2-3 and α2-6 Sia expressed in the porcine respiratory epithelium [ 88 , 133 ] , and HPLC and matrix-assisted laser desorption/ionization time-of-fl ight mass spectrometry (MALDI-TOF-MS) analyses showed gradually increased molar ratios of α2-6/α2-3-linked sialyl glycans of 3.2-, 4.9-, and 13.2fold for Neu5Ac and 1.8-, 2.7-, and 5.9-fold for Neu5Gc from the upper trachea and the lower trachea towards the lungs (the major replication site of swine-adapted infl uenza viruses) of a pig, respectively [ 77 ] . Neu5Ac/Neu5Gc ratios are 24.8/4.3 in the swine upper trachea, 27.1/4.1 in the swine lower trachea, 40.5/4.2 in the swine lung [ 77 ] , and 98/2 in the duck intestine [ 89 ] , whereas normal human tissues carrying nonfunctional hydroxylase to produce Neu5Gc [ 138 ] possess only Neu5Ac if Neu5Gc-containing food such as pork has not been eaten. Most duck-derived and swine-derived infl uenza A viruses displayed marked binding to Neu5Gc, related to the presence of V/I155 in H1 swine-adapted HAs [ 139 ] , but they preserved preferential binding to Neu5Ac glycoconjugates, whereas human-adapted infl uenza viruses showed preferential binding to only Neu5Ac glycoconjugates [ 89 , 140 ] . The swine-origin pandemic H1N1 2009 virus containing V155 rapidly spreads worldwide. Either T155Y or E158G mutation generated by a reverse genetics system in human H3 HA facilitates virus binding to Neu5Gc but retains strong binding affi nity to Neu5Ac [ 141 ] . HAlo virus (A/Vietnam/1203/04 (H5N1) virus with removal of the multibasic cleavage site, responsible for high pathogenicity) with Y161A mutation generated by a reverse genetics system showed change of preferential binding from Neu5Ac to Neu5Gc with a fi ve-to tenfold growth defect on MDCK cells [ 142 ] . It is still uncertain whether different ratios of Neu5Ac/Neu5Gc among animal species affect potential infection of infl uenza A viruses. Clearly, avian viruses with α2-3 binding preference would not overcome the interspecies barrier for efficient transmission in humans unless its binding preference is switched to α2-6. Thus, fi ndings that classical swine infl uenza A viruses bind preferentially to Neu5Acα2-6Gal [ 88 , 118 , 143 , 144 ] and that avian-like swine viruses acquired higher binding affi nity for Neu5Acα2-6Gal over time [ 88 , 118 , 143 ] suggest that pigs

provide a great source of natural selection of virus variants with α2-6 receptor-binding HAs, a prerequisite for a human pandemic.

Epithelial cells of the horse trachea showed prevalence of Siaα2-3Gal using lectin staining with Neu5Gc accounting for more than 90 % of Sia by HPLC analysis. Although most equine infl uenza viruses display high recognition of Neu5Gcα2-3Gal, they still prefer binding to Neu5Acα2-3Gal [ 82 ] .

Seal and whale lung cells contain predominately Siaα2-3Gal over Siaα2-6Gal by lectin staining, and both seal and whale viruses prefer to recognize Siaα2-3Gal [ 145 ] .

Changes in amino acid(s) in the RNA polymerase PB2 subunit resulting in different surface shape and/or charge affecting its protein's interaction with cellular factors have been thought to contribute to effi cient transmission of infl uenza viruses in humans, a characteristic of an infl uenza virus in a pandemic outbreak. T271A plays roles in (1) acquisition of HA mutation conferring recognition of a human-type receptor and (2) effi cient respiratory droplet transmission [ 146 , 147 ] . E627K/Q591R/D701N facilitates (3) effi cient infl uenza virus replication in the upper respiratory tract of humans and (4) effi cient infl uenza virus replication at 33 °C in the human upper part airway [ 102 ] .

Changes in amino acids in other viral proteins, such as PA and NS1, interacting with cellular factors could contribute to the emergence of an infl uenza pandemic, and further studies are therefore needed to clarify viral factors involved in generation of a potential pandemic virus.

Of the three types of infl uenza viruses, only type A can lead to a pandemic, possibly due to the variety of subtypes originating from wild water fowls that harmoniously interact with the virus in cooperation with the virus's ability to cross the species barrier to infect a variety of animals ( see Fig. 8 ) . A virus crossing the species barrier to infect a new host species must experience a new environment in the host body including cellular receptors, host factors supporting/ against virus replication, and local temperature, and thus is limited unless there is transmission evolution to surmount the species barrier. Of the infl uenza A viruses crossing into and establishing in

terrestrial poultry, some of the LPAI H5 and H7 subtypes have evolved into HPAI viruses with a universal pathogenic marker of a multibasic cleavage site causing systemic infection with a mortality rate as high as 100 % in poultry [ 152 ] . It was virtually unknown what factors in poultry drive the virus to acquire an HPAI property and why the HPAI viruses have continued to circulate in poultry despite the fact that a rapid and high fatality rate due to the HPAI property could result in a dead end for virus transmission. These questions challenge researchers to unravel the ultimate selection parameter for survival of the fi ttest during virus-host co-evolution,

Classified into types based on serological cross-reactivity of M proteins and NPs Fig. 8 A summary of human infection with infl uenza virus and emergence of an infl uenza pandemic. Airborne infl uenza in humans is caused by type A, B, or C, prevalent in the rainy season in several tropical regions, such as Thailand, Vietnam, and Brazil, and in winter (November-April for the Northern Hemisphere and May-October for the Southern Hemisphere), due to virus stability and host vulnerability to infection. Types A and B cause annual epidemic infl uenza typically due to virus antigenic variation (minor change); generally, they do not cause severe disease except in people under conditions as indicated [ 115 , 116 ] . Type C usually causes only mild illness in humans [ 148 ] . Type B is restricted to humans, though occasionally found in seals [ 149 ] and ferrets [ 150 ] , type C can be isolated from humans and pigs and is found in seropositive dogs [ 151 ] , and type A viruses have natural reservoirs in the intestinal tract (40 °C) of wild birds (harmonious interactions with the virus allowing the production of variety of subtypes without selective pressure) and are transmitted via feces to domestic birds (intestinal tract, 40 °C) and from domestic birds to other animals (respiratory tract of pigs, 39 °C) if they are able to fi ne-tune for transmission to and replication in the new host. So far, only H1N1 Spanish/18, H2N2 Japan/57, H3N2 Hong Kong/68, and H1N1 Swine/09 have been successfully established in humans. Currently only H3N2 Hong Kong/68 and H1N1/09 variants are circulating in humans. Epizootic infl uenza A viruses still cross to humans occasionally, and thus qualitative surveillance of the next pandemic zoonoses is needed. See color fi gure in the online version each of which has evolved to prevail with the host attempting to escape from or restrict the infection by various means including host immunity and apoptosis and the virus evolving strategies to block/evade host clearance mechanisms and to support its propagation including entry to the host cell and use of the host cellular machinery for each step of its life cycle [ 153 ] . LPAI and HPAI viruses can sometimes infect other animals but with limited transmission, including wild birds [ 154 ] , pigs [ 75 ] , and humans [ 155 ] , and cause mild to severe and fatal diseases (assessed by the number of severe cases and deaths) depending on the followings: (1) environmental factors, such as cold weather facilitating viral infection, (2) susceptibility and response of each host, which are accounted for by host genetics and other factors including age and health status, and (3) virus strain, with each virus strain having distinct pathogenic profi les in different host species: more research is needed to understand viral pathogenesis. For effi cient transmission in humans, a nonhuman virus acquires mutations through either an adaptation or reassortment mechanism for suffi cient human-tohuman transmission, providing a chance for the virus to further evolve until it can achieve suitable interactions with host factors for effi cient replication and transmission in human populations and eventually leading to a pandemic. Viral factors believed to play key roles in generating an infl uenza virus with pandemic potential are HA and PB2. Homotrimeric HA carries the followings: (1) antigenic sites, which if new, are not recognized by the host immune system, (2) receptor binding sites, which if they have 190D and 225D in H1 HA, 226L together with 228S (or 224K in H5 HA) in H2, H3, H5, or H7 HA, and 226L in H9 HA, are believed to confer virus preferential binding to a human-type receptor, (3) glycosylation sites, which if there is loss of glycosylation at 158-160, are believed to enhance HPAI H5N1 virus binding to a humantype receptor, and (4) stalk domains with T318A or H107Y, possibly being acquired for sustained and effi cient human-to-human transmission of the H5 HA variants. Viral PB2 with 271A together with 627K/591R/701N enhances HA binding to human-type receptors, enhances respiratory droplet transmission, supports viral growth in a mammalian host at 33 °C, and facilitates effi cient viral replication in the human upper respiratory tract. Investigation should be continued to identify other factors involved in effi cient infl uenza virus replication and transmission in humans for use as viral genetic markers for early surveillance of the emergence of a pandemic.

Although there has been an accumulation of information on infl uenza; (1) new variants have emerged, (2) avian viruses have occasionally infected other animals including intermediate hosts carrying both avian-type and human-type receptors, such as pheasants, turkeys, quails, and guinea fowls, with pigs in particularly tending to drive the virus binding to α2-6 human-type receptors, and (3) an infl uenza pandemic is still unpredictable. HPAI H5N1 viruses have caused sporadic human infections since 1997, and the pandemic phase is currently at level 3 ( see Table 2 , small clusters of disease in people). A new LPAI H7N9 virus reassorted between avian viruses in poultry just crossed species to infect humans in February 2013 and is now (January 2014) at pandemic level 3. Except for the pandemic 1918 virus, past pandemics emerged from an existing human strain picking up new genes (new to human immunity but effi cient replication in the human upper respiratory tract) from an avian and/or swine virus(es). This evidence suggested that new variants that have emerged via reassortment acquire major change in their genetic materials fi tting with a new host condition more easily and faster than do variants that have emerged via point mutations alone, which have gradual changes in their genetic materials. Thus, avoiding intermediate host infection with more than one infl uenza virus strain should be important for preventing/delaying the next pandemic. More knowledge of the molecular requirements of reassortment at levels of viral and host factors could lead to a better understanding of how appropriate viruses emerge, leading to strategies for effi cient prevention and antiviral interventions. Identifying viral and host factors, especially knowledge gained from their interaction structures, required for effi cient replication in each host species may be a key for understanding virus-host determinants and surveillance of viral host jumps and pathogenesis of infl uenza virus infection leading to the disease.

Available data have suggested that an infl uenza pandemic has never emerged through direct viral mutations alone. However, highly mutable avian infl uenza A viruses that have sporadically continued direct transmission to and infection in humans have raised concerns for pandemic potential with unpredictable pathogenesis (depending on virus-host interactions). HPAI H5N1 viruses have continued to infect humans with high morbidity and mortality rates, some isolates showing increased binding to α2-6 human-type receptors, and they are able to infect a variety of animals including wild birds and pigeons, which are responsible for introduction of the viruses they carry into different areas, pigs, which are mixing vessels driving the virus to bind to human-type receptors, and cats, which are in close contact with human beings [ 156 ] . Also, novel reassortant LPAI (invisible disease in domestic poultry) H7N9 viruses contain some mammalian fl u adaptations, PB2-627K and 226L, and they target upper and lower respiratory tracts of infected primates [ 157 ] and cause severe illness with a high death rate in humans. In addition to surveillance of human infection, extensive surveillance of infection of these viruses to other animals and back to migratory birds should be carried out since control of the viral spread into other regions relies on early recognition. Continuing surveillance is important for understanding how a pandemic emerges and establishing strategies for effi cient control and treatment if a pandemic arises as well as for prevention and control of the next pandemic. The best way for preventing infl uenza spread and a pandemic is to avoid direct contact with materials having suspected contamination as well as hygiene in healthcare for both animals and farmers, especially in mixed duck-poultry-pig farms.

Variation in infl uenza virus genes: epidemiological, pathogenic, and evolutionary consequences

A distinct lineage of infl uenza A virus from bats

New world bats harbor diverse infl uenza a viruses

Bat-derived infl uenza hemagglutinin H17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism

Infl uence of PB2 host-range determinants on the intranuclear mobility of the infl uenza A virus polymerase

The PB2 subunit of the infl uenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon

The infl uenza virus nucleoprotein: a multifunctional RNAbinding protein pivotal to virus replication

Infl uenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

Nuclear transport of infl uenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import

Mechanism for inhibition of infl uenza virus RNA polymerase activity by matrix protein

Infl uenza virus assembly: effect of infl uenza virus glycoproteins on the membrane association of M1 protein

Infl uenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1

Infl uenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential

A complicated message: Identifi cation of a novel PB1-related protein translated from infl uenza A virus segment 2 mRNA

The multifunctional NS1 protein of infl uenza A viruses

Cellular RNA binding proteins NS1-BP and hnRNP K regulate infl uenza A virus RNA splicing

A single-aminoacid substitution in the NS1 protein changes the pathogenicity of H5N1 avian infl uenza viruses in mice

Control of apoptosis in infl uenza virus-infected cells by up-regulation of Akt and p53 signaling

Adaptive mutation in infl uenza A virus nonstructural gene is linked to host switching and induces a novel protein by alternative splicing

Induction and evasion of type I interferon responses by infl uenza viruses

Amino acid residues contributing to the substrate specifi city of the infl uenza A virus neuraminidase

Neuraminidase is important for the initiation of infl uenza virus infection in human airway epithelium

Evasion of innate and adaptive immune responses by infl uenza A virus

Swine fl u pandemic. What's old is new: 1918 virus matches 2009 H1N1 strain

Structure of infl uenza hemagglutinin in complex with an inhibitor of membrane fusion

Crystallographic detection of a second ligand binding site in infl uenza virus hemagglutinin

The antigenicity and evolution of infl uenza H1 haemagglutinin, from 1950-1957 and 1977-1983: two pathways from one gene

Subtype-and antigenic site-specifi c differences in biophysical infl uences on evolution of infl uenza virus hemagglutinin

Structural identifi cation of the antibodybinding sites of Hong Kong infl uenza haemagglutinin and their involvement in antigenic variation

Structural basis of immune recognition of infl uenza virus hemagglutinin

Evolution of the hemagglutinin of equine H3 infl uenza viruses

Hemagglutinin mutations related to antigenic variation in H1 swine infl uenza viruses

Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and infl uenza virus

Evolution and ecology of infl uenza A viruses

Comparative histopathological characteristics of highly pathogenic avian infl uenza (HPAI) in chickens and domestic ducks

Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian infl uenza viruses in Italy

Infl uenza virus A pathogenicity: the pivotal role of hemagglutinin

Generation of a highly pathogenic avian infl uenza A virus from an avirulent fi eld isolate by passaging in chickens

Virulence-associated sequence duplication at the hemagglutinin cleavage site of avian infl uenza viruses

Update on avian infl uenza A (H5N1) virus infection in humans

WHO website. Updated unifi ed nomenclature system for the highly pathogenic H5N1 avian infl uenza viruses

Wild ducks as long-distance vectors of highly pathogenic avian infl uenza virus (H5N1)

Susceptibility of North American ducks and gulls to H5N1 highly pathogenic avian infl uenza viruses

Ducks: the "Trojan horses" of H5N1 infl uenza

Differential immune response of mallard duck peripheral blood mononuclear cells to two highly pathogenic avian infl uenza H5N1 viruses with distinct pathogenicity in mallard ducks

Avian fl u: H5N1 virus outbreak in migratory waterfowl

Infl uenza A (H5N1) viruses from pigs

N -Glycans from porcine trachea and lung: predominant NeuAcα2-6Gal could be a selective pressure for infl uenza variants in favor of human-type receptor

Evolutionary dynamics and emergence of panzootic H5N1 infl uenza viruses

WHO website. Current WHO phase of pandemic alert for avian infl uenza H5N1

Human infection with avian infl uenza A(H7N9) virus -update

Sialic acid species as a determinant of the host range of infl uenza A viruses

The role and potential of sialic acid in human nutrition

Sialobiology of infl uenza: molecular mechanism of host range variation of infl uenza viruses

H5N1 receptor specifi city as a factor in pandemic risk

Anatidae migration in the western Palearctic and spread of highly pathogenic avian infl uenza H5N1 virus

Differences in infl uenza virus receptors in chickens and ducks: implications for interspecies transmission

Molecular basis for the generation in pigs of infl uenza A viruses with pandemic potential

Recognition of N -glycolylneuraminic acid linked to galactose by the α2,3 linkage is associated with intestinal replication of infl uenza A virus in ducks

Receptor specifi city of infl uenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain

Substitution of amino acid residue in infl uenza A virus hemagglutinin affects recognition of sialyl-oligosaccharides containing N -glycolylneuraminic acid

Infl uenza virus entry and infection require host cell N -linked glycoprotein

Analysis of N -glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian infl uenza viruses

Evolution of the receptor binding phenotype of infl uenza A (H5) viruses

Receptor-binding profi les of H7 subtype infl uenza viruses in different host species

Is the gene pool of infl uenza viruses in shorebirds and gulls different from that in wild ducks

Avian infl uenza in the Western Hemisphere including the Pacifi c Islands and Australia

Epidemiology and control of H5N1 avian infl uenza in China

Structure and receptor binding specifi city of hemagglutinin H13 from avian infl uenza A virus H13N6

Replication and adaptive mutations of low pathogenic avian infl uenza viruses in tracheal organ cultures of different avian species

Acquisition of human-type receptor binding specifi city by new H5N1 infl uenza virus sublineages during their emergence in birds in Egypt

Biological and structural characterization of a host-adapting amino acid in infl uenza virus

Differences between infl uenza virus receptors on target cells of duck and chicken

Differences between infl uenza virus receptors on target cells of duck and chicken and receptor specifi city of the 1997 H5N1 chicken and human infl uenza viruses from Hong Kong

The quail and chicken intestine have sialylgalactose sugar chains responsible for the binding of infl uenza A viruses to human type receptors

Species and age related differences in the type and distribution of infl uenza virus receptors in different tissues of chickens, ducks and turkeys

Quail carry sialic acid receptors compatible with binding of avian and human infl uenza viruses

Infl uenza A virus receptors in the respiratory and intestinal tracts of pigeons

Cells in the respiratory and intestinal tracts of chickens have different proportions of both human and avian infl uenza virus receptors

Avian fl u: infl uenza virus receptors in the human airway

Human and avian infl uenza viruses target different cell types in cultures of human airway epithelium

Infection of human airway epithelium by human and avian strains of infl uenza a virus

Human and avian infl uenza viruses target different cells in the lower respiratory tract of humans and other mammals

Glycomic analysis of human respiratory tract tissues and correlation with infl uenza virus infection

Animal models for infl uenza virus pathogenesis and transmission

Complications of viral infl uenza

Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic infl uenza A(H1N1) virus affects receptor binding

Receptor-binding specifi city of pandemic infl uenza A (H1N1) 2009 virus determined by carbohydrate microarray

Pandemic infl uenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States

Avian infl uenza virus (H5N1): a threat to human health

Infl uenza A H5N1 replication sites in humans

Avian infl uenza and sialic acid receptors: more than meets the eye?

Outbreak of low pathogenicity H7N3 avian infl uenza in UK, including associated case of human conjunctivitis

Avian infl uenza A/ (H7N2) outbreak in the United Kingdom

Pandemic infl uenza viruses-hoping for the road not taken

Contemporary North American infl uenza H7 viruses possess human receptor specifi city: Implications for virus transmissibility

Multifocal avian infl uenza (H5N1) outbreak

Characterization of a human H9N2 infl uenza virus isolated in Hong Kong

Airborne transmission of infl uenza A/ H5N1 virus between ferrets

Experimental adaptation of an infl uenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Quantitative description of glycanreceptor binding of infl uenza A virus H7 hemagglutinin

Amino acid 226 in the hemagglutinin of H9N2 infl uenza viruses determines cell tropism and replication in human airway epithelial cells

Potential for transmission of avian infl uenza viruses to pigs

The epidemiology and evolution of infl uenza viruses in pigs

Evidence for the natural transmission of infl uenza A virus from wild ducts to swine and its potential importance for man

Human infl uenza A viruses in pigs: isolation of a H3N2 strain antigenically related to A/ England/42/72 and evidence for continuous circulation of human viruses in the pig population

Origins and evolutionary genomics of the 2009 swine-origin H1N1 infl uenza A epidemic

The molecular basis for the absence of N -glycolylneuraminic acid in humans

Receptor specifi city, host-range, and pathogenicity of infl uenza viruses

Swine infl uenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium

Identifi cation of amino acid residues of infl uenza A virus H3 HA contributing to the recognition of molecular species of sialic acid

Residue Y161 of infl uenza virus hemagglutinin is involved in viral recognition of sialylated complexes from different hosts

Early alterations of the receptorbinding properties of H1, H2, and H3 avian infl uenza virus hemagglutinins after their introduction into mammals

Receptor-binding properties of swine infl uenza viruses isolated and propagated in MDCK cells

Receptor specifi city of infl uenza A viruses from sea mammals correlates with lung sialyloligosaccharides in these animals

residue 271 plays a key role in enhanced polymerase activity of infl uenza A viruses in mammalian host cells

Key molecular factors in hemagglutinin and PB2 contribute to effi cient transmission of the 2009 H1N1 pandemic infl uenza virus

Infl uenza C virus infection in military recruitssymptoms and clinical manifestation

Recurring infl uenza B virus infections in seals

A new type of virus from epidemic infl uenza

Rescue of infl uenza C virus from recombinant DNA

A review of avian infl uenza in different bird species

Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope

Highly pathogenic avian infl uenza (H5N1) outbreaks in wild birds and poultry

WHO website. Infl uenza at the humananimal interface (HAI)

Characterization of H7N9 infl uenza A viruses isolated from humans