key: cord-0844045-ecie0dls
authors: Kumlin, Urban; Olofsson, Sigvard; Dimock, Ken; Arnberg, Niklas
title: Sialic acid tissue distribution and influenza virus tropism
date: 2008-08-08
journal: Influenza Other Respir Viruses
DOI: 10.1111/j.1750-2659.2008.00051.x
sha: 13b2340ab0e81bc4d29b8c0fb0a6894490c003b1
doc_id: 844045
cord_uid: ecie0dls

Abstract Avian influenza A viruses exhibit a strong preference for using α2,3‐linked sialic acid as a receptor. Until recently, the presumed lack of this receptor in human airways was believed to constitute an efficient barrier to avian influenza A virus infection of humans. Recent zoonotic outbreaks of avian influenza A virus have triggered researchers to analyse tissue distribution of sialic acid in further detail. Here, we review and extend the current knowledge about sialic acid distribution in human tissues, and discuss viruses with ocular tropism and their preference for α2,3‐linked sialic acid.

Influenza viruses, members of the family Orthomyxoviridae, are enveloped viruses with segmented, single-stranded, negative-sense RNA packaged in virions as ribonucleoproteins. Influenza viruses are further classified into three genera, A, B and C, on the basis of the antigenic diversity of virion proteins such as the matrix protein (M1) and nucleocapsid protein (NP). Further classification is based on the haemagglutinin (HA) and neuraminidase (NA) glycoproteins protruding from the viral envelope of influenza A and B viruses. Human influenza viruses mainly cause acute respiratory disease with high incidence and mortality, usually as epidemics but sometimes as worldwide pandemics. To date, 16 different influenza A virus HA subtypes (H1-H16) and nine different NA subtypes (N1-N9) have been identified in wild waterfowl, which are the major reservoir of all influenza A viruses. All mammalian influenza viruses are believed to originate from avian influenza A viruses, and only three subtypes of influenza A virus, H1N1, H2N2 and H3N2, have adapted to humans since 1918. 1 The causative viruses of the 1957 Asian (H2N2) and 1968 Hong Kong (H3N2) pandemics were found to be reassortants of human and avian influenza A viruses, 2,3 whereas the 1918 Spanish (H1N1) influenza A virus was likely to be entirely of avian origin but adapted to humans. 4 When H1N1 reappeared in 1977, H3N2 did not disappear but continued to circulate; therefore, currently, together with their H1N2 reassortant, three subtypes of influenza A virus are associated with epidemics. The ongoing zoonotic outbreaks of H5N1 avian influenza A virus have raised concerns that the situation may develop into a pandemic comparable in magnitude with the Spanish influenza virus pandemic.

Recently it become evident that other pathogenic viruses, including adenovirus type 37 (Ad37, family Adenoviridae), enterovirus type 70 (EV70, family Picornaviridae) and Newcastle disease virus (NDV, family Paramyxoviridae), all with a pronounced tropism for the human eye, share receptor preference with avian influenza A viruses for glycans terminating with a2,3-linked sialic acid (SAa2,3), [5] [6] [7] [8] [9] which is distinct from the SAa2,6-terminated receptor preferred by human influenza A viruses. Here, we review differences in sialic acid preferences of viruses that cause disease in humans and in the distribution of different sialic acids in tissues. Given the ocular tropism of Ad37, EV70 and NDV, and their preference for SAa2,3, we also discuss the human eye as a potential portal of entry for viruses such as the avian influenza A viruses.

Vertebrate cell surface glycans consist mainly of five-and six-carbon monosaccharides. One remarkable exception is the family of sialic acids, whose members have nine-carbon backbones. 10 Other important features of sialic acids are: (i) the high level of diversity -currently the family comprises over 50 different members, with neuraminic acid considered as the prototype, and includes important variants such as N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) (Figure 1 ), (ii) their presence mainly in higher animals and in certain bacteria, and (iii) their location, typically at the termini of glycan chains, and usually linked to galactose via a2,3-or a2,6glycosidic bonds, or to sialic acid via a2,8-glycosidic bonds. Not surprisingly, their expression on the surface of cells makes sialic acids easily accessible as attachment molecules for a wide variety of microbial pathogens, including many viruses.

Until recently, little was known about the distribution of sialic acid in humans, although the expression of SAa2,6 on the surface of tracheal epithelial cells was demonstrated more than 15 years ago, which, at that time 11,12 correlated perfectly with the known receptor preference, tropism and host range of influenza A viruses. Thus, human influenza A viruses bind preferentially to cell surface SAa2,6 and replicate in the respiratory tract, whereas avian influenza A viruses bind preferentially to SAa2,3 and only recently were reported to target the human airway. In this context, it should be noted that humans, but not primates, express SAa2,6 on cells in the upper respiratory tract, 13 which is in agreement with the restricted replication of human influenza A virus in airways of chimpanzees. 14 Another important observation is that Neu5Gc is a frequent constituent of glycans in most birds and mammals, but the gene encoding the hydroxylase enzyme necessary for the conversion of Neu5Ac to Neu5Gc was inactivated in our human ancestors, but not in other primates, approximately 2AE7 Ma, 15 possibly as a response to a microbial threat. 16 Some avian influenza A viruses replicate only or mainly in cells that express Neu5Gc. 17 Along with the current attention paid to sialic acid-binding viruses causing disease in humans, interest in sialic acid biology and its tissue distribution has intensified.

Recent research has more carefully documented the distribution of SAa2,3 and SAa2,6 in human tissues and more particularly in human airways. SAa2,3 is found on alveolar cells, whereas SAa2,6, with a few exceptions, is predominantly found on non-alveolar cells (Table 1 ). This correlates reasonably well with the predominant manifestations caused by human influenza A virus infection (rhinitis, pharyngitis, tracheobronchitis, bronchitis and bronchiolitis), indicating replication in the respiratory tract. 18 Importantly, the distribution of SAa2,3 and SAa2,6 also correlates with reports of lower respiratory tract symptoms and the difficulty in detecting H5N1 in nasopharyngeal samples, 19 and with the replication of H5N1 in cells lining the alveoli. 20 Specifically, SAa2,6 predominates on M cells in nasopharyngeal lymphoid tissue 21 and on epithelial cells in the nasal mucosa, 22 paranasal sinuses, 22 pharynx, 22 larynx, 23 trachea, [11] [12] [13] [22] [23] [24] trachea ⁄ bronchi, 25,26 bronchi 22,24,27 and in bronchioles, 13, 22 but not on alveolar cells. 24, 28 Goblet cells in the larynx, 23 trachea 13,23 and bronchi 13,24 may also contain SAa2,6. Conversely, SAa2,3 is primarily found on alveolar cells 22, 24, 28 and on bronchiolar epithelial cells at the junction between bronchioles and alveoli, 22 and has been found on bronchial 27 and tracheal 24 epithelial cells. Epithelial tracheobronchial cells 25, 26 and epithelial cells in the nasal mucosa 22 may also express SAa2,3, albeit at lower levels. Goblet cells in the larynx, 23 trachea [11] [12] [13] 23 and bronchial 13 epithelium also contain SAa2,3. Nasopharyngeal and bronchial epithelial cells abundantly express SAa2,3 linked via galactose to N-acetylglucosamine (SAa2,3Galb1,4GlcNAc), but not SAa2,3 linked via galactose to N-acetylgalactosamine (SAa2,3Galb1,3GalNAc). 28 Both trisaccharides, however, are abundantly expressed on alveolar pneumocytes.

The role of ciliated versus non-ciliated cells as targets for influenza A viruses has also been investigated. In the upper respiratory tract, SAa2,6 seems to be expressed on both ciliated and non-ciliated cells, whereas SAa2,3 tends to be expressed mainly on ciliated cells. Specifically, SAa2,6 is found not only on non-ciliated tracheal cells, 24 and non-ciliated tracheobronchial cells, 25, 26 but also on ciliated cells in the upper respiratory tract, 28 ciliated tracheal cells, 11, 12, 24 ciliated bronchial cells, 24 the ciliated borders of bronchial epithelial cells 13 and to a lesser extent on ciliated tracheobronchial cells. 25, 26 SAa2,3 has been reported to be expressed on ciliated nasopharyngeal, 28 ciliated tracheal, 24 ciliated tracheobronchial cells 25, 26 and ciliated bronchial cells, 24, 28 and also on non-ciliated cuboidal bronchial cells at the junctions between bronchioles and alveoli. 22 Thus, the distribution of SAa2,3 and SAa2,6 in human cells and tissues (Table 1 ) generally parallels the cell and tissue tropism of influenza A viruses, 22, 25, 26, 28, 29 and emphasizes sialic acid's role as a critical determinant of influenza A virus tropism.

With few exeptions, sialic acid-binding viruses isolated from humans exhibit a clear preference for two organs, the respiratory tract and the eye (Table 2 ). SAa2,6-binding viruses, such as human influenza A and B viruses, coronavirus OC43, polyomavirus JC and human parainfluenza virus 3, are more frequently isolated from the respiratory tract than from the eye. The human eye, on the other hand, apparently constitutes a very efficient replication site for at least three unrelated viruses with specificity for SAa2,3. Ad37 was isolated for the first time in 1976 30 and has been demonstrated to be one of the most frequent agents of epidemic keratoconjunctivitis (EKC). 31 The large amounts of Ad37 detected in eye swabs from EKC patients 32 are consistent with the observations that Ad37 is readily transmitted via direct or indirect contact 33 and that EKC is common in densely populated areas of the world. 34 EV70 was first isolated during a pandemic of a highly contagious form of acute haemorrhagic conjunctivitis (AHC) that originated in Ghana in 1969 35 and subsequently spread through Africa to Asia. EV70 was also responsible for a second pandemic of AHC in the early 1980s 36 that included both the eastern and western hemispheres, and for numerous regional outbreaks. Like Ad37, EV70 also uses SAa2,3 as a cellular receptor. 7 The third virus that binds to SAa2,3 is NDV, 9 a virus of domestic fowl that has been associated with several zoonotic outbreaks of conjunctivitis among poultry and kitchen workers and with case reports of laboratory infection. 37 The ability of these three viruses (Ad37, EV70 and NDV) to utilize the eye as a primary site of replication further emphasizes the potential role of the eye as a gateway for infections caused by other viruses that use SAa2,3 as a receptor. Therefore, it is important to highlight the potential of ocular SA, in general, as a receptor and of ocular SAa2,3 in particular, as a primary target of previously unrecognized importance for human infection by other viruses, including avian influenza A virus. It should also be noted that the human eye is an organ in which many other viruses replicate. For example, herpes simplex virus, measles virus, respiratory syncytial virus, echoviruses and species B adenoviruses have all been isolated from the human eye, but use cellular receptors other than SA; therefore, they are not discussed further in this review. For other SAa2,3-binding viruses with respiratory tropism, such as human parainfluenza viruses and polyomavirus BK, it will also be of interest to identify the cells in which these viruses replicate, and to determine if these cells express SAa2,3.

Relatively little is known about the levels and distribution of sialic acid on cells in the human eye. Sialyl Lewis X (sLe x ; SAa2,3Galb1,4(Fuca1,3)GlcNAc) and Sialyl Lewis A (sLe a ; SAa2,3Galb1,3(Fuca1,4)GlcNAc) have both been identified on conjunctival epithelial cells using antibodies, 38, 39 and both SAa2,3 and SAa2,6 have been detected on transformed, normal human conjunctival epithelial cells (NHC) using lectins. 40 Thus, the presence of SAa2,3 on conjunctival cells is in agreement with the ocular tropism of SAa2,3-binding viruses such as Ad37, EV70 and NDV. This also suggest that human conjunctival cells are susceptible to both human and avian influenza A viruses, which may be of importance when considering potential sites where recombinant influenza A viruses with pandemic potential may be generated. The role of a 'mixing vessel' has previously been allotted to the pig, 41 an animal known to express both SAa2,3 and SAa2,6 in the respiratory tract and susceptible to both human and avian influenza A viruses. The presence of both SAa2,3 and SAa2,6 in human conjunctival cells indicates that these cells should not be ignored as candidate mixing vessels.

A major zoonotic outbreak caused by avian influenza A virus occurred in 1997, when at least 18 people in Hong Kong were infected by the H5N1 virus and six of these 18 individuals died, mainly due to respiratory failure. 42 Since 2003, recurrent outbreaks of zoonotic H5N1 virus have been reported from Asia and Africa, where this subtype may now be enzootic. 43 Recently, it has become evident that not only human influenza A virus but also avian influenza A virus infection may give rise to serious CNS manifestations. 44 In this context, it is interesting that several viruses that replicate in the human eye (including EV70 and NDV) have also been associated with CNS complications, 45 suggesting that the CNS is an accessible secondary target for SAa2,3-binding viruses that replicate in the human eye.

Multiple reports describe the eye as an intial site of replication in humans for measles virus, 46 respiratory syncytial virus, 47 and CVA24v, 48 and in experimentally infected animals for ebola virus, 49 human influenza A virus, 50 and importantly, avian influenza A virus including H5N1, 51 followed by further transmission to non-ocular sites, including airways. Conjunctivitis caused by H7 subtypes of avian influenza A virus has been reported sporadically over the years, [52] [53] [54] [55] and both before and after the zoonotic outbreak of H7N7 in the Netherlands in 2003. 56 During this outbreak, one person died of respiratory failure; however, 59 Additional information indicates that the eye may be an initial site of replication for H5N1 virus in humans. For example, a Belgian veterinarian who investigated two H5N1-positive Thai eagles developed conjunctivitis 2 days after examination of the eagles, but no respiratory symptoms. 60 This raises the question whether H5N1 may use the human eye as a portal of entry preceeding transmission to the respiratory tract. 5 This possibility is supported by the relatively longer incubation period in humans for H5N1 avian influenza A virus (normally 4-7 days) 58 than for human H3N2 (normally 1-4 days), 18 avian H7N3 (1-3 days) 61 and avian H7N7 (1 day), 62,63 as well as the low frequency of virus detection and the relatively low viral load in nasopharyngeal samples from zoonotic H5N1 patients. 19 Also consistent with this idea is that one of the Turkish patients who developed respiratory symptoms of zoonotic H5N1 in the 2005-2006 outbreak presented with conjunctivitis. 58, 64 Recent work in animal models has confirmed that avian H7 viruses that replicate in mouse eyes (but not H5 or human H3 viruses) cause lethal disease following ocular infection of mice. 65 At present, it is unclear why H5 viruses are much less frequently associated with conjunctivitis in humans than are H7 viruses. Glycan arrays can be used to identify sialylated glycans recognized by influenza virus and other viruses. [66] [67] [68] A recent study using glycan arrays 66 showed differences in the sialic acid-binding specificity of influenza virus H7 isolates. European and North American isolates associated with conjunctivitis exhibited a strong preference for binding SAa2,3; however, the pattern of binding for the European isolates more closely paralleled binding of classic avain influenza viruses, while the North American isolates showed reduced binding to glycans containing SAa2,3 and increased binding to glycans containing SAa2,6. Virus binding was skewed even more towards glycans containing SAa2,6 for North American H7 isolates that were not associated with conjunctivitis. Glycan array analysis will be an important tool for unravelling the complex inter-relationship between sialic acid-binding specificity and influenza virus tropism and pathogenicity. However, for identification of the specific sialic acid-containing cellular receptors used by influenza A viruses, additional experimental approaches are required. The possibility exists that influenza virus replication in the human eye may occur asymptomatically. It has been suggested that asymptomatic replication occurred frequently during the zoonotic outbreak of H7N7 in the Netherlands in 2003 69 and during the zoonotic outbreaks of H5N2 in Japan in 2005 (http://www.mhlw.go.jp/english/ topics/influenza/avian02.html) and H5N1 in South Korea in 2006 (http://www.promedmail.org/pls/otn/f?p=2400:1202: 857450566246460::NO::F2400_P1202_CHECK_DISPLAY, F2400_P1202_PUB_MAIL_ID:X,34431). Whether or not patients or their relatives were asked about a history of conjunctivitis during outbreaks of H5N1 has not been reported, and there is no evidence that ocular samples from infected patients or from individuals exposed to H5N1 were tested for the presence of virus. Moreover, human-tohuman transmission of avian H5N1 influenza A virus is inefficient, 70 which is compatible with virus replication that is limited to the lower respiratory tract, 20 and there are no reported cases of human-to-human transmission by smallparticle aerosols. 70 However, influenza infection in the human eye represents a potential source for transmission between humans, but not necessarily a signal of an imminent pandemic. Mild or subclinical ocular infections may explain the prolonged incubation time before respiratory symptoms appear in zoonotic H5N1 cases, and raise the concern that unrecognized cases with mild or inapparent respiratory infection is common.

Not long ago, differences in the receptor specificity of human and avian influenza A viruses and a presumed lack of sialic acid receptors for avian influenza A viruses in humans was believed to constitute an almost absolute barrier to human infection by avian influenza viruses. Recent zonootic outbreaks of avian influenza A viruses triggered research on sialic acid distribution in both humans and birds, leading to evidence that this early picture was invalid. It is now clear that SAa2,3 is expressed on both alveolar and conjunctival cells in humans, which are important targets for viruses such as avian influenza A virus. From these findings it can be concluded that sialic acid is a critical determinant of influenza A virus tropism and that the human eye is a potential portal of entry for emerging sialic acid-binding viruses. However, further investigation of sialic acid distribution and more extensive diagnostic efforts, which should include ocular sampling, are required in order to better understand the tropism and transmission of zoonotic influenza A viruses.

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The Authors