Science in China Series C: Life Sciences www.scichina.com life.scichina.com © 2009 SCIENCE IN CHINA PRESS www.springer.com/scp www.springerlink.com Review Tissue and of influenza : Importance of quantitative analysis

ZHANG Hong1,2

1 Z-BioMed, Inc., Rockville, MD 20855, USA; 2 Department of Respiratory Medicine, Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China It is generally accepted that human influenza viruses preferentially bind to cell-surface glycoproteins/ glycolipids containing sialic acids in α2,6-linkage; while avian and equine influenza viruses preferen- tially bind to those containing sialic acids in α2,3-linkage. Even though this generalized view is accurate for H3 subtype isolates, it may not be accurate and absolute for all subtypes of influenza A viruses and, therefore, needs to be reevaluated carefully and realistically. Some of the studies published in major scientific journals on the subject of tissue tropism of influenza viruses are inconsistent and caused confusion in the scientific community. One of the reasons for the inconsistency is that most studies were quantitative descriptions of sialic acid receptor distributions based on lectin or influenza immunohistochemistry results with limited numbers of stained cells. In addition, recent studies indicate that α2,3- and α2,6-linked sialic acids are not the sole receptors determining tissue and host tropism of influenza viruses. In fact, determinants for tissue and host tropism of human, avian and animal influenza viruses are more complex than what has been generally accepted. Other factors, such as glycan topology, concentration of invading viruses, local density of receptors, lipid raft microdomains, coreceptors or sialic acid-independent receptors, may also be important. To more efficiently control the global spread of pandemic influenza such as the current circulating influenza A H1N1, it is crucial to clarify the determinants for tissue and host tropism of influenza viruses through quantitative analysis of experimental results. In this review, I will comment on some conflicting issues related to tissue and host tropism of influenza viruses, discuss the importance of quantitative analysis of lectin and influenza virus immunohistochemistry results and point out directions for future studies in this area, which should lead to a better understanding of tissue and host tropism of influenza viruses. tissue and host tropism, influenza viruses, sialic acid receptors, quantitative analysis

The influenza viruses belong to the Orthomyxoviridae associated with high morbidity and mortality in people family and are enveloped animal viruses containing a of all ages, caused millions of deaths in the previous segmented single-stranded RNA genome[1,2]. They are three influenza pandemics, is the cause of the current classified into three types, A, B and C, based on their influenza H1N1 pandemic and has the potential to cause immunologically distinct nucleoprotein and matrix more pandemics in the future[3,4]. protein . Type A influenza viruses are further A new human influenza virus could initiate as an grouped into antigenic subtypes according to their avian or animal influenza virus that adapts to humans specific surface glycoproteins hemagglutinin (HA) and through accumulation of mutations or as a hybrid influ- neuraminidase (NA). Influenza A occurs in humans, other mammals, and birds and sixteen distinct HA Received October 1, 2009; accepted November 11, 2009 subtypes and nine NA subtypes are currently recognized. doi: 10.1007/s11427-009-0161-x Influenza A attracts the most attention because it is †Corresponding author (email: [email protected])

Citation: ZHANG Hong. Tissue and host tropism of influenza viruses: Importance of quantitative analysis. Sci China Ser C-Life Sci, 2009, 52(12): 1101-1110, doi: 10.1007/s11427-009-0161-x

enza virus containing a combination of genes derived At this stage, the influenza H1N1 pandemic can still be from an avian, an animal and a human influenza virus. characterized as being moderate in terms of severity and Therefore, influenza pandemics arise when a new mortality rate. The epidemiology, molecular biology, influenza virus emerges from human, avian or animal pathology, and pathogenic factors of influenza viruses influenza viruses or from reassortants of them, infects including H5N1 in humans have been comprehensively humans, and spreads efficiently among people. The first reviewed recently[8−14]. This article will briefly review transmission of the highly pathogenic H5N1 avian the generalized view of receptors for influenza viruses, influenza A virus directly from chickens to humans in comment on some conflicting issues related to tissue and Hong Kong was reported in 1997[5]. As of 31 August host tropism of influenza viruses, discuss the importance 2009, a total of 440 laboratory-confirmed human cases of quantitative analysis of lectin and influenza virus of avian influenza A (H5N1) virus from 15 immunohistochemistry results and point out directions countries have been reported to WHO and 262 of them for future studies in this area. (59.5%) have been fatal (www.who.int). In China, 25 of the 38 confirmed cases (65.8%) have been fatal. Among 1 Generalized view of receptors for the 45 confirmed cases (12 deaths) reported to WHO in influenza viruses 2009, 7 were from China and 4 of them were fatal. The generalized view is that avian influenza viruses These events demonstrate that avian influenza viruses replicate in the gastrointestinal tract of the host and can infect human directly and China is an important preferentially bind to cell-surface glycoproteins or country for the surveillance, prevention and control of glycolipids containing sialic acids in α2,3-linkage potential influenza pandemics. Continued circulation of (SAα2,3); while human influenza viruses replicate in the H5N1 and other avian influenza viruses in Asia shows host’s respiratory tract and bind to respiratory epithelial that a future pandemic from avian influenza is a real cells via sialic acids attached to glycoproteins or glycoli- threat. pids containing sialic acids in α2,6-linkage (SAα2,6). The current circulating influenza A H1N1 viruses Hundreds of articles including many reviews related to were probably originated from the combination of six influenza virus receptors have been published during last gene segments (PB2, PB1, PA, HA, NP, and NS1) from 20 years. Many review articles and several articles swine influenza H1N2 viruses circulating in the USA published in major scientific journals such as Science, from 1999 to 2001 (nucleotide sequence similarities Nature and Nature Medicine made generalized statemen- >96%) and two gene segments (NA and M1) from swine ts about influenza virus receptors and cited two original influenza viruses circulating in Europe from 1985 to articles published in 1983[15,16]. Here are some examples: 1999 (nucleotide sequence similarities >93%)[6]. The “Human and avian influenza A viruses differ in their gene segments of these influenza A H1N1 viruses could recognition of host cell receptors: the former preferential- also be traced back to human or avian origins if those ly recognize receptors with saccharides terminating in nucleotide sequences with similarities lower than 93% were considered. Since the first report of the swine sialic acid-α2,6-galactose (SAα2,6Gal), whereas the [17] influenza H1N1 in April 2009[7], there have been over latter prefer those ending in SAα2,3Gal” ; “Avian 526060 cases and at least 6770 deaths (1.29%) reported influenza viruses bind to cell-surface glycoproteins or to the WHO from all regions of the world (www. who. glycolipids containing terminal sialyl-galactosyl resi- int). The WHO Director-General raised the level of dues linked by 2-3-linkages [Neu5Ac(α2-3)Gal], whereas influenza pandemic alert from phase 5 to phase 6 on human viruses, including the earliest available isolates June 11, 2009 following the advice from the Emergency from 1957 and 1968 pandemics, bind to receptors that Committee which concluded that the criteria for a contain terminal 2-6-linked sialyl-galactosyl moieties [18] pandemic had been met. Currently, pandemic H1N1 [Neu5Ac(α2-6)Gal]” ; “HA binds to receptors influenza virus continues to be the predominant circula- containing glycans with terminal sialic acids, where ting virus of influenza in the world and all pandemic their precise linkage determines species preference. A H1N1 2009 influenza viruses sequenced so far have switch in receptor specificity from sialic acids connected been antigenically and genetically very similar to the to galactose in α2-3 linkages (avian) to α2-6 linkages first sequenced new influenza virus (A/California/7/2009). (human) is a major obstacle for influenza A viruses to

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cross the species barrier and to adapt to a new host”[19]; extent, to be dependent on the species…” (true for H3 “Human viruses of the H1, H2 and H3 subtypes that are subtype isolates only) could have evolved to become known to have caused pandemics in 1918, 1957, and generalized statements such as “human influenza viruses 1968, respectively, recognize α2,6-linked sialic acid”[20]; preferentially recognize receptors with SAα2,6Gal, and “Human influenza strains preferentially bind to whereas avian influenza viruses preferentially recognize sialic acid residues linked to galactose by the α2,6 receptors with SAα2,3Gal” (implying isolates of all HA linkage, while avian and equine influenza strains subtypes). recognize sialic acid linked to galactose by α2,3 In another article of Rogers et al., the sequencing linkage.”[21] bands showing mutations of CTG-leucine (human specific) In one of their original articles published in 1983, to CAG-glutamine (avian specific) or ATG-methionine Rogers et al.[15] modified human type B erythrocytes to (specific for both human and avian receptors) at amino contain sialyloligosaccharides of defined sequence with acid 226 of HA1 were not clear enough to make defini- different sialyltransferases purified from porcine tive conclusions[16]. In conclusion, the generalized view submaxillary glands and rat liver, and examined that avian and human influenza viruses preferentially differential adsorption of influenza virus strains isolated bind to cell-surface glycoproteins or glycolipids contain- from a variety of hosts. Hemagglutination results of ing sialic acids in α2,3-linkage and α2,6-linkage respec- human and animal strains (Table 1 of the article by tively may not be accurate and absolute for all subtypes Rogers et al.[15]) indicated that receptor determinants of of influenza A viruses, and therefore, would need to be human (SAα2,6Gal) and avian (SAα2,3Gal) influenza reevaluated carefully and realistically with quantitative virus isolates were accurate only for H3 subtype analysis of more experimental results. influenza A viruses[15 ]. In contrast, subtype H2 human strain (A/Japan/305/57) showed the same HA titers 2 Distribution of sialic acid receptors and (1024) for both SAα2,6Gal and SAα2,3Gal receptors; cell/tissue tropism of influenza viruses subtype H1 human strains A/PR/8/34 and A/FM/1/47 Lectins such as Sambucus nigra agglutinin (SNA) and preferentially bound to avian specific receptor SAα2, Maackia amurensis agglutinin (MAA) have been used to 3Gal (HA titers of 256 and 512) instead of human measure the distribution of SAα2,3Gal or SAα2,6 specific receptor SAα2,6Gal (HA titers of 128 and 256); terminated glycoprotein or glycolipids in tissue sections and subtype H1 avian strain (A/duck/Alberta/513/78) by immunohistochemistry methods. Labeled influenza bound to both human and avian receptors (HA titers of viruses have also been used to determine the cell and 512 for SAα2,6Gal and 1024 for SAα2,3Gal). Therefore, tissue tropism of human, avian and animal influenza their results with H1 and H2 subtype influenza viruses viruses. To illustrate that some of the studies published were contradictory to those with H3 subtype influenza in major scientific journals are inconsistent and caused viruses and did not support the generalized view of confusion on the subject of cell and tissue distribution of influenza virus receptor specificities for either human SAα2,3 and SAα2,6 receptors as well as tropism of (SAα2,6Gal) or avian (SAα2,3Gal) receptors. Rogers et influenza viruses, those related studies have been al. were probably aware of their contradictory results of compared and summarized in the Table 1. H1 and H2 subtypes at the time and cautiously Matrosovich et al. demonstrated in 2004 that human concluded in the abstract of the article “Receptor and avian influenza viruses target different cell types in specificity appeared, to some extent, to be dependent on cultures of human airway epithelium: human influenza the species from which the virus was isolated. In viruses preferentially infected non-ciliated cells, while particular, human isolates of the H3 serotype all avian influenza viruses mainly infected ciliated cells[18]. agglutinated cells containing the SAα2,6Gal linkage, but They found that tropism for a specific type of airway not cells bearing the SAα2,3Galβ1,3GalNAc sequence. epithelial cells depends on the virus host species, rather In contrast, antigenically similar (H3) isolates from than on virus type, subtype, or strain and this pattern avian and equine species preferentially bound correlated with the predominant localization of human erythrocytes containing the SAα2,3Gal linkage”[15]. It is influenza receptors (α2-6-linked sialic acids) on non-ci- unclear how “Receptor specificity appeared, to some liated cells and avian influenza receptors (α2-3-linked

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Table 1 Summary of recently published studies on the subject of tissue distribution of SAα-2,3 and SAα-2,6 receptors and in6fection of human tissues with influenza viruses Published PNAS 2004[18] Nature 2006[22] Science 2006[23] Respir Res 2007[24] FASEB J 2008[25] Studies Distribution of SAα-2,3 and SAα-2,6 Receptors In Tissues ΜΑΑ Binding for SAα-2,3Gal receptor (avian)

Nasal/ Bound to some ciliated Occasionally detected on epithelial Strong MMA1 binding but no Detected on a small proportion of primary epithelial cells cells in nasal mucosa MMA2 binding in the epithelium non-ciliated and a few ciliated epithelial cells

Trachea/ Bound to some ciliated Found on non-ciliated cuboidal MAA1 bound to bronchial cells; Detected on non-ciliated and a few bronchus primary epithelial cells bronchiolar cells at the junction Strong MMA binding but no ciliated epithelial cells and between bronchiole and alveolus MMA2 binding to ciliated and non- endothelial cells (EC) ciliated cells

Lung Found on cells lining the alveolar MAA1 bound to alveolar Detected on type II cells and aveolus wall but not pneumocytes; endothelial cells MAA2 bound to pneumocytes Other tissues Detected on brain neuron and EC; EC; liver Kupffer cells; kidney glomerular EC; heart EC; intestines neurons and EC; and T cells in spleen

SΝΑ Binding for SAα-2,6Gal receptor (human) Nasal/ Strong staining on Dominant on epithelial cells in Strong binding of SNA in the Detected on ciliated and pharynx non-ciliated nasal mucosa epithelium non-ciliated epithelial cells primary epithelial cells and EC

Trachea/ Strong staining on Found on trachea and bronchus Strong binding of SNA in ciliated Detected on ciliated and non- bronchus nonciliated epithelia cells and non-ciliated cells ciliated epithelial cells and EC primary epithelial cells Lung/aveolus Found on alveolar cells Minimal SNA binding Detected on pneumocytes and EC

Other tissues Detected on brain EC; placenta Hofbauer cells and EC; liver bile duct epithelial cells and EC; kidney glomerular EC and distal tubule epithelial cells; heart EC; intestines EC; and B cells in spleen (to be continued on the next page)

(Continued)

Tissues Infected by Influenza Viruses

Human Preferentially infected Human A/Kawasaki/173/01 viruses non- (H1N1) bound extensively to Human A/Netherlands/213/03 ciliated cells epithelial cells in the bronchi (H3N2) bound to epithelial cells in and, to a lesser degree, to trachea alveolar cells; H5N1 virus (A/Hong Kong/213/03) infected both alveolar cells and bronchial epithelial cells

Avian Mainly infected ciliated Avian “H5N1 virus viruses cells A/duck/Mongolia/301/01 (A/Vietnam/1194/04) attached (H3N2) and predominantly to type II A/duck/Vietnam/5001/05 pneumocytes, alveolar (H5N1) viruses didn’t infect macrophages, and nonciliated bronchial epithelial cells; but cuboidal epithelial cells in terminal they bound extensively to bronchioles”; alveolar cells; “Attachment became progressively A/duck/Vietnam/5001/05 did rarer toward the trachea” infect alveolar cells

Conclusions 1. The predominant 1. “Avian and human flu 1. “The findings fit with the 1. MAA binding pattern may be 1. “HuIV-Rs were abundantly localization of human viruses seem to target different limited pathology data for H5N1 dependent on the supplier and present in the respiratory tract and receptors (SAα2,6) regions of a patient’s virus in humans”; different isoforms show a different lungs. They were also detected on localized predominantly respiratory tract”; tissue distribution; Hofbauer cells, glomerular cells, on non-ciliated 2. The findings “contrast with the splenic B cells, and in the liver”; epithelial cells; 2. “Although not quantitative, idea that avian influenza viruses 2. Identified a heterogeneous these results indicate that there generally have little affinity for distribution of SNA and MAA 2. Endothelial cells of all organs 2. Avian receptors could be functional human respiratory tissues”; binding in bronchial epithelium with examined expressed both human (SAα2,3) localized significance in the preferential no clear distinction between ciliated and avian receptors; predominantly on expression of SAα2,3Gal and 3. “The predilection of H5N1 virus and non-ciliated cells; ciliated epithelial cells; SAα2,6Gal molecules on for type II pneumocytes and 3. “The distribution pattern of human airway cells”; alveolar macrophages may 3. “SNA binding was widespread in AIV-Rs is partially inconsistent 3. Ciliated cells most contribute to the severity of the the upper than the lower respiratory with the pattern of infected cells likely serve as primary 3. Findings indicate that H5N1 pulmonary lesion.” tract”. as detected in previous studies, target cells in cases viruses “can replicate which suggests there may be other where avian viruses efficiently only in cells in the receptors or mechanisms involved cause human disease. lower region of the respiratory in H5N1 infection”. tract”.

SNA, a lectin from the elderberry plant Sambucus nigra agglutinin; MAA, Maackia amurensis agglutinin; EC, endothelial cells.

sialic acids) on ciliated cells. Based on quantitative However, they contrast with the idea that avian influenza analysis of immunostaining of influenza virus-infected viruses generally have little affinity for human cells, Matrosovich et al. concluded that ciliated cells respiratory tissues.” They also suggested “The most likely serve as primary target cells in those rare predilection of H5N1 virus for type II pneumocytes and cases where avian viruses cause human disease[18]. In alveolar macrophages may contribute to the severity of contrast, Shinya et al. reported in their 2006 article that the pulmonary lesion.”[23] α2-3-linked sialic acids (avian influenza receptors) were Using Maackia amurensis lectin II (MAL-II, MAA2) found on non-ciliated cuboidal bronchiolar cells and and Sambubus nigra agglutinin (SNA), Yao et al. α2-6-linked sialic acids (human influenza receptors) investigated the distribution of α2,3-linked and were dominant on epithelial cells in nasal mucosa, with α2,6-linked sialic acids in various organs including α2-3-linked sialic acids being occasionally detected[22]. upper and lower respiratory tracts of two H5N1 cases However, a substantial number of cells lining the and 14 control cases[24]. They observed that avian alveolar wall also expressed α2-3-linked sialic acids. influenza receptors (α2,3-linked sialic acids) were Shinya et al. observed that human-derived viruses (such detected on different cells including type II pneumocytes, as A/Kawasaki/173/01) bound extensively to epithelial a limited number of epithelial cells of the upper cells in the bronchi and, to a lesser degree, to alveolar respiratory tract, the bronchi, bronchioli, and trachea, cells (3 stained cells shown in Figure 2b of the article); splenic T cells, and neurons in the brain and intestines. by contrast, avian viruses (such as A/duck/ Mongolia/301/ They suggested, “the relative lack of AIV-Rs in the 01) bound extensively to alveolar cells (8 stained cells upper airway may be one of the factors preventing shown in Figure 2d) but less widely to bronchial efficient human-to-human transmission of H5N1 epithelial cells based on qualitative analysis of their influenza”[24]. Yao et al. found that human influenza immunohistochemical results. They concluded that receptors (α2,6-linked sialic acids) were abundantly A/Hong Kong/213/03 virus infected bronchial epithelial present in the respiratory tract and lungs and were cells by showing about 9 stained cells (Supplementary detected on Hofbauer cells, glomerular cells, splenic B Figure 4c) and alveolar cells by showing about 12 cells, and in the liver. Yao et al. also found that stained cells (Supplementary Figure 4d). Shinya et al. endothelial cells of all organs examined expressed both suggested, “Although not quantitative, these results avian and human receptor types which “may account for indicate that there could be functional significance in the the multiple organ involvement in H5N1 influenza”[24]. preferential expression of SAα2,3Gal and SAα2,6Gal In their 2007 article, Nicholls et al. compared the molecules on human airway cells” and “Our findings lectin binding properties of MAA from different indicate that …H5N1 viruses… can replicate efficiently suppliers and found “that there were differences in tissue only in cells in the lower region of the respiratory distribution of the α2,3 linked SA when 2 different tract”.[22] isomers of MAA (MAA1 for SAα2,3Galb1,4GlcNac In their article published in 2006, van Riel et al.[23] and MAA2 for SAα2,3Galb1,3GlcNac) lectin were used. identified the cell types in the lower respiratory tract of MAA1 had widespread binding throughout the upper humans and four animal species (mouse, ferret, macaque, and lower respiratory tract. By comparison, MAA2 and cat) to which H5N1 virus (A/Vietnam/1194/04) binding was mainly restricted to the alveolar epithelial attached by showing 1 to 3 stained cells in each of the cells of the lung”[25]. They suggested in the article “We tissue sections using immunohistochemistry technique. also urge attention to the exact isoform of MAA present They found that H5N1 virus attached mainly to type II in lectins supplied by different manufacturers. These pneumocytes, alveolar macrophages, and non-ciliated results imply a need for a re-evaluation of the findings cuboidal epithelial cells in terminal bronchioles; and reported in previous studies on the tissue distribution of attachment became progressively rarer toward the SA receptor types”. They also mentioned, “Since MAA1 traches. They concluded, “These findings fit with the bound to non-SAα2,3 glycans it has not been used as limited pathology data for H5N1 virus infection in extensively by some researchers as MAA2. For instance, humans, which show diffuse alveolar damage and the Shinya et al. have recently demonstrated SAα2,3Gal presence of H5N1 virus in type II pneumocytes. expression only in the lung but not the bronchus or

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upper respiratory tract[7]. Since they only used MAA2 of influenza virus receptors (Table 1)[18,22−26]. As Yao et lectin binding (Y. Kawaoka, personal communication) al. concluded after investigating the expression of avian our MAA2 results are in accord with theirs. But our (AIV-Rs) and human influenza receptors (HuIV-Rs) in finding of MAA1 binding in the upper and lower various organs (upper and lower respiratory tracts, brain, respiratory tract does have implications for the possible kidney and intestines, etc.) of two H5N1 cases and distribution of receptors for avian influenza viruses control cases, “the distribution pattern of AIV-Rs is including the currently circulating H5N1 viruses”. It is partially inconsistent with the pattern of infected cells as clear that results by Shinya et al. “SAα2,6Gal is detected in previous studies, which suggests there may dominant on epithelial cells in nasal mucosa, with be other receptors or mechanisms involved in H5N1 SAα2,3Gal being occasionally detected”[22] and their infection” and “The absence of AIV-Rs in several cell conclusion “Our findings may provide a rational types, including ciliated epithelial cells of the trachea, explanation for why H5N1 viruses at present rarely intestinal epithelial cells, cytotrophoblasts, and Hofbauer infect and spread between humans although they can cells appears to be in contrast with the detected replicate efficiently in the lungs”[22] were incomplete H5N1-infection of these cells. This suggests that other because they only used MAA2. receptors or coreceptors might be involved in virus- [25] As summarized in Korteweg and Gu’s review article, target-cell interaction in H5N1 avian influenza” . They “Conflicting results have been reported as to the cell also mentioned that “It is remarkable, however, that type expressing avian influenza virus receptors in the despite the widespread and abundant expression of trachea and bronchi.”[8]. Tollis et al. also stated “the AIV-Rs in the lungs, only a limited number of pneumoc- determinants involved in host range restriction have not ytes have been found to be infected in previous studies, been completely clarified… The H5N1 viruses isolated implying that the presence of AIV-Rs is not the only from human in Hong Kong were shown to retain the factor affecting the capability of AIV to infect cells.”[25] receptor binding properties of avian viruses, thus Recent studies published by other groups also indicate suggesting that gene products other than HA may that α2,3- and α2,6-linked sialic acids are not the sole contribute to host range restriction”[12]. The summarized receptors determining tissue tropism of influenza results of recently published studies on the subject of viruses[26−32]. cell and tissue distribution of SAα2,3 and SAα2,6 The recent article by Nicholls et al. demonstrated that receptors and tissue tropism of influenza viruses show ex vivo cultures of human nasopharyngeal, adenoid and the inconsistency of these studies, because some of the tonsillar tissues can be infected with H5N1 viruses in studies were qualitative descriptions of receptor spite of an apparent lack of sialic acid α2-3 virus distributions based on immunohistochemistry results receptors[26]. Previous studies by other groups using with limited numbers of stained cells and without MDCK cells[27] and human airway tracheobronchial common standard controls (Table 1). To make published epithelial cells[28] have shown that removal of sialic acid results repeatable and comparable among different from cultured cells by broad-spectrum sialidases does laboratories, immunohistochemistry results should be not abolish infection by human influenza viruses. Using analyzed quantitatively. human A549 and Hela cells with high levels of α2-6 sialic acid and CHO cells that have only α2-3 sialic acid, 3 Alpha 2-3 and alpha 2-6 linked sialic acids Kumari et al. found that absence of α2-6 sialic acid does are not the sole receptors determining cell not protect a cell from influenza infection and the and host tropism for influenza viruses presence of high levels of α2-6 SA on a cell surface More than 750 articles related to influenza virus receptors does not guarantee productive replication of a virus with [29] have been published during last 10 years and attention α2-6 receptor specificity . Binding studies of influen- has primarily been given to the role of alpha 2-3 and za viruses to sialic acids show that reassortant viruses [30] alpha 2-6 linked sialic acids as receptors of human, with A/NWS/33 HA bind to α2,8-linked sialic acid . [31] avian and animal influenza viruses. However, conflic- In addition, Glaser et al. have demonstrated that ting results have been reported in major scientific human influenza viruses can infect knock-out mice journals on the subject of the cell and tissue distribution lacking a major α2,6 sialyltransferase and grow to

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similar titers in the lung and trachea as compared to viruses, especially A/Chicken/Indonesia/ BL/03, have wild-type mice. Results from these studies indicate that the same cellular tropism as that of A/Vietnam/3046/04[26]. α2-3 and α2-6 linked sialic acids are not the sole In the same article, the thermal inactivation curve receptors determining cell and host tropism of influenza shown in Figure 1e indicated that it took 8 hours to 4 viruses and suggest that the absence of these receptors reduce the virus (A/Vietnam/3046/04) titer from 10 to 0 does not necessarily mean cells are protected from 10 50% tissue culture infectious doses per mL (TCID50/ infection by influenza viruses automatically. Therefore, mL). However, as shown in Figure 1h, it took 18 hours 5 4 it is over simplified to assume that removal of sialic acid to reduce the virus titer from 10 to 10 TCID50/mL for receptors from human respiratory tracts would eliminate the same influenza virus. If the thermal inactivation infections by H5N1, H1N1 and other influenza viruses[33]. curves in Figures 1e and h were switched, then the Other factors, such as glycan topology[34], local density results of Figure 1e might have not supported the of receptors, concentration of invading viruses, lipid raft conclusion that “productive viral infection in the tissue microdomains[35,36], coreceptors or sialic acid-indepen- fragments from the nasopharynx was demonstrated by an dent receptors, may also be important. In light of the increase in the viral yield in culture supernatants (Figure findings discussed above, more attention should be 1e)”. To show productive viral infection of influenza given to other cell surface components in the human viruses in primary cultured cells, Gu et al. carried out respiratory tract important for interactions with avian, three independent experiments with each experiment in [37] swine and human influenza viruses, which will lead to a duplicate , therefore, their data could be averaged and better understanding of cell and host tropism of expressed as a mean±SD which demonstrated that virus influenza viruses and provide new targets for more titer was increased continuously from 24 to 48 and 72 effective prevention and treatment of seasonal and hours after infection. In contrast, Nicholls et al. showed pandemic influenza. decreased virus titers from 20 to 24 hours after infection in Figure 1e and from 24 to 48 hours after infection in 4 Quantitative analysis of cell and tissue Figure 1h using the same virus at the same input dose 10 [26] tropism of influenza viruses (10 TCID50/mL) . It would be helpful if the authors explained why the thermal inactivation curves for the Three of the articles published in major scientific journa- same virus are different between the two experiments ls were selected as examples to illustrate why it is and why virus titers did not continuously increase from important to quantitatively analyze immunohistochemical 20 to 24 hours in Figure 1e and from 24 to 48 hours in staining results. In the article published in 2007, Figure 1h after infection. One of the possible Nicholls et al. designed a study to determine the tissue explanations is that each experiment was carried out tropism of seven influenza A viruses (one H1N1, one only once and the results may not be reproducible. H3N2 and five H5N1) in the upper and lower respiratory Furthermore, the authors mislabeled the influenza virus tracts[26]. These seven influenza viruses included A/Hong Kong/54/98 (H1N1) used in the study as 54/98 A/Hong Kong/54/98 (H1N1), A/Hong Kong/1174/99 (H5N1) in Figure 2d. (H3N2), A/Vietnam/3046/04 (H5N1), A/Hong Nicholls et al. also reported results of lectin histoche- Kong/483/95 (H5N1), A/Hong Kong/213/03 (H5N1), mistry on the ex vivo biopsies and archival tissues (Sup- A/Vietnam/1203/04 (H5N1) and A/Chicken/Indonesia/ plementary Figure 2), although they realized “lectin BL/03 (H5N1). Nicholls et al. qualitatively reported the histochemistry should not be regarded as definitive”[26]. cellular location of influenza A nucleoprotein in different There are some concerns regarding their lectin histoche- tissues and in primary nasopharyngeal cells infected mistry studies: (i) Published data are not quantitative; with influenza viruses H1N1, H3N2 and one of the five and (ii) the definition of “strong positive staining” is not H5N1 viruses, A/Vietnam/3046/04. These results would given. As described by Matrosovich et al. in their 2004 be more convincing if the authors had performed a article[18] and Gu et al. in their 2007 article[37], which quantitative analysis of the data by scanning more fields quantitatively described immunostaining studies of inf- under a microscope to count more stained epithelial cells. luenza virus-infected cells, it is feasible to analyze the It would be interesting as well for the scientific lectin histochemistry results quantitatively. Matrosovich community to know whether the other four H5N1 et al. counted cells by observing en face with

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oil-immersion at 1000 magnification, analyzed 25—35 generalized view is accurate for H3 subtype isolates, it fields for each sample, averaged the results and may not be accurate and absolute for all subtypes of calculated the percentages of specific cells with respect influenza A viruses based on the analysis of the original to the total amount of cells. However, Nicholls et al. results published in 1983 and, therefore, needs to be choose to qualitatively describe their lectin histochemis- reevaluated carefully and realistically. Recent published try results as “strong” or “poor” staining instead of studies indicate that α2-3 and α2-6 linked sialic acids quantitatively counting lectin stained/unstained cells and are not the sole receptors determining tissue and host comparing the percentages of lectin stained cells tropism of human and avian influenza viruses[26−32] between tissues from the upper and lower respiratory Other factors, such as glycan topology[34], lipid raft tracts. Without quantitative analysis, it is possible that microdomains[35,36], local density of sialic acid receptors, one group could report the presence of specific receptors concentration of invading viruses, coreceptors and sialic by showing 3 or more lectin or virus bound epithelial acid-independent receptors, may also be important in cells in one tissue section and another group could report determining tissue and host tropism of human, avian and the absence of the same receptors by showing 2 or less animal influenza viruses. In light of the findings lectin or virus bound epithelial cells in another tissue discussed above, more attention should be given to other section. The above examples have been used to show the cell surface components in the human respiratory tract importance of quantitative descriptions of sialic acid important for interactions with influenza viruses. Some receptor distributions based immunohistochemistry of the studies published in major scientific journals are results. In order to make published results comparable inconsistent and caused confusion on the subject of among different laboratories and avoid potential tissue tropism of influenza viruses. To be more effective confusion in the field of tissue and host tropism of in controlling the global spread of pandemic influenza influenza viruses, every laboratory should analyze lectin such as the current circulating influenza A H1N1, it is and virus immunohistochemistry results quantitatively crucial to clarify the determinants for tissue and host before reporting in any scientific journals. tropism of influenza viruses through quantitative analysis of experimental results, which should lead to a 5 Conclusion remarks better understanding of tissue and host tropism of Determinants of tissue and host tropism of human, avian influenza viruses and provide new targets for effective and animal influenza viruses are more complex than prevention and treatment of seasonal and pandemic what has been generally accepted. Even though the influenza.

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