Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.     

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.     

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA₁ allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Activation of Influenza A Viruses by Host Proteases from Swine Airway Epithelium

Pigs are important natural hosts of influenza A viruses, and due to their susceptibility to swine, avian, and human viruses, they may serve as intermediate hosts supporting adaptation and genetic reassortment. Cleavage of the influenza virus surface glycoprotein hemagglutinin (HA) by host cell proteases is essential for viral infectivity. Most influenza viruses, including human and swine viruses, are activated at a monobasic HA cleavage site, and we previously identified TMPRSS2 and HAT to be relevant proteases present in human airways. We investigated the proteolytic activation of influenza viruses in primary porcine tracheal and bronchial epithelial cells (PTEC and PBEC, respectively). Human H1N1 and H3N2 viruses replicated efficiently in PTECs and PBECs, and viruses containing cleaved HA were released from infected cells. Moreover, the cells supported the proteolytic activation of HA at the stage of entry. We found that swine proteases homologous to TMPRSS2 and HAT, designated swTMPRSS2 and swAT, respectively, were expressed in several parts of the porcine respiratory tract. Both proteases cloned from primary PBECs were shown to activate HA with a monobasic cleavage site upon coexpression and support multicycle replication of influenza viruses. swAT was predominantly localized at the plasma membrane, where it was present as an active protease that mediated activation of incoming virus. In contrast, swTMPRSS2 accumulated in the trans-Golgi network, suggesting that it cleaves HA in this compartment. In conclusion, our data show that HA activation in porcine airways may occur by similar proteases and at similar stages of the viral life cycle as in human airways.     

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the alpha2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the alpha2,6 and the alpha2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.     

Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases.

Cleavage of influenza A virus hemagglutinin (HA) is required for expression of fusion activity and virus entry into cells. Extracellular proteases are responsible for the proteolytic cleavage activation of avirulent avian and mammalian influenza viruses and contribute to pathogenicity and tissue tropism. The relative contributions of host and microbial proteases to cleavage activation in natural infection remain to be established. We examined 23 respiratory bacterial pathogens and 150 aerobic bacterial isolates cultured from the nasal cavities of pigs for proteolytic activity. No evidence of secreted proteases was found for the bacterial pathogens, including Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Streptococcus suis. Proteolytic bacteria were isolated from 7 of 11 swine nasal samples and included Staphylococcus chromogenes, Staphylococcus hyicus, Aeromonas caviae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Enterococcus sp. Only P. aeruginosa secreted a protease, elastase, that cleaved influenza virus HA. However, compared to trypsin, the site of cleavage by elastase was shifted one amino acid in the carboxy-terminal direction and resulted in inactivation of the virus. Under the conditions of this study, we identified several bacterial isolates from the respiratory tracts of pigs that secrete proteases in vitro. However, none of these proteolytic isolates demonstrated direct cleavage activation of influenza virus HA.     

Role of receptor binding specificity in influenza A virus transmission and pathogenesis

The recent emergence of a novel avian A/H7N9 influenza virus in poultry and humans in China, as well as laboratory studies on adaptation and transmission of avian A/H5N1 influenza viruses, has shed new light on influenza virus adaptation to mammals. One of the biological traits required for animal influenza viruses to cross the species barrier that received considerable attention in animal model studies, in vitro assays, and structural analyses is receptor binding specificity. Sialylated glycans present on the apical surface of host cells can function as receptors for the influenza virus hemagglutinin (HA) protein. Avian and human influenza viruses typically have a different sialic acid (SA)‐binding preference and only few amino acid changes in the HA protein can cause a switch from avian to human receptor specificity. Recent experiments using glycan arrays, virus histochemistry, animal models, and structural analyses of HA have added a wealth of knowledge on receptor binding specificity. Here, we review recent data on the interaction between influenza virus HA and SA receptors of the host, and the impact on virus host range, pathogenesis, and transmission. Remaining challenges and future research priorities are also discussed.     

Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Matriptase,HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.     

Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium

Host cell proteases that cleave the hemagglutinin (HA) of influenza viruses in the human respiratory tract are still not identified. Here we cloned two human type II transmembrane serine proteases with known airway localization, TMPRSS2 and HAT, into mammalian expression vector. Cotransfection of mammalian cells with plasmids encoding HA and either protease resulted in HA cleavage in situ. Transient expression of either protease in MDCK cells enabled multicycle replication of influenza viruses in these cells in the absence of exogenous trypsin. These data suggest that TMPRSS2 and HAT are candidates for proteolytic activation of influenza viruses in vivo.     

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.     

Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong

An H5N1 avian influenza A virus was transmitted to humans in Hong Kong in 1997. Although the virus causes systemic infection and is highly lethal in chickens because of the susceptibility of the hemagglutinin to furin and PC6 proteases, it is not known whether it also causes systemic infection in humans. The clinical outcomes of infection in Hong Kong residents ranged widely, from mild respiratory disease to multiple organ failure leading to death. Therefore, to understand the pathogenesis of influenza due to these H5N1 isolates, we investigated their virulence in mice. The results identified two distinct groups of viruses: group 1, for which the dose lethal for 50% of mice (MLD50) was between 0.3 and 11 PFU, and group 2, for which the MLD50 was more than 10(3) PFU. One day after intranasal inoculation of mice with 100 PFU of group 1 viruses, the virus titer in lungs was 10(7) PFU/g or 3 log units higher than that for group 2 viruses. Both types of viruses had replicated to high titers (>10(6) PFU/g) in the lungs by day 3 and maintained these titers through day 6. More importantly, only the group 1 viruses caused systemic infection, replicating in nonrespiratory organs, including the brain. Immunohistochemical analysis demonstrated the replication of a group 1 virus in brain neurons and glial cells and in cardiac myofibers. Phylogenetic analysis of all viral genes showed that both groups of Hong Kong H5N1 viruses had formed a lineage distinct from those of other viruses and that genetic reassortment between H5N1 and H1 or H3 human viruses had not occurred. Since mice and humans harbor both the furin and the PC6 proteases, we suggest that the virulence mechanism responsible for the lethality of influenza viruses in birds also operates in mammalian hosts. The failure of some H5N1 viruses to produce systemic infection in our model indicates that multiple, still-to-be-identified, factors contribute to the severity of H5N1 infection in mammals. In addition, the ability of these viruses to produce systemic infection in mice and the clear differences in pathogenicity among the isolates studied here indicate that this system provides a useful model for studying the pathogenesis of avian influenza virus infection in mammals.     

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.     

Effects of receptor binding specificity of avian influenza virus on the human innate immune response

Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.     

Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals

Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.     

Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.     

Receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential

The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.