The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses.

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.The ability of the hemagglutinin protein (HA) of influenza viruses to mediate fusion between viral and endosomal membranes during virus entry into the cell depends on cleavage of fusion-incompetent precursor HA0 into disulfide-linked subunits HA1 and HA2 by a host endoprotease. Cleavage of HA is essential for infection and determines viral pathogenicity and tissue tropism (reviewed in references 8, 10, 11, and 22). Thus, the highly pathogenic avian influenza viruses of subtypes H5 and H7 are cleaved at the multibasic motif R-X-R/K-R by ubiquitous subtilisin-like cellular proteases (11, 23) and cause lethal systemic infection in birds. All other influenza A viruses, including human epidemic and pandemic strains, have a single arginine at the HA cleavage site; these viruses can only be cleaved in a limited number of tissues, such as the intestinal tract in birds and the respiratory tract in birds and mammals (11,22).Early studies demonstrated that influenza viruses with monobasic cleavage site can be proteolytically activated in cell culture by the addition of trypsin (12, 13). Less is known about proteases that cleave influenza viruses under conditions of natural infection. Several trypsin-like proteases isolated from rat and swine lung were shown to support replication of influenza viruses in vitro (3,9,18,25). However, it remains unclear whether these proteases play a role in in vivo infection. In the case of human influenza, specific proteases responsible for HA cleavage in the human respiratory tract have not been identified thus far.In search of such proteases, we use a new approach. Instead of isolating and characterizing influenza virus-activating enzymes from respiratory tissues, we clone and express genes of trypsin-like proteases known to be present in the human airway epithelium and test them for cleavage of HA. We have analyzed here two such proteases, TMPRSS2 (5, 14, 21) and HAT (human airway trypsin-like protease) (4, 24, 27, 28), given their previous detection in the human airways and the availability of their full-length coding sequences. As the source of human genetic material for cloning, we used differentiated cultures of human airway epithelial cells grown at the air-liquid interface in serum-free, hormone-and growth factor-supplemented medium as previously described (6,15). These cultures support multicycle replication of human influenza virus...

Influenza virus neuraminidase (NA) plays an essential role in release and spread of progeny virions, following the intracellular viral replication cycle. To test whether NA could also facilitate virus entry into cell, we infected cultures of human airway epithelium with human and avian influenza viruses in the presence of the NA inhibitor oseltamivir carboxylate. Twenty-to 500-fold less cells became infected in drug-treated versus nontreated cultures (P < 0.0001) 7 h after virus application, indicating that the drug suppressed the initiation of infection. These data demonstrate that viral NA plays a role early in infection, and they provide further rationale for the prophylactic use of NA inhibitors.It is believed that the major function of viral neuraminidase (NA) is at the final stage of infection when NA cleaves sialic acid from cell surface and progeny virions facilitating virus release from infected cells (1, 2). Less is known about NA functions during virus entry into the cell. It has long been assumed that NA promotes virus access to target cells in airways by mucus degradation (3). However, this concept has never been formally proven due to the lack of an adequate experimental system. Moreover, some evidence arguing against the role of NA at the early stages of infection has been reported (reviewed in reference 2).To address this issue, we studied the effects of the NA inhibitor oseltamivir carboxylate (OC) (9) on influenza virus entry into cultures of human airway epithelium. Primary human tracheobronchial epithelial cells (HTBE; Clonetics) and primary nasal epithelial cells (PromoCell GmbH) were grown on membrane supports (12-mm Transwell-Clear; Corning, Inc.) at the air-liquid interface in serum-free growth factor and hormone-supplemented medium (6, 8). Fully differentiated 4-to 8-week-old cultures were used for all experiments. These cultures were pseudostratified and polarized; contained basal, ciliated, and mucus-secreting cells; and closely resembled human airway epithelium in vivo (Fig. 1). OC (1 M, if not indicated otherwise) was added to virus suspensions and to basolateral compartments of the cultures shortly before infecting two replicate cultures from the apical side. Two control cultures were infected in the absence of inhibitor. One hour postinfection, we removed the virus inoculum and incubated cultures at the air-liquid interface for additional 6 h to allow intracellular virus replication. The cultures were then fixed, and infected cells were identified by staining with polyclonal antisera to whole viruses followed by corresponding peroxidase-labeled secondary antibodies (Dianova) and aminoethylcarbazole substrate (Sigma). Positive staining indicated successful virus entry in the cell. The cultures were analyzed en face at a magnification of ϫ300 (Olympus IMT-2). A total number of cells expressing viral antigen was counted in the epithelial segment that included all consecutive microscopic views (0.28 by 0.42 mm) along the diameter of the culture (segment surface area, 3 mm 2 ; number of...

No reliable cell culture assay is currently available for monitoring human influenza virus sensitivity to neuraminidase inhibitors (NAI). This can be explained by the observation that because of a low concentration of sialyl-␣2,6-galactose (Sia[␣2,6]Gal)-containing virus receptors in conventional cell lines, replication of human virus isolates shows little dependency on viral neuraminidase. To test whether overexpression of Sia(␣2,6)Gal moieties in cultured cells could make them suitable for testing human influenza virus sensitivity to NAI, we stably transfected MDCK cells with cDNA of human 2,6-sialyltransferase (SIAT1). Transfected cells expressed twofold-higher amounts of 6-linked sialic acids and twofold-lower amounts of 3-linked sialic acids than parent MDCK cells as judged by staining with Sambucus nigra agglutinin and Maackia amurensis agglutinin, respectively. After transfection, binding of a clinical human influenza virus isolate was increased, whereas binding of its egg-adapted variant which preferentially bound 3-linked receptors was decreased. The sensitivity of human influenza A and B viruses to the neuraminidase inhibitor oseltamivir carboxylate was substantially improved in the SIAT1-transfected cell line and was consistent with their sensitivity in neuraminidase enzyme assay and with the hemagglutinin (HA) receptor-binding phenotype. MDCK cells stably transfected with SIAT1 may therefore be a suitable system for testing influenza virus sensitivity to NAI.The neuraminidase (NA) of influenza A and B viruses cleaves the ␣-glycosidic linkages between sialic acid and the adjacent sugar and thus destroys virus receptors on the cell surface, extracellular inhibitors, and viral glycoproteins (reviewed in references 2 and 8). The NA activity is believed to be particularly important at the late stages of infection by preventing hemagglutinin (HA)-mediated self-aggregation and facilitating release of progeny virions from cells. Interaction of virions with cell-associated and soluble sialylglycoconjugates of the host is mediated by HA and NA in an antagonistic manner, which has to be carefully balanced to allow efficient virus replication (reviewed in reference 36).With increasing use of neuraminidase inhibitors (NAI) for influenza treatment, there is a need for a suitable methodology to monitor for emergence of virus resistance (32,34,38). In cell culture experiments, resistance to NAI results from mutation of either HA, NA, or both glycoproteins. Mutations in HA usually precede NA mutations and reduce virus affinity for sialic acid-containing receptors, thereby decreasing the dependency of the virus on NA function, whereas mutations in NA decrease the binding affinity of the inhibitor to the catalytic site (reviewed in references 19, 29, and 30). In a clinical setting, NA-mediated resistance seems to be the primary mechanism of resistance to NAI and can be easily and reliably monitored using an in vitro enzyme inhibition assay (32,34,38). Since the possibility cannot be excluded that the loss of sensitivit...

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