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.

A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.

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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