Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs.

HA-Dependent Tropism of H5N1 and H7N9 Influenza Viruses to Human Endothelial Cells Is Determined by Reduced Stability of the HA, Which Allows the Virus To Cope with Inefficient Endosomal Acidification and Constitutively Expressed IFITM3.

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1).

Receptor-binding properties of influenza viruses isolated from gulls.

Ducks, gulls and shorebirds represent the major hosts of influenza A viruses (IAVs) in nature, but distinctions of IAVs in different birds are not well defined. Here we characterized the receptor specificity of gull IAVs with HA subtypes H4, H6, H14, H13 and H16 using synthetic sialylglycopolymers. In contrast to duck IAVs, gull IAVs efficiently bound to fucosylated receptors and often preferred sulfated and non-sulfated receptors with Galβ1-4GlcNAc cores over the counterparts with Galβ1-3GlcNAc cores.

pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH.

Species-specific and individual differences in Nipah virus replication in porcine and human airway epithelial cells.

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species.

Role of Substitutions in the Hemagglutinin in the Emergence of the 1968 Pandemic Influenza Virus.

Hemagglutinin (HA) of H3N2/1968 pandemic influenza viruses differs from the putative avian precursor by seven amino acid substitutions. Substitutions Q226L and G228S are known to be essential for adaptation of avian HA to mammals. We found that introduction of avian-virus-like amino acids at five other HA positions (positions 62, 81, 92, 144, and 193) of A/Hong Kong/1/1968 virus decreased viral replication in human cells and transmission in pigs.

Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells.

The epithelium of conducting airways represents the main target for influenza virus in mammals. However, the peculiarities of virus interactions with differentiated airway epithelial cells remain largely unknown. Here, influenza virus budding was studied in differentiated cultures of human tracheobronchial epithelial cells using transmission electron microscopy.

Receptor-binding profiles of H7 subtype influenza viruses in different host species.

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.

Aprotinin, a protease inhibitor, suppresses proteolytic activation of pandemic H1N1v influenza virus.

Background: The recent emergence of pandemic influenza virus H1N1v stresses the need for the development of new anti-influenza drugs.

Methods: Host proteases responsible for viral haemagglutinin (HA) cleavage are attractive targets for such drugs. Aprotinin, a natural 58-amino-acid polypeptide from bovine lungs, was chosen for this purpose because it is a drug already approved for human use as an antiprotease compound to treat pancreatitis and bleeding, and because it inhibits a wide spectrum of serine proteases, some of which are involved in influenza virus activation.

Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus.

Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells.

Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses.

Human influenza viruses derive their genes from avian viruses. The neuraminidase (NA) of the avian viruses has, in addition to the catalytic site, a separate sialic acid binding site (hemadsorption site) that is not present in human viruses. The biological significance of the NA hemadsorption activity in avian influenza viruses remained elusive.

Avian-virus-like receptor specificity of the hemagglutinin impedes influenza virus replication in cultures of human airway epithelium.

A non-optimal receptor-binding specificity of avian influenza viruses is believed to hamper their replication in humans; however, the magnitude of this restriction remains undefined. Here we generated recombinant viruses, R1 and R2, that differed solely by two amino acids in the receptor-binding site of their hemagglutinin (HA). R1 harbored the original HA of the pandemic human virus A/Hong Kong/1/68 (H3N2), whereas R2 was the L226Q/S228G HA mutant with avian-virus-like receptor specificity.

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.

New low-viscosity overlay medium for viral plaque assays.

Background: Plaque assays in cell culture monolayers under solid or semisolid overlay media are commonly used for quantification of viruses and antiviral substances. To overcome the pitfalls of known overlays, we tested suspensions of microcrystalline cellulose Avicel RC/CLtrade mark as overlay media in the plaque and plaque-inhibition assay of influenza viruses.

Results: Significantly larger plaques were formed under Avicel-containing media, as compared to agar and methylcellulose (MC) overlay media.

Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium.

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.

Human and avian influenza viruses target different cell types in cultures of human airway epithelium.

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.

Overexpression of the alpha-2,6-sialyltransferase in MDCK cells increases influenza virus sensitivity to neuraminidase inhibitors.

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-alpha2,6-galactose (Sia[alpha2,6]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(alpha2,6)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).

The long noncoding region of the human parainfluenza virus type 1 f gene contributes to the read-through transcription at the m-f gene junction.

Sendai virus (SV) and human parainfluenza virus type 1 (hPIV1) have genomes consisting of nonsegmented negative-sense RNA in which the six genes are separated by well-conserved intergenic (IG) sequences and transcriptional start (S) and end signals. In hPIV1-infected cells, transcriptional termination at the M-F gene junction is ineffective; a large number of M-F read-through transcripts are produced (T. Bousse, T.