Viral and host factors required for avian H5N1 influenza A virus replication in mammalian cells

Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions

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/CL™ 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. The plaque size increased with decreasing Avicel concentration, but even very diluted Avicel overlays (0.3%) ensured formation of localized plaques. Due to their low viscosity, Avicel overlays were easier to use than methylcellulose overlays, especially in the 96-well culture plates. Furthermore, Avicel overlay could be applied without prior removal of the virus inoculum thus facilitating the assay and reducing chances of cross-contamination. Using neuraminidase inhibitor oseltamivir carboxylate, we demonstrated applicability of the Avicel-based plaque reduction assay for testing of antiviral substances. CONCLUSION: Plaque assay under Avicel-containing overlay media is easier, faster and more sensitive than assays under agar- and methylcellulose overlays. The assay can be readily performed in a 96-well plate format and seems particularly suitable for high-throughput virus titrations, serological studies and experiments on viral drug sensitivity. It may also facilitate work with highly pathogenic agents performed under hampered conditions of bio-safety labs

H5N1 influenza vaccine quality is affected by hemagglutinin conformational stability

Since 1997, highly pathogenic H5N1 avian influenza viruses have circulated in wild and domestic birds and sporadically have infected humans. Conventional inactivated vaccines made from these viruses were shown to have decreased HA content and immunogenicity compared to seasonal preparations. We assumed that the high pH threshold (5.6-6.0) known for the HA conformational change (pH of fusion or activation) of avian highly pathogenic influenza viruses resulted in the low stability of native HA conformation and affected the vaccine quality. The 58Lys→Ile mutation introduced into the HA2 subunit of the HA of A/chicken/Kurgan/5/05 (H5N1) virus decreased the pH threshold of the HA activation. The mutant virus demonstrated increased HA stability toward acidic pH and elevated temperature, decreased binding efficiency to the monoclonal antibody IIF4 that recognizes the HA low pH form, and increased HA resistance to trypsin digestion. Virus with modified HA was less susceptible to freezing stress and showed an increased content of immunocompetent HA in inactivated vaccine preparation compared to the analogous virus with original HA. Therefore, we have shown a way to increase the quality of inactivated vaccines made from highly pathogenic avian influenza viruses.Since 1997, highly pathogenic H5N1 avian influenza viruses have circulated in wild and domestic birds and sporadically have infected humans. Conventional inactivated vaccines made from these viruses were shown to have decreased HA content and immunogenicity compared to seasonal preparations. We assumed that the high pH threshold (5.6-6.0) known for the HA conformational change (pH of fusion or activation) of avian highly pathogenic influenza viruses resulted in the low stability of native HA conformation and affected the vaccine quality. The 58Lys→Ile mutation introduced into the HA2 subunit of the HA of A/chicken/Kurgan/5/05 (H5N1) virus decreased the pH threshold of the HA activation. The mutant virus demonstrated increased HA stability toward acidic pH and elevated temperature, decreased binding efficiency to the monoclonal antibody IIF4 that recognizes the HA low pH form, and increased HA resistance to trypsin digestion. Virus with modified HA was less susceptible to freezing stress and showed an increased content of immunocompetent HA in inactivated vaccine preparation compared to the analogous virus with original HA. Therefore, we have shown a way to increase the quality of inactivated vaccines made from highly pathogenic avian influenza viruses

H9N2 Influenza A Viruses from Poultry in Asia Have Human Virus-like Receptor Specificity

AbstractH9N2 influenza A viruses are currently widespread in chickens, quail, and other poultry in Asia and have caused a few cases of influenza in humans. In this study, we found that H9N2 viruses from Hong Kong live bird markets have receptor specificity similar to that of human H3N2 viruses. In addition, the neuraminidase of poultry H9N2 viruses has mutations in its hemadsorbing site, a characteristic resembling that of human H2N2 and H3N2 viruses but differing from that of other avian viruses. Peculiar features of surface glycoproteins of H9N2 viruses from Hong Kong suggest an enhanced propensity for introduction into humans and emphasize the importance of poultry in the zoonotic transmission of influenza viruses

One-way trip: Influenza virus' adaptation to gallinaceous poultry may limit its pandemic potential

We hypothesise that some influenza virus adaptations to poultry may explain why the barrier for human-to-human transmission is not easily overcome once the virus has crossed from wild birds to chickens. Since the cluster of human infections with H5N1 influenza in Hong Kong in 1997, chickens have been recognized as the major source of avian influenza virus infection in humans. Although often severe, these infections have been limited in their subsequent human-to-human transmission, and the feared H5N1 pandemic has not yet occurred. Here we examine virus adaptations selected for during replication in chickens and other gallinaceous poultry. These include altered receptor binding and increased pH of fusion of the haemagglutinin as well as stalk deletions of the neuraminidase protein. This knowledge could aid the delivery of vaccines and increase our ability to prioritize research efforts on those viruses from the diverse array of avian influenza viruses that have greatest human pandemic potential

Host Range Restriction and Pathogenicity in the Context of Influenza Pandemic

Certain viral factors determine the host range restriction and pathogenicity of influenza A viruses

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. A sequence database analysis revealed that the NAs of the majority of human H2N2 viruses isolated during the influenza pandemic of 1957 differ from their putative avian precursor by amino acid substitutions in the hemadsorption site. We found that the NA of a representative pandemic virus A/Singapore/1/57 (H2N2) lacks hemadsorption activity and that a single reversion to the avian-virus-like sequence (N367S) restores hemadsorption. Using this hemadsorption-positive NA, we generated three NA variants with substitutions S370L, N400S and W403R that have been found in the hemadsorption site of human H2N2 viruses. Each substitution abolished hemadsorption activity. Although, there was no correlation between hemadsorption activity of the NA variants and their enzymatic activity with respect to monovalent substrates, all four hemadsorption-negative NAs desialylated macromolecular substrates significantly slower than did the hemadsorption-positive counterpart. The NA of the 1918 pandemic virus A/Brevig Mission/1/18 (H1N1) also differed from avian N1 NAs by reduced hemadsorption activity and less efficient hydrolysis of macromolecular substrates. Our data indicate that the hemadsorption site serves to enhance the catalytic efficiency of NA and they suggest that, in addition to changes in the receptor-binding specificity of the hemagglutinin, alterations of the NA are needed for the emergence of pandemic influenza viruses

H5N1 chicken influenza viruses display a high binding affinity for Neu5Acα2-3Galβ1-4(6-HSO3)GlcNAc-containing receptors

AbstractTo characterize differences in the receptor-binding specificity of H5N1 chicken viruses and viruses of aquatic birds, we used a panel of synthetic polyacrylamide (PAA)-based sialylglycopolymers that carried identical terminal Neu5Acα2-3Gal fragments but varied by the structure of the next saccharide residues. A majority of duck viruses irrespective of their HA subtype, bound with the highest affinity to trisaccharide Neu5Acα2-3Galβ1-3GlcNAc, suggesting that these viruses preferentially recognize sialyloligosaccharide receptors with type 1 core (Galβ1-3GlcNAc). Substitution of 6-hydroxyl group of GlcNAc residue of tested sialylglycopolymers by 6-sulfo group had little effect on receptor binding by duck viruses. By contrast, H5N1 chicken and human viruses isolated in 1997 in Hong Kong preferred receptors with type 2 core (Galβ1-4GlcNAcβ) and bound sulfated trisaccharide Neu5Acα2-3Galβ1-4(6-HSO3)GlcNAcβ (6-Su-3′SLN) with the extraordinary high affinity. Another chicken virus, A/FPV/Rostok/34 (H7N1), and several mammalian viruses also displayed an increased affinity for sulfated sialyloligosaccharide receptor. The binding of chicken and mammalian viruses to tracheal epithelial cells of green monkey decreased after treatment of cells with glucosamine-6-sulfatase suggesting the presence of 6-O-Su-3′SLN determinants in the airway epithelium. It remains to be seen whether existence of the 6-O-Su-3′SLN groups in the human airway epithelial cells might facilitate infection of humans with H5N1 chicken viruses

Influenza Viruses Display High-Affinity Binding to Human Polyglycosylceramides Represented on a Solid-Phase Assay Surface

AbstractPolyglycosylceramides (PGCs), complex glycolipids containing up to 50 or more sugar residues, are recognized as the minor components of the cell-surface membranes, but a knowledge on their tissue distribution, structure, and function is limited. In this study, the binding of influenza viruses to preparations of PGCs was investigated using a TLC overlay assay and a microwell adsorption assay. The ability of PGCs to bind influenza virus was dependent on the source from which they were derived. Preparations of PGCs from human erythrocytes were found to support binding of A and B influenza virus strains at a much lower concentration than sialyl-6-paragloboside and to be somewhat better receptors for these viruses compared to the sialylglycoprotein fetuin. A high virus-binding activity of PGCs suggests that these species could potentially serve as biologically important cell-surface receptors for influenza viruses

1918 Influenza Pandemic and Highly Conserved Viruses with Two Receptor-Binding Variants

The “Spanish influenza pandemic swept the globe in the autumn and winter of 1918–19, and resulted in the deaths of approximately 40 million people. Clinically, epidemiologically, and pathologically, the disease was remarkably uniform, which suggests that similar viruses were causing disease around the world. To assess the homogeneity of the 1918 pandemic influenza virus, partial hemagglutinin gene sequences have been determined for five cases, including two newly identified samples from London, United Kingdom. The strains show 98.9% to 99.8% nucleotide sequence identity. One of the few differences between the strains maps to the receptor-binding site of hemagglutinin, suggesting that two receptor-binding configurations were co-circulating during the pandemic. The results suggest that in the early stages of an influenza A pandemic, mutations that occur during replication do not become fixed so that a uniform “consensus” strain circulates for some time