Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

Abstract

A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses.

Risk assessment of recent Egyptian H5N1 influenza viruses

“…The HA proteins of the Egyptian H5N1 viruses analyzed here encode the avian virus-characteristic N219, Q221, and G223 residues. Viruses of subclades 2.2.1 and 2.2.1.2 possess characteristic D43N, S120N/D, ΔS129 (Δ indicates the deletion of an amino acid compared with the H3 HA reference sequence), and I150T mutations in HA417; the ΔS129/I150T double mutation confers binding to Siaα2,6 Gal while retaining Siaα2,3 Gal binding181920. The viruses analyzed here encode D43N, S120D, ΔS129, and I150T, suggesting that they bind to human-type receptors.…”

“…All inoculum viruses and samples isolated from exposed animals possess amino acids that facilitated the binding of Egyptian HPAI H5N1 viruses to human-type receptors181920. To assess the receptor-binding specificity of the viruses tested here, we performed a solid phase binding assay in which α2,3- or α2,6-linked sialylglycopolymers were coated onto a microtiter…”

Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes

“…In ferrets inoculated by the URT route, however, systemic spread was delayed, and this factor may provide a longer therapeutic window for effective oseltamivir treatment. An additional application of the URT infection route, due to focal replication within the low-pH conditions of the mammalian nasal passage (54), includes growth characterization and selection of H5N1 viruses with acidstable hemagglutinins, such as those mutants identified in previous transmission studies (55) or mutants isolated from infected patients (56). This study suggests that exposure route is an important factor that needs to be taken into consideration when evaluating antiviral regimes and investigating antiviral and vaccine efficacy in the ferret model.…”

Evolution of H5-Type Avian Influenza A Virus Towards Mammalian Tropism in Egypt, 2014 to 2015

“…However, previous studies rarely predicted changes, which could potentiate the interspecies transmission of these AIVs. Instead, such studies discussed the shift of HPAIV H5N1 from avian species to mammals by focusing mainly on the acquired aa mutations and variations following transmission to and adaptation in mammals (e.g., human isolates) [22]. In contrast, this study used a novel and economic bioinformatics tool, the ISM, to predict and identify variations in the HA of avian H5N1 isolates from the 2014 to 2015 season that had the ability to increase the human propensity of these virus strains.…”