Decreased Neuraminidase Activity Is Important for the Adaptation of H5N1 Influenza Virus to Human Airway Epithelium

Abstract

Highly pathogenic avian H5N1 influenza viruses remain a pandemic threat. Antiviral drugs such as neuraminidase (NA) inhibitors will be crucial for disease control in the event of a pandemic. Should drug-resistant H5N1 viruses develop, all defense strategies will be compromised. To determine the likelihood and mechanisms of emergence of NA inhibitor-resistant H5N1 variants in humans, we serially passaged two H5N1 viruses, A/Hong Kong/213/03 and A/Turkey/65-1242/06, in normal human bronchial epithelial (NHBE) cells in the presence of oseltamivir, zanamivir, or peramivir. To monitor the emergence of changes associated with the adaptation of H5N1 viruses to humans, we passaged the strains in the absence of drugs. Under pressure of each NA inhibitor, A/Turkey/65-1242/06 developed mutations in the hemagglutinin (HA) (H28R and P194L/T215I) and NA (E119A) proteins that reduced virus binding to α2,3-sialyl receptor and NA activity. Oseltamivir pressure selected a variant of A/Hong Kong/213/03 virus with HA P194S mutation that decreased viral binding to α2,6 receptor. Under peramivir pressure, A/Hong Kong/213/03 virus developed a novel NA mutation, R156K, that reduced binding to all three drugs, caused about 90% loss of NA activity, and compromised replication in NHBE cells. Both strains were eliminated in NHBE cells when they were cultivated in the absence of drugs. Here, we show for the first time that decreased NA activity mediated through NA inhibitors is essential for the adaptation of pandemic H5N1 influenza virus to humans. This ability of decreased NA activity to promote H5N1 infection underlines the necessity to optimize management strategies for a plausible H5N1 pandemic.

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility

“…Segment-specific regulation is particularly important for the HA and NA genes, because balancing the relative activity levels of these two molecules is key to maintaining optimal fitness across different host contexts (20,21,29). Previously described mechanisms of NA regulation in response to selective pressure entail cis-acting mutations that modulate NA activity in cells or virions (22,(30)(31)(32).…”

The Universal Epitope of Influenza A Viral Neuraminidase Fundamentally Contributes to Enzyme Activity and Viral Replication

“…NA Enzymatic Activity-NA enzymatic activity was measured by standardizing the samples with pfu as described (24). Virus was aliquoted at a titer of 1 ϫ 10 7 pfu and then incubated at 37°C with 2-(4-methylumberlliferyl)-␣-D-N-acetylneuraminic acid (MUNANA).…”

Fitness Costs for Influenza B Viruses Carrying Neuraminidase Inhibitor-Resistant Substitutions: Underscoring the Importance of E119A and H274Y

“…The rg-E119A virus uniquely possessed a decrease in NA enzyme activity while retaining V max and K m values similar to those of the rg-WT virus, suggesting that reduction of NA enzyme activity does not preclude impairment to overall viral NA function. Indeed, this dichotomy was previously shown to be advantageous for adaptation of avian influenza A (H5N1) viruses to NHBE cells (44).…”