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.

Fig 1

NA inhibitors (oseltamivir, zanamivir, and peramivir) reduce the influenza virus yield in NHBE cells. Cells were infected with A/Hong Kong/213/03 (H5N1) (A) or A/Turkey/65–1242/06 (H5N1) (B) virus at an MOI of 0.1 in the presence of NA inhibitors. After a 1-h incubation, the inoculum was removed, and the cells were incubated for another 24 h. Viruses released into the apical compartment of NHBE cells were harvested by the apical addition and collection of 300 μl of medium allowed to equilibrate for 30 min. The virus titer was determined by plaque assay in MDCK cells. The drug concentration that caused a 50% decrease in the PFU titer in comparison to control wells without drug was defined as the EC₅₀. Values are the means ± standard deviations of three independent experiments.

Fig 2

Generation of variant influenza viruses with decreased susceptibility to NA inhibitors in NHBE cells. Highly pathogenic H5N1 influenza viruses were cultivated in NHBE cells in the presence or absence of increasing or decreasing concentrations of NA inhibitor (oseltamivir, zanamivir, or peramivir). A/Hong Kong/213/03-like (H5N1) (A) and A/Turkey/65-1242/06-like (H5N1) (B) influenza virus variants with decreased susceptibility to NA inhibitors in NHBE cells were generated. At each passage, plaque assays were performed in MDCK cells to test the infectivity (as log₁₀ PFU/ml) of the viruses.

Fig 3

Replication and receptor specificity of drug-resistant H5N1 viruses.(A) Replication of HK/213, HK/213-HA^P194S, TK/65, TK/65-HA^H28R,P194L, and TK/65-HA^H28R,T215I viruses in NHBE cells. NHBE cell cultures were infected via the apical side with each virus at an MOI of 0.1. The progeny viruses released from the apical surface of infected cultures were collected at the indicated time points and titrated in MDCK cells by plaque assay. Representative results expressed as log₁₀ PFU/ml from three independent experiments are shown. *, P < 0.05; °, P < 0.01 (compared with the value for respective wild-type virus, unpaired t test, or one-way ANOVA). (B) Receptor specificity of HK/213, HK/213-HA^P194S, TK/65, TK/65-HA^H28R,P194L, and TK/65-HA^H28R,T215I viruses. Association constants (K_ass) of virus complexes with synthetic sialylglycopolymers conjugated to 3′SL(N) and 6′SL are shown. Higher K_ass values indicate stronger binding. Values are the means ± standard deviations of four independent experiments.*, P < 0.05; °, P < 0.01 (compared with the value for respective wild-type virus, unpaired t test, or one-way ANOVA).

Fig 4

Replication and neuraminidase activity in drug-resistant H5N1 viruses. (A) Replication of HK/213, HK/213-NA^R156K, TK/65, and TK/65-NA^E119A viruses in NHBE cells. NHBE cell cultures were infected via the apical side with each virus at an MOI of 0.1. The progeny viruses released from the apical surface of infected cultures were collected at the indicated time points and titrated in MDCK cells by plaque assay. Representative results expressed as log₁₀ PFU/ml from three independent experiments are shown. °, P < 0.01, compared with the value for respective wild-type virus by an unpaired t test. (B) NA enzyme kinetics of the recombinant HK/213, HK/213-NA^R156K, TK/65, and TK/65-NA^E119A viruses. Substrate conversion velocity (V₀) of NA is shown as a function of substrate concentration. Fluorogenic MUNANA substrate was used at a final concentration of 0 to 2,000 μM. The viruses were standardized to an equivalent dose of 10^7.5 PFU/ml. Fluorescence was measured every 92 s for 45 min at 37°C, using excitation and emission wavelengths of 355 and 460 nm, respectively.

Fig 5

Polymerase activity of ribonucleoprotein complexes of wild-type A/Hong Kong/213/03 (H5N1) (A) and A/Turkey/65–1242/06 (H5N1) (B) viruses and their variants with single point mutations. Amino acid mutations are indicated by shading of a residue below each plot. The polymerase activities were determined by dual-luciferase reporter assay in three independent experiments. The 293T cells were transfected in triplicate with luciferase and Renilla reporter plasmids, with plasmids expressing PB2, PB1, PA, and NP from HK/213, TK/65, or mutated viruses. Cells were incubated at 33°C, 37°C, or 39°C for 24 h, and cell lysates were analyzed to measure firefly luciferase and Renilla activities. The latter was used to normalize transfection efficiency. Values shown represent the means ± standard deviations of activities of each RNP complex relative to that of the respective wild-type virus. *, P < 0.05; °, P < 0.01 (compared with the value for respective wild-type virus by one-way ANOVA).