I223R mutation in influenza A(H1N1)pdm09 neuraminidase confers reduced susceptibility to oseltamivir and zanamivir and enhanced resistance with H275Y.

BACKGROUND: Resistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility. METHODS: The NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80). RESULTS: Compared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin. CONCLUSIONS: Reduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised

Molecular cloning of the NA gene from the D25 nasal swab.

<p>The NA region between residues 201 and 300 was amplified, cloned and sequenced. Frequencies of clones harboring various patterns of amino acids at positions 223 and 275 in the NA are represented. For each pattern, frequencies of clones with additional mutations are indicated.</p

Enzymatic characteristics of the NA of virus isolates.

a<p>results of pyrosequencing quantification on isolates obtained after 2 passages on MDCK cells.</p>b<p>mean±standard deviation of 3 independent determinations.</p>c<p>Ki for oseltamivir carboxylate.</p>d<p>Ki for zanamivir.</p>e<p>na: non applicable.</p>*<p>significantly different Km or Ki value as compared to the D0 isolate (Student t test. p<0.05).</p

Additional mutations in neuraminidase and hemagglutinin.

<p>Only amino acid mutations or mixture of wild type and mutations are indicated.</p>a<p>HA and NA sequence only from isolate.</p>b<p>HA sequence only from isolate.</p>*<p>: detected only on isolates.</p>#<p>: detected only on clinical specimens.</p>x<p>: relative to the sequence of A/NewYork/18/2009.</p

Enzymatic characteristics of the NA of viruses produced by reverse genetics.

a<p>mean±standard deviation of 2 independent determinations.</p>b<p>Ki for oseltamivir carboxylate.</p>c<p>Ki for zanamivir.</p>d<p>na: non applicable.</p>*<p>significantly different Km or Ki value as compared to RG wild type (Student t test. p<0.05).</p

Replication kinetics of recombinant viruses in MDCK-SIAT1.

<p>MDCK-SIAT1 cells were inoculated at a multiplicity of infection (MOI) of 0.001 PFU with the engineered viruses. Supernatants were harvested after at 12, 24, 36, 48, 60 and 72 hours post infection and were titrated on MDCK-SIAT1 cells. The results shown are from two independent experiments. Mean titers and standard deviations are shown.</p

Treatment history and viral evolution.

<p><b>A</b>. Evolution of nasal H1N1pdm09 RNA levels and neuraminidase inhibitors treatment history. For each sequential nasopharyngeal sample the level of H1N1pdm09 RNA determined by real-time RT-PCR targeting the M gene is expressed as the log of copies per mL. Neuraminidase inhibitor treatment is represented by boxes: oseltamivir 75 mg×1/d (grey box), oseltamivir 75 mg×2/d (black box), and inhaled zanamivir 10 mg×2/d (stippled box). <b>B</b>. Based on the pyrosequencing quantification performed on primary specimens, the proportion of amino acids at positions 223 and 275 in the NA is indicated as a percentage below each sample. The level of the predominant residue is indicated in bold.</p