I222 Neuraminidase Mutations Further Reduce Oseltamivir Susceptibility of Indonesian Clade 2.1 Highly Pathogenic Avian Influenza A(H5N1) Viruses

<div><p>We have tested the susceptibility to neuraminidase inhibitors of 155 clade 2.1 H5N1 viruses from Indonesia, isolated between 2006–2008 as well as 12 clade 1 isolates from Thailand and Cambodia from 2004–2007 using a fluorometric MUNANA-based enzyme inhibition assay. The Thailand and Cambodian clade 1 isolates tested here were all susceptible to oseltamivir and zanamivir, and sequence comparison indicated that reduced oseltamivir susceptibility we observed previously with clade 1 Cambodian isolates correlated with an S246G neuraminidase mutation. Eight Indonesian viruses (5%), all bearing I222 neuraminidase mutations, were identified as mild to extreme outliers for oseltamivir based on statistical analysis by box plots. IC₅₀s were from 50 to 500-fold higher than the reference clade 1 virus from Viet Nam, ranging from 43–75 nM for I222T/V mutants and from 268–349 nM for I222M mutants. All eight viruses were from different geographic locales; all I222M variants were from central Sumatra. None of the H5N1 isolates tested demonstrated reduced susceptibility to zanamivir (IC₅₀s all <5 nM). All I222 mutants showed loss of slow binding specifically for oseltamivir in an IC₅₀ kinetics assay. We identified four other Indonesian isolates with higher IC₅₀s which also demonstrated loss of slow binding, including one virus with an I117V mutation. There was a minimal effect on the binding of zanamivir and peramivir for all isolates tested. As H5N1 remains a potential pandemic threat, the incidence of mutations conferring reduced oseltamivir susceptibility is concerning and emphasizes the need for greater surveillance of drug susceptibility.</p></div

Box plots of means of IC₅₀s for zanamivir and oseltamivir for Indonesian HPAI H5N1 isolates.

<p>Means were calculated from the log₁₀ transformed duplicate values, then back transformed. Boxes represent the 25th to 75th percentiles, and horizontal lines within the boxes represent the median values. The difference between the 25^th–75^th percentiles is defined as the interquartile range (IQR). The ends of the solid lines extending either side of the boxes represent the approximate 95% confidence limits. Mild and extreme outliers lie outside these 95% confidence limits at 1.5x or 3x the IQR respectively from the 75^th percentile. Viet clade 1 is the pooled results of all the assays using the reference clade 1.1 A/chicken/Vietnam/08/2004, Indon#1 = clade 2.1 Indonesian samples from batch 1, and Indon #2 = samples from batch 2. (A) Only one outlier was identified for zanamivir whereas there were 8 mild or extreme outliers for oseltamivir (B).</p

Comparison of 60 min IC₅₀ values for enzyme inhibition assays with and without preincubation with inhibitor for wild type and mutant viruses.

a<p>(−)Virus, inhibitor and MUNANA substrate were added simultaneously with no preincubation. (+) virus and inhibitor were preincubated for 30 min, then MUNANA was added. Both reactions were followed for 60 min. Values are the means of duplicate reactions.</p>b<p>Slow binding is demonstrated by a higher IC₅₀ without preincubation compared to with preincubation; ratio of (−)/(+) >2.0.</p>c<p>(−)/(+) ratio ∼1 shows changed kinetics, which can be due to fast binding and fast dissociation, as seen for all the I222 mutants with oseltamivir, or slow binding, but fast dissociation as seen for the P154S mutant with zanamivir and oseltamivir, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066105#pone-0066105-g002" target="_blank">Fig. 2</a>.</p

Susceptibility of clade 1.1 HPAI H5N1 isolates from Thailand to zanamivir and oseltamivir in the enzyme inhibition assay.

a<p>Samples were all assayed with a 30 min preincubation with inhibitor, and then MUNANA was added and reactions were stopped after 60 min and read. Samples were tested in duplicate and means were calculated from the log₁₀ transformed values, then back transformed.</p>b<p>Virus used as zanamivir and oseltamivir sensitive reference. Mean and standard deviation of six H5N1 assays carried out during the same period.</p

Maximum clade credibility phylogenetic tree based on 67 Arctic-related RABV N gene sequences.

<p>The horizontal branches are drawn to a scale of estimated year of divergence. Upper and lower limits of the 95% highest posterior density (HPD) estimates, and the corresponding divergence dates for the selected nodes are shown. Posterior probability values are shown above the branch for relevant key nodes only. Isolates for which the complete N gene sequence was obtained in this study are indicated by an asterisk.</p

Clade specific amino acid differences in the nucleoprotein of Nepalese RABV.

a<p>Excludes virus sample 3878-10 which adopts Val at position 179.</p

A: Magnified phylogeny of the Indian subcontinent clade.

<p>B: Magnified phylogeny of the Arctic-like 1 and 3 sub-clades. Branches are labeled with bootstrap values generated with both the neighbor-joining (1,000 bootstrap replications) and maximum likelihood (100 bootstrap replications, number in brackets) algorithms. Bootstrap values are given for selected relevant nodes only. A scale indicating genetic distance is presented by the horizontal bar. Designations of the different RABV phylogroups (clade, sub-clade and lineage) are as indicated, based on previously defined assignments <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002560#pntd.0002560-Bourhy1" target="_blank">[5]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002560#pntd.0002560-Kuzmin1" target="_blank">[25]</a>. Isolates for which the complete N gene sequence was obtained in this study are indicated by an asterisk with the viruses from Nepal underscored.</p

Clade specific amino acid differences in the glycoprotein of Nepalese RABV.

a<p>Amino acid positions exclude the 19aa signal peptide sequence.</p>b<p>Virus samples 3878-09 and 3878-78 have the Indian subcontinent residue.</p>c<p>Virus samples 3878-09 and 3878-78 have Thr substitution.</p>d<p>Virus sample 3878-78 has the Indian subcontinent residue.</p

Maximum likelihood phylogenetic tree based on 173 complete RABV N gene nucleotide sequences.

<p>Branches are labeled with bootstrap values generated with both the neighbor-joining (1,000 bootstrap replications) and maximum likelihood (100 bootstrap replications, number in brackets) algorithms. Bootstrap values are given for selected relevant nodes only. A scale indicating genetic distance is presented by the horizontal bar. Designations of the different RABV phylogroups (clade, sub-clade and lineage) are as indicated, based on previously defined assignments <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002560#pntd.0002560-Bourhy1" target="_blank">[5]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002560#pntd.0002560-Kuzmin1" target="_blank">[25]</a>. Isolates for which the complete N gene sequence was obtained in this study are indicated by an asterisk with the viruses from Nepal underscored.</p

Identification of additional clade 2.1 viruses with altered oseltamivir binding by IC₅₀ kinetics.

<p>Comparison of IC₅₀s after each 10 min without preincubation of virus with inhibitor (−) and with a 30 min preincubation (+) of virus with inhibitor. After addition of MUNANA substrate both assays were incubated for 60 min. Lower initial 10 min IC₅₀s in the (+) reaction compared to the final 60 min IC₅₀s in the (−) reaction indicates slow binding. Similar IC₅₀s in both assays demonstrate both fast binding and dissociation. These four isolates all demonstrated further loss of slow binding compared to the wild type clade 2.1 reference virus, although only one had a known mutation conferring reduced oseltamivir susceptibility. <b>Cl 2 wt</b> = Clade 2.1 wild type A/chicken/Bangli/BBVD-563/2007, <b>Tang</b> = A/chicken/West Java Tangerang/PTB6/2008, <b>Tanjun = </b>A/chicken/West Java/Tja-31/2008, <b>Bangli</b> = A/chicken/Bangli/BBVD-562/2007 (I117V mutation), <b>Paya</b> = A/chicken/Payakumbuh/BPPVRII-307/2007.</p