• NAI resistance mutations

Influenza virus neuraminidase inhibitor (NI) resistance is defined as a significantly raised IC50 (drug concentration inhibiting neuraminidase (NA) activity by 50%) value, coupled with a characterised mutation in the NA gene.  The table below shows the NAI resistance mutations that have been generated and classified in vitro.

Susceptibility to NI drugs is not absolute, ranging from an alteration in susceptibility to full resistance.  There are typical ranges of IC50 values that differ between influenza subtypes and between oseltamivir and zanamivir for each subtype.  It is not, therefore, possible to compare subtype and drug specific data. 

Influenza B viruses tend to have IC50 values 10-100 fold higher than influenza A viruses. This is normal for influenza B and this lower susceptibility does not appear to have a significant clinical impact.  IC50 values for influenza B viruses tend to be higher for oseltamivir than zanamivir.  IC50 values for H1N1 viruses tend to be higher for zanamivir than oseltamivir, whereas the inverse is true for H3N2 viruses where IC50 values tend to be higher for oseltamivir than zanamivir.

Furthermore, values generated by fluorescence and chemiluminescence methods should not be compared as values generated by chemiluminescence are typically lower than those for the same virus and drug in the fluorescence test.  IC50 values generated from the different assay methods should not be directly compared.

Whilst there is no absolute definition of resistance, due, in part, to lack of global agreement, true resistance is usually classified as an IC50 value at least 10 times greater than the mean IC50 for similar viruses in the same influenza season. 

The phylogenetic tree of NA genes of seasonal H1N1 viruses shows the relationships of oseltamivir-resistant viruses (in red), due to the H275Y substitution, which emerged during late 2007 – 2008 to contemporary oseltamivir-sensitive viruses (in black). Reference vaccine viruses are in blue. Common amino acids which distinguish the different clades are indicated (Collins et al., 2009)

Figure 1. Locations of amino acid substitutions which reduce oseltamivir sensitivity by altering drug interaction with the hydrophobic pocket of the NA active site.

Structures of the open (gold) and closed (green) conformations of N1, and of N2 and B NAs are superimposed.  A deletion of residues 244-247 in N2 is shown in red.  Residues are numbered according to N2 numbering. (Hay et al., 2008).

       
Figure 1a and 1b:  Structures of the effects of the H275Y and N295S mutations.

X-ray structures of complexes of oseltamivir (a) and zanamivir (b) with wild-type (yellow) and mutant (green)  N1 NAs superimposed, showing the effects of the H275Y (N1 numbering) substitution on drug interaction (Collins et al., 2009 ).

Figure 2:  Structures of the effects of the N295S mutations.

 X-ray structures of complexes of oseltamivir with wild-type (yellow) and mutant (green) N1 NAs showing the effect of the N295S (N1 numbering) substitution on drug interaction (Collins et al., 2009).