Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia

Abstract

Background: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported.

Methods: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses.

Results: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution.

Conclusions: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Figure 1.

Map of the Hunter New England (HNE) health district and Australia. HNE health district is colored in red.

Figure 2.

Number of laboratory-confirmed influenza positive specimens detected in the Hunter New England health district during 2011.

Figure 3.

Frequency of detection of oseltamivir-resistant H275Y variants in Australia in 2011. A red dot represents the sample date of an individual oseltamivir-resistant A(H1N1)pdm09 H275Y case, and a blue dot indicates the sample date of an individual oseltamivir-sensitive A(H1N1)pdm09 case detected in the states and territories of Australia in 2011.

Figure 4.

Phylogenetic trees of hemagglutinin (A) and neuraminidase (B) sequences of oseltamivir-resistant H275Y variants and oseltamivir-sensitive wild type A(H1N1)pdm09 viruses. Hunter New England health district and other NSW and Western Australian H275Y variants are shown in red, sporadic H275Y mutants from other regions in green, and Hunter New England health district sensitive viruses in blue. Amino acid mutations common to each clade and bootstrap confidence values >75 are indicated on the trees.

Figure 5.

All human A(H1N1)pdm09 NA sequences containing date information (at least year/month) were downloaded from the National Center for Biotechnology Information influenza virus resource [41] (http://www.ncbi.nlm.nih.gov/genomes/FLU/) and GISAID (http://www.gisaid.org) and merged with the ones reported in this study (keeping only 1 per unique strain identifier). Sequences shorter than 90% of the median length are considered as fragmentary and were removed from the analysis. The resulting 8085 sequences were then aligned with the multiple sequence alignment program MAFFT [42] and mutation frequencies relative to the vaccine reference strain A/California/07/2009 counted with a custom perl script. All mutations with at least 50 occurrences since March 2009 and global frequencies >20% in any month are shown, plus L415M, which is characteristic for an outbreak with H275Y in Japan in January. Data collected in the month of March 2009 were merged with April 2009, while data collected from September 2011 were merged with August 2011 as there were less than 10 sequences in those months.

Figure 6.

Computational structural analysis of combinations of candidate permissive mutations in the neuraminidase (NA). Mutations were modeled with FoldX in the A(H1N1)pdm09 NA crystal structure (PDB:3nss) after minimization and using 5 repetitions.