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Intra-host viral variability in children clinically infected with H1N1 (2009) pandemic influenza

https://doi.org/10.1016/j.meegid.2015.04.009Get rights and content

Highlights

  • Intra-host viral variability was studied in patients infected with H1H1 influenza.

  • One three-week old toddler harboured a mixed infection.

  • This mixed infection originated from complex and rapid local reassortment phenomena.

  • Mutations conferring resistance to neuraminidase inhibitors were suspected.

  • Technical challenges in interpretation of NGS data are discussed.

Abstract

Recent in-depth genetic analyses of influenza A virus samples have revealed patterns of intra-host viral genetic variability in a variety of relevant systems. These have included laboratory infected poultry, horses, pigs, chicken eggs and swine respiratory cells, as well as naturally infected poultry and horses. In humans, next generation sequencing techniques have enabled the study of genetic variability at specific positions of the viral genome. The present study investigated how 454 pyrosequencing could help unravel intra-host genetic diversity patterns on the full-length viral hæmagglutinin and neuraminidase genes from human H1N1 (2009) pandemic influenza clinical cases. This approach revealed unexpected patterns of co-infection in a 3-week old toddler, arising from rapid and complex reassortment phenomena on a local epidemiological scale. It also suggested the possible existence of very low frequency mutants resistant to neuraminidase inhibitors in two untreated patients. As well as revealing patterns of intra-host viral variability, this report highlights technical challenges in the appraisal of scientifically and medically relevant topics such as the natural occurrence of homologous recombination or very low frequency drug-resistant variants in influenza virus populations.

Introduction

Influenza A viruses probably exist within individual hosts as swarms of many variant viruses whose genomes closely conform to a population consensus sequence. This was highlighted by recent in-depth analyses of samples revealing patterns of intra-host viral genetic variability in a variety of systems. These have included laboratory infected poultry (Iqbal et al., 2009), horses (Murcia et al., 2010, Murcia et al., 2013), pigs (Murcia et al., 2012), chicken eggs and swine respiratory cells (Bourret et al., 2013) as well as naturally infected poultry (Croville et al., 2012, Monne et al., 2014) and horses (Hughes et al., 2012). In humans, the emergence of the 2009 H1N1 pandemic strain has coincided in time with the increased use of next generation sequencing techniques to investigate intra-host viral variability. Such techniques have often been used to assess the occurrence of polymorphisms at specific positions of the viral genome. For instance, a customized pyrosequencing assay was specifically designed to detect intra-host polymorphisms at hæmagglutinin amino acid 222 (H1 numbering excluding the signal peptide), deemed to alter receptor binding (Levine et al., 2011). In an epidemiological study, intra-host polymorphism at the same site was positively correlated with severity of the associated clinical condition (Resende et al., 2014). Others have studied the emergence of an oseltamivir resistance mutation in the neuraminidase gene and distinct neuraminidase haplotypes over the course of a prolonged infection in an immunocompromised patient (Ghedin et al., 2011). Finally, some reported on the emergence of the same oseltamivir resistance mutation (H275Y, N1 numbering) in a patient suspected to have been infected by a donor harbouring a pure oseltamivir-sensitive viral population (Fordyce et al., 2013).

The influenza A virus hæmagglutinin (HA) and neuraminidase (NA) proteins have essential biological functions and are considered the most protective antigens and most variable proteins of this virus (Ellebedy and Webby, 2009). Here, we applied next-generation sequencing on a limited number of clinical specimens to unravel intra-host viral genetic diversity patterns over the complete hæmagglutinin and neuraminidase genes in young patients clinically infected with H1N1 pandemic viruses.

Section snippets

Case history and sampling

Seven patients aged 3 weeks–23 months were admitted at Toulouse children hospital between 26th December 2010 and 16th January 2011 for clinical signs compatible with influenza (Table 1). These patients were confirmed with influenza A virus infection using the RealTime Ready Influenza A/H1N1 Detection Set (Roche diagnostics, Meylan, France) as described in Mansuy et al. (2012). Nasal swabs were taken 0–5 days after the onset of illness, except for one patient for which the interval was 34 days and

Coverage overview

We used 454 pyrosequencing to describe intra-host viral variability patterns in the hæmagglutinin (HA) and neuraminidase (NA) genes of influenza virus samples from seven H1N1 (2009) pandemic patients aged 3 weeks–23 months. Mean read length obtained was 436.6 nucleotides. After complete sequence clean-up and bioinformatic treatment (cf. Section 2), 12,461,774 and 20,615,839 nucleotides were included in the viral genome analyses of the HA and NA genes respectively. Sequences obtained were mapped

Discussion

We investigated intra-host viral variability in seven children infected with the influenza A H1N1 (2009) pandemic virus using pyrosequencing, with the aim to provide a description of some intra-host viral variability patterns in these human patients. Global levels of intra-host viral diversity were not clearly correlated with age in these very young patients, though a dataset consisting of a larger number of patients of different ages would provide greater statistical power for such analysis.

As

Acknowledgement

We are grateful to Laurence Tiley for comments on the manuscript. This work was funded by the French Région Midi-Pyrénées, Ministry of Agriculture, and INRA. The funding sources had no involvement in study design, collection, analysis and interpretation of data, writing of the report, or decision to submit the article for publication.

The authors declare that there are no competing interests.

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