Phenotypic and genotypic profile of susceptibility to neuraminidase inhibitors of influenza A(H3) circulating in Portugal during 2016/2017 season

Background: The influenza antiviral surveillance is one of the key areas for influenza control. The neuraminidase inhibitors (NAI) play an important role in flu treatment especially for high risk patients. This study aims to determine the NAI susceptibility profile of influenza A(H3) detected during 2016/2017 season in Portugal and to evaluate the emergence of new variants by deep sequencing analysis related to NAI resistance. Methods: Nasopharyngeal swabs were collected from patients with influenza like illness (ILI) selected in primary care settings in the scope of the National Influenza Surveillance Program during 2016/2017 season. For 134 A(H3) viruses, isolated in MDCK-Siat1 cells, the phenotypic antiviral drug susceptibility assay to NAI (Oseltamivir and zanamivir) was performed. Viral RNA was extracted directly from biological samples and after multiplex PCR amplification, the whole genome was sequenced for 84 influenza A(H3) viruses by deep sequencing on a MiSeq platform. The neuraminidase gene sequences were assembled using a in-house multi-software pipeline with a mean depth of coverage of 1144x. Multiple gene alignments and mutational analysis was performed on MEGA software 6.0. All neuraminidase sequences were submitted to Flusurver(http://flusurver.bii.a-star.edu.sg/) in order to detect any mutation associated with susceptibility to neuraminidase inhibitors. Results: All 134 A(H3) strains had IC50 compatible with susceptibility to both NAI. The IC50 mean values were 0,30 (IQR 0,22-0,36) for oseltamivir and 0,49 (IQR 0,40-0,59) for zanamivir. The IC50 were unchanged for viruses that belonged to both co-circulating clades 3C.2a (A/Hong Kong/4801/2014-like) and 3C.2a1 (A/Bolzano/7/2016-like) although amino acid replacements were observed in NA. By deep sequencing analysis no variations were identified in any of the known resistant markers for NAI. Intrahost single nucleotide variants (iSNV) (frequency above 20% and under 95%) were detected in 4/84 (5%) viruses in nucleotide positions not related with NAI susceptibility. A further synonymous iSNV was observed in 23/84 (27%),with a frequency range 97,8-100,0%, at position 666 (amino acid 222). Curiously, non-synonymous changes affecting this codon ( I222V/L substitution) have been associated with reduced susceptibility to oseltamivir and NA activity, although wild-type I222 was observed in 100% of viruses in this study. Majority of these viruses belonged to the 3C.2a1clade(A/Bolzano/7/2016-like). Conclusions: All of the tested A(H3)viruses were susceptible to oseltamivir and zanamivir. Isolates possessing iSNV`s expressed the wild-type aa residue and had IC50 compatible with NAI susceptible. NAI`s should be considered a good therapeutic options for influenza A(H3) infection, prioritized to high risk groups. Whole genome deep sequencing, directly from the biological samples, proved to be a good laboratory method to monitor the emergence of iSNV that could lead to a resistant genotype. This approach could be used in management of influenza severe infections under NAI treatment.