Genetic variability of the neuraminidase gene of influenza A(H1N1)pdm09 viruses circulating from the 2012/2013 to 2017/2018 season in Vojvodina Province, Serbia

https://doi.org/10.1016/j.mcp.2020.101557Get rights and content

Highlights

  • Substantial genetic diversity of the NA gene of influenza A(H1N1)pdm09 viruses was found.

  • Permisive mutations V241, N369K, N386K and K432E were found.

  • Five potential T-cell epitopes within the NA were detected.

  • Fourteen sequences within the NA were identified as potential B-cell epitopes.

Introduction

Influenza A viruses are enveloped, single-stranded negative-sense RNA viruses. Their genome consists of eight distinct segments which code for at least one essential protein [1]. Sixth genomic segment codes the neuraminidase (NA), one of two major glycoproteins embedded in the virus envelope. The NA is an exosialidase which enzimatically cleaves the glycosidic bonds between the terminal sialic acid and adjacent sugar residue from cellular glycoprotein receptors and both of the virus surface glycoproteins [2]. It allows the detachment of the newly synthesized virus from the surface of infected cells and prevents the self-aggregation of viral particles [3]. Thereby, the NA is essential for viral progeny release and for spreading the infectious virus particles to new cells. The NA also promotes virus access to the epithelial cells by the degradation of respiratory mucus by cleaving sialylated mucin glycoconjugates [4].

There are 11 phylogenetically and antigenically distinct NA subtypes (N1–N11) but human influenza A viruses that currently circulate in the human population carry only two, N1 or N2 [5]. Influenza A(H1N1)pdm09 virus emerged in 2009 and since then it has been causing seasonal epidemics of flu along with A(H3N2) influenza subtype which was introduced in the human population in 1968.

Influenza A virus NA is a mushroom-shaped tetramer of four identical polypeptide chains [2]. In influenza A(H1N1)pdm09 viruses, each NA monomer consists of four domains: an short N-terminal cytoplasmic sequence (residues 1 to 6), a transmembrane domain (residues 7 to 34), a thin stalk (residues 35 to 82) and a globular “head” domain (residues 83 to 469) that carries the enzyme active site [6]. The catalytic site has highly conserved primary and secondary structure among all known influenza A virus NA subtypes. Eight amino acid residues (R118, D151, R152, R225, E277, R293, R368, Y402 in N1 numbering here and through the text) are involved in direct interaction with the substrate, while 11 framework residues (E119, R156, W179, S180, D/N199, I223, E228, H275, E278, N295, E425) are responsible for stabilizing the active site [7]. The catalytic site is surrounded by amino acid residues which form the antibody binding site (antigenic epitope).

At present, the NA is a target of the main class of antivirals currently available worldwide for the prevention and treatment of influenza. Those drugs bind the enzyme active site and inhibit NA enzymatic activity preventing effective viral replication. Neuraminidase inhibitors (NAIs) have a major role in controlling influenza virus infections in patients with or who are at greater risk for severe, complicated forms of illness [8]. According to WHO [9], annual epidemics of influenza result in about 3–5 million cases of severe illness, and about 290 000 to 650 000 deaths.

As a result of error-prone RNA-dependent RNA polymerase, influenza A virus genomic segments continuously acquire mutations which may lead to amino acid sequence changes. The influenza A virus surface glycoproteins hemagglutinin and NA are subjected to the strongest selective pressure of the immune system [10]. A gradual accumulation of amino acid substitutions in antigenic epitopes of the HA and NA (antigenic drift) enables the virus to escape from the immunity induced by vaccination or previous infection, while amino acid substitutions within or near the NA enzyme active site may lead to reduced NAIs sensitivity [11].

The aim of this study was to gain insight into the variability of the NA gene of influenza A(H1N1)pdm09 viruses collected during six post-pandemic seasons (2012/2013 - 2017/2018) in comparison with influenza A/California/07/2009 virus, which was one of the earliest virus isolated at the beginning of the 2009/2010 pandemic.

Section snippets

Specimen collection

Combined nasal and throat swab samples were simultaneously collected from patients suspected of having influenza virus infections using commercial polyester swabs (Copan, Italy) and analysed for the presence of influenza A(H1N1)pdm09 viruses at the Center of Virology at Institute of Public Health of Vojvodina, Serbia as part of the routine influenza surveillance program during the 2012/2013, 2013/2014, 2014/2015, 2015/2016, 2016/2017 and 2017/2018 winter seasons (from October 1 of one year to

Detection and phylogenetic analysis of influenza A(H1N1)pdm09 viruses

Out of the 3730 samples analysed during six consecutive seasons, 411 (11.1%) were positive for the presence of influenza A(H1N1)pdm09 viruses (Table 1). The frequencies of influenza A(H1N1)pdm09 viruses varied between 0.5% in season 2016/2017 and 19.3% in season 2015/2016.

The phylogenetic analysis of NA sequences showed that influenza A(H1N1)pdm09 viruses from same season clustered together with viruses from other European countries (Fig. 1). Except for one virus from season 2012/2013, all

Discussion

This study describes the genetic variability of the NA gene of influenza A(H1N1)pdm09 viruses over the course of six consecutive influenza seasons in the post-pandemic period. Phylogenetic analysis of NA sequences showed the circulation of genetic groups 6 and 7 in season 2012/2013 and emergence and dominance of new subgroups 6B and 6B.1 in the 2013/2014 and 2015/2016 season, respectively. Since the pandemic 2009/2010, the NA gene of influenza A(H1N1)pdm09 viruses has diverged into at least

Conclusions

Results of this study show substantial genetic diversity and continuous evolution of the NA gene of influenza A(H1N1)pdm09 viruses since the 2009/2010 pandemic. The accumulation of mutations within NA antigenic regions and the presence of permissive mutations raise the possibility of emergence and global spread of antigenically new strain with the NA H275Y mutation. In order to develop the best strategies for epidemic and pandemic preparedness and management, the genetic variability of the NA

Author's contribution

J.R. designed the study, performed the analysis of the data and wrote the manuscript. G.K., N.N., N.D. and A.P. performed the RT-PCR assays and interpreted the results. S.M. and M.R. coordinated and performed the sample collection and revised the manuscript. I.H.C. and V.P. revised the manuscript. All authors approved the final version of the manuscript submitted to the journal.

Declaration of competing interest

This study was partially supported by a research grant from Foundation for Influenza Epidemiology (FIE), which is partly founded by Sanofi Pasteur, under the Global Influenza Hospital Surveillance Network. FIE provided support in the form of a research funding and personal fees for J.R., S.M. and M.R. The sponsor did not participate in the study design, data collection, analysis and interpretation, in the writing of the manuscript or in the decision to submit the manuscript for publishing. All

Acknowledgements

Our special thanks to John McCauley PhD and all colleagues from The Crick Worldwide Influenza Centre, The Francis Crick Institute, United Kingdom, for performing a nucleic acid sequencing. We would also like to thank Ikuyo Takayama PhD from the National Institute of Infectious Diseases, Influenza Virus Research Center, Tokyo, Japan, for all the help during the implementation of the neuraminidase inhibitor susceptibility testing in our laboratory. We acknowledge the Foundation for Influenza

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References (38)

  • N. Korsun et al.

    Antigenic and genetic characterization of influenza viruses circulating in Bulgaria during the 2015/2016 season

    Infect. Genet. Evol.

    (2017)
  • J. Nordholm et al.

    Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases

    J. Biol. Chem.

    (2013)
  • W.K. Dawson et al.

    RNA structure interactions and ribonucleoprotein processes of the influenza A virus

    Briefings Funct. Genomics

    (2018)
  • J.L. McAuley et al.

    Influenza virus neuraminidase structure and functions

    Front. Microbiol.

    (2019)
  • S. Von Grafenstein et al.

    Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

    J. Biomol. Struct. Dyn.

    (2015)
  • X. Yang et al.

    A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus

    PloS One

    (2014)
  • (2019)
  • S. Maurer-Stroh et al.

    Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites

    Biol. Direct

    (2009)
  • H. Zhang et al.

    Evolutionary analysis of the antigenic determinant, glycosylation, and sialidase sites of neuraminidase from the human influenza A virus isolated in China from 1995 to 2012

    Biomed. Res.

    (2017)
  • T.M. Uyeki et al.

    Clinical practice guidelines by the infectious diseases society of America: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenza

    Clin. Infect. Dis.

    (2019)
  • World Health Organization

    Influenza (seasonal). Fact sheet, 15.04

  • A.D. Neverov et al.

    Coordinated evolution of influenza A surface proteins

    PLoS Genet.

    (2015)
  • M.C. Jespersen et al.

    BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes

    Nucleic Acids Res.

    (2017)
  • M. Bhasin et al.

    Prediction of CTL epitopes using QM, SVM and ANN techniques

    Vaccine

    (2004)
  • R. Gupta et al.

    Prediction of glycosylation across the human proteome and the correlation to protein function

    Pac. Symp. Biocomputing

    (2002)
  • S. Liu et al.

    Susceptibility of influenza A(H1N1)/pdm2009, seasonal A(H3N2) and B viruses to Oseltamivir in Guangdong, China between 2009 and 2014

    Sci. Rep.

    (2017)
  • D.V. da Silva et al.

    The influenza virus neuraminidase protein transmembrane and head domains have coevolved

    J. Virol.

    (2015)
  • M. Zanin et al.

    An amino acid in the stalk domain of N1 neuraminidase is critical for enzymatic activity

    J. Virol.

    (2017)
  • W. Zhu et al.

    From variation of influenza viral proteins to vaccine development

    Int. J. Mol. Sci.

    (2017)
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