Dataset Information

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus.

ABSTRACT: Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, zanamivir, and peramivir. Replicative capacity was analyzed by viral yield assays in ST6GalI-MDCK cells. Infectivity and transmission of the WT, H275Y, and I223V-H275Y recombinant viruses were evaluated in ferrets. As expected, the H275Y mutation conferred resistance to oseltamivir (982-fold) and peramivir (661-fold) compared to the drug-susceptible recombinant WT. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold), whereas the I223V virus had reduced susceptibility to oseltamivir (6-fold) only. Interestingly, enhanced levels of resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir (1,647-, 17,347-, and 16-fold increases in IC(50)s, respectively) were observed for the I223R-H275Y recombinant, while the I223V-H275Y mutant exhibited 1,733-, 2,707-, and 2-fold increases in respective IC(50)s. The I223R and I223V changes were associated with equivalent or higher viral titers in vitro compared to the recombinant WT. Infectivity and transmissibility in ferrets were comparable between the recombinant WT and the H275Y or I223V-H275Y recombinants. In conclusion, amino acid changes at residue I223 may alter the NAI susceptibilities of pH1N1 variants without compromising fitness. Consequently, I223R and I223V mutations, alone or with H275Y, need to be thoroughly monitored.

SUBMITTER: Pizzorno A

PROVIDER: S-EPMC3294894 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus.

Pizzorno Andrés A Abed Yacine Y Bouhy Xavier X Beaulieu Edith E Mallett Corey C Russell Rupert R Boivin Guy G

Antimicrobial agents and chemotherapy 20111227 3

Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, ...[more]

PMID: 22203589

Similar Datasets

Project description:During the first wave of the 2009 pandemic, caused by a H1N1 influenza virus (pH1N1) of swine origin, antivirals were the only form of therapeutic available to control the proliferation of disease until the conventional strain-matched vaccine was produced. Oseltamivir is an antiviral that inhibits the sialidase activity of the viral neuraminidase (NA) protein and was shown to be effective against pH1N1 viruses in ferrets. Furthermore, it was used in humans to treat infections during the pandemic and is still used for current infections without reported complication or exacerbation of illness. However, in an evaluation of the effectiveness of oseltamivir against pH1N1 infection, we unexpectedly observed an exacerbation of disease in virus-infected mice treated with oseltamivir, transforming an otherwise mild illness into one with high morbidity and mortality. In contrast, an identical treatment regime alleviated all signs of illness in mice infected with the pathogenic mouse-adapted virus A/WSN/33 (H1N1). The worsened clinical outcome with pH1N1 viruses occurred over a range of oseltamivir doses and treatment schedules and was directly linked to a reduction in NA enzymatic activity. Our results suggest that the suppression of NA activity with antivirals may exacerbate disease in a host-dependent manner by increasing replicative fitness in viruses that are not optimally adapted for replication in that host. IMPORTANCE:Here, we report that treatment of pH1N1-infected mice with oseltamivir enhanced disease progression, transforming a mild illness into a lethal infection. This raises a potential pitfall of using the mouse model for evaluation of the therapeutic efficacy of neuraminidase inhibitors. We show that antiviral efficacy determined in a single animal species may not represent treatment in humans and that caution should be used when interpreting the outcome. Furthermore, increased virulence due to oseltamivir treatment was the effect of a shift in the hemagglutinin (HA) and neuraminidase (NA) activity balance. This is the first study that has demonstrated that altering the HA/NA activity balance by reduction in NA activity can result in an increase in virulence in any animal model from nonpathogenic to lethal and the first to demonstrate a situation in which treatment with a NA activity inhibitor has an effect opposite to the intended therapeutic effect of ameliorating the infection.

Project description:Analyzing the evolutionary pattern of the influenza A(H1N1)pdm09 strain in different regions is important for understanding its diversification. We therefore conducted this study to elucidate the genetic variability and molecular evolution of the influenza A(H1N1)pdm09 strains that circulated during the 2009-2010 and 2010-2011 influenza seasons in Sendai, Japan. Nasopharyngeal swab specimens were collected from patients with influenza-like illnesses who visited outpatient clinics in Sendai City, Japan, from September 2009 to April 2011. A total of 75 isolates were selected from September 2009 to April 2011 to analyze the genetic changes in the entire hemagglutinin 1 (HA1) segment of the HA gene and the neuraminidase (NA) gene based on sequence analysis. Bayesian coalescent Markov chain Monte Carlo analyses of HA1 and NA gene sequences were performed for further analysis. High sequence identities were observed for HA1 and NA in influenza A(H1N1)pdm09, displaying 99.06 and 99.33 % nucleotide identities, respectively, with the A(H1N1)pdm09 vaccine strain A/California/07/2009. The substitution rates of nucleotides for HA1 in the 2009-2010 and 2010-2011 were 1.5 × 10-3 and 1.6 × 10-3 substitutions per site per year, respectively. Phylogenetic tree analysis demonstrated that Sendai isolates were clustered into global clade 7, which is characterized by an S203T mutation in the HA1 gene. Moreover, two distinct circulation clusters were present in the 2010-2011 season. Mutations were present in antigenic or receptor-binding domains of the HA1 segment, including A141V, S143G, S183P, S185T, and S203T. The Bayesian skyline plot model illustrated a steady rate for the maintenance of genetic diversity, followed by a slight increase in the later part of the 2010-2011 season. Selection analysis revealed that the HA1 (position 197) and NA (position 46) sites were under positive selection; however, no known mutation conferring resistance to NA inhibitors such as H275Y was observed. The effect on control of the influenza A(H1N1)pdm09 virus, including vaccine strain selection, requires continuous monitoring of the strain by genetic surveillance.

Project description:BackgroundExposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.Methods and resultsAntibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.ConclusionsThe NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1.

Dataset Information

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus.

Publications

Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets