A Novel Neuraminidase Deletion Mutation Conferring Resistance to Oseltamivir in Clinical Influenza A/H3N2 Virus
- 1 Research Center in Infectious Diseases, Centre Hospitalier Universitaire de Québec-Centre Hospitalier de l'Université Laval and Laval University, Québec City, Québec, Canada
- Reprints or correspondence: Dr. Guy Boivin, CHUQ-CHUL, Rm. RC-709, 2705 blvd Laurier, Sainte-Foy, Québec, Canada G1V 4G2 (Guy.Boivin{at}crchul.ulaval.ca)or Dr. Yacine Abed, CHUQ-CHUL, Rm. RC-709, 2705 blvd Laurier, Sainte-Foy, Québec, Canada G1V 4G2 (Yacine.Abed{at}crchul.ulaval.ca).
Abstract
Neuraminidase (NA) mutations responsible for influenza resistance to oseltamivir vary according to the NA subtype; in influenza A/H3N2 viruses, NA-gene mutations occur predominantly at codons E119 and R292. In an oseltamivir-resistant influenza A/H3N2 virus isolated from an immunocompromised child after 107 days of cumulative treatment, the NA gene contained 3 aa substitutions (N146K, S219T, and A272V) and a 4-aa deletion (Del245–248); reversion mutation experiments using recombinantNAproteins determined that the deletion was the sole change responsible for resistance to oseltamivir. This study highlights the diversity of mechanisms of resistance to oseltamivir in clinical settings, reinforcing the need for novel anti-influenza strategies.
Influenza A viruses are a major public health problem worldwide. In immunocompromised hosts, the course of influenza infections is usually more severe, with prolonged viral shedding that could last several months [1–4]. Therefore, the use of potent anti-influenza agents may play an important role in the control of influenza infections in this population. Neuraminidase inhibitors (NAIs) such as inhaled zanamivir and orally administered oseltamivir phosphate are the cornerstone of anti-influenza therapy because the virus has a high level of resistance to the adamantanes (amantadine and rimantadine) and toxicity is associated with the latter. These 2 NAIs have demonstrated clinical benefits in the prevention and treatment of seasonal influenza infections [5]. Other investigational NAIs, including a cyclopentaneanalogue compound (peramivir) and a pyrrolidine-based drug (A-315675) also have been found to have inhibitory activities against influenza A and B viruses, both in vitro and in animal studies [6, 7]. As with other antiviral agents, there is a growing concern with regard to the emergence of NAI-resistant influenza viruses and their consequences in terms of treatment failure and viral transmission. Therefore, understanding the mechanisms of resistance to NAIs and implementing a surveillance program regarding resistance to NAIs are important for the preservation of long-lasting therapeutic benefits.
The incidence of resistance to oseltamivir, the most frequently prescribed NAI, has been low in clinical trials but has been found to be substantial in hospitalized children and immunocompromised patients [8]. Of greater concern, oseltamivir-resistant A/H1N1 viruses were recently detected worldwide in untreated immunocompetent individuals [9]. The type of neuraminidase (NA) mutations responsible for the NAI-resistance phenotype was found to vary according to the drug and the NA subtype [10]. For oseltamivir, NA-gene mutations at codons E119 and R292 are most frequently detected in influenza viruses of the N2 subtype, whereas the H274Y mutation predominates in the N1 subtype [8, 10]. So far, resistance to zanamivir has not been reported in clinical influenza A viruses. The present report of a clinical influenza A/H3N2 virus recovered from an immunocompromised child describes a novel NA mutation conferring resistance to oseltamivir.
Subject, Materials, and Methods
The detailed clinical history of the patient has been reported elsewhere [1]. In brief, severe combined immunodeficiency disease was diagnosed at age 4 months (in April 2002), and the child underwent allogeneic haploidentical stem-cell transplantation. Infection with influenza A virus was diagnosed in April 2005 and progressed to chronic pneumonitis of the lingula. The child initially was treated with oseltamivir (24 mg [2 mg/kg] twice a day) for 3 months but, because of both progression of respiratory symptoms and cultures persistently positive for influenza virus, was switched to amantadine (30 mg twice a day) and zanamivir (10–20 mg, via nebulizer, twice a day). The patient shed influenza virus for 1 year, after which viral cultures were found to be negative while the child was being treated with zanamivir.
Nasopharyngeal aspirates or brochoalveolar-lavage samples were tested for the presence of influenza viruses, by culturing the virus on Madin-Darby canine kidney (MDCK) cells; a maximum of 3 passages were performed prior to phenotypic and genotypic analyses. RNA was isolated from cell-culture supernatants by use of the QIAamp Viral RNA kit (Qiagen), and cDNA was synthesized by use of random hexamer primers (Amersham Pharmacia Biotech) and SuperScript II reverse transcriptase (GIBCO-BRL). Viral cDNA was used to amplify the NA and hemagglutinin (HA) genes, by PCR using Pfu Turbo Polymerase (Stratagene) and gene-specific primers. PCR products were directly sequenced and also were cloned by blunt-end ligation into the pBluescript II KS vector (Stratagene), with at least 8 recombinant plasmids carrying the NA gene being randomly selected and subjected to sequence analysis. Selected influenza viruses also were plaque-purified once before sequence analysis.
The NA gene of 2 clinical isolates of influenza A/H3N2—specifically, sample 2 recovered after 2 weeks of antiviral therapy and sample 21 recovered after 107 days of cumulative oseltamivir therapy—was amplified by PCR using primers containing a BsaI restriction-enzyme site, followed by ligation into the pHH21 plasmid linearized with the BsmBI restriction enzyme, allowing cloning of influenza genomic segments containing BsmBI or BsaI restriction sites (a gift from Dr. Earl Brown, University of Ottawa). The plasmid containing the NA gene of isolate 21, which, compared with the initial wild-type (WT) strain, harbored 3 substitutions (N146K, S219T, and A272V) and a 4-aa deletion (Del245–248), was used for site-directed mutagenesis experiments. Reversion of the 3 substitutions to the WT sequence was performed by use of appropriate primers and the QuickChange Site-Directed Mutagenesis kit (Stratagene), whereas reversion of the deletion was performed by the inverse PCR method and appropriate back-to-back primers. Plasmids were sequenced to ensure that unwanted mutations had not been introduced. For expression of recombinant NA proteins, 293T (human embryonic kidney) cells were cotransfected with 1 µg of each of the 4 expression plasmids (pCAGGS-PA, -PB1, -PB2, and -NP) of A/WSN/33 (H1N1) and the respective pHH21-NA plasmid. At 48 h after transfection, cells were treated with 0.02% EDTA in PBS and were harvested. The cells were resuspended in PBS containing CaCl2 at 3.5 mmol/L and were stored at −80 °C. Total proteins from transfected cells were quantified by the BCA protein assay (Pierce), and NA-inhibition assays were performed as described below, with an equivalent quantity of expressed proteins [11].
NA-inhibition assays were performed with 800–1200 fluorescence units of a plaque-purified virus or recombinant NA protein in the presence of serial half-log dilutions (0.038-10,540 nmol/L) of zanamivir, oseltamivir carboxylate (both synthesized by GlaxoSmithKline), peramivir/BCX-1812 (BioCryst), or A-315675 (Abbott Laboratories). Methylumbelliferyl-N-acetylneuraminic acid (Sigma) was used as the fluorescent substrate [1].
Results
A total of 17 isolates of influenza A/H3N2, from 27 nasopharyngeal aspirates and 2 bronchoalveolar-lavage samples, were recovered from the immunocompromised child during a period of 12 months (table 1). Most isolates of oseltamivir-resistant influenza that were recovered during and after oseltamivir therapy harbored the well-known resistance mutation E119V [1]. However, one isolate (sample 21), which was an A/California/7/2004-like strain recovered almost 3 months after the end of 107 days of cumulative oseltamivir therapy and while the patient was receiving nebulized zanamivir and orally administered amantadine, did not harbor the E119V mutation but, instead, contained 3 substitutions (N146K, S219T, and A272V) and a 4-aa deletion (Del245–248) in its NA gene, compared with the initial WT strain; on the other hand, both strains had identical HA genes. This mutant isolate was resistant to oseltamivir (the IC50 value was increased 175-fold, compared with that of the WT strain) but remained susceptible to zanamivir and peramivir and had reduced susceptibility to A-315675 (the IC50 value was increased 3-, 1- and 12-fold, respectively, compared with those of the WT strain) (table 2). A similar pattern of resistance to NAIs was seen for recombinant NA proteins expressed in 293T cells (table 2).
Table 1.
Genotypic characterization of influenza A/H3N2 viruses recovered from an immunocompromised child treated with oseltamivir, zanamivir, and amantadine.
Table 2.
Susceptibility to neuraminidase (NA) inhibitors, as assessed by NA-inhibition assays.
As shown in table 2, the reversion of substitutions at residues 146, 219, or 272 in the recombinant mutant protein (revertants 1, 2, and 3, respectively) to theWTsequence did not significantly alter the phenotype of resistance to oseltamivir and to A-315675; conversely, the addition of 4 aa, at codons 245–248, restored the phenotype of susceptibility (table 2).
To investigate in vitro the stability of the NA substitutions and deletion, the resistant strain was subjected to 4 passages in MDCK cells in the absence of drug pressure. Sequence analysis showed that the passaged mutant virus conserved the same NA genotype, with the substitutions and the deletion. When an MOI of 0.01 was used, virus yields of the mutant isolate inMDCKcells were 3- and 1.5-log lower than those of theWTstrain, during the early stages of the viral cycle (i.e., at 24 h and 36 h after infection) [11]; in contrast, the 2 virus strains generated similar titers at later stages (i.e., at 48, 60, and 72 h after infection) (data not shown).
Discussion
Previous studies have demonstrated that resistance to NAIs may result from mutations in the receptor-binding site of the HA protein and/or in the active center of theNAenzyme [8]. Besides these substitution mutations, massive deletions of 270 aa, including most residues that constitute the active center of the NA enzyme, have been identified also in NAI-resistant influenza A/H1N1 variants obtained after in vitro passages under NAI pressure and in a volunteer treated with peramivir [12, 13]. These massive deletions were found to alter NA enzymatic activity and resulted in a reduced dependence on NA. In the present report, we have described a novel oseltamivir-resistant influenza A/H3N2 variant with an NA gene that contains 3 substitutions (N146K, S219T, and A272V) and a small, 4-aa deletion (residues 245–248). This genotype was confirmed in plaque-purified viruses and by sequence analysis of cloned PCR products. It is unlikely that the NA-gene deletion was selected by in vitro passages, because it was found in 2 different isolates (samples 20 and 21) (table 1). Notably, this variant exhibited NA activity that was comparable to that of the WT strain, as demonstrated in NA enzymatic assays using both the virus and the recombinant NA proteins expressed in 293T cells. Also, this unusual genotype conferred a high level of resistance to oseltamivir—but not to zanamivir—when both the mutant isolate and the recombinant mutant NA protein were tested by NA-inhibition assays. Using reversion mutation experiments, we furthermore have demonstrated that the 4-aa deletion is the sole mutation responsible for the oseltamivir-resistance phenotype. We also introduced the 3 substitutions into the NA protein of influenza A/Hong Kong/1/68 (H3N2), and we found that there was no impact on the phenotype of resistance to oseltamivir (data not shown). Residues 146, 219, and 272 are not part of framework or catalytic residues of the influenza NA enzyme [14]. However, residue 146N is involved in an N-linked glycosylation site that is highly conserved in all influenza A and B viruses [14]. The loss of this glycosylation site has been found to impact the transport and/or folding of the NA protein of influenza A/Tokyo/3/67 (H3N2) [15]. Similarly, the deleted 4 aa (residues 245–248) in the resistant variant that we studied are not framework or catalytic residues. However, this deletion may have caused a conformational change that resulted in decreased binding of the NA enzyme to oseltamivir. Further structural- and binding-affinity experiments are needed to clarify the precise mechanisms by which this deletion alters the susceptibility to some, but not all, NAIs.
Antiviral therapy using the potent influenza NAIs is thought to play a major role in the immunocompromised host by reducing the duration of viral shedding, the risk of progression to pneumonia, and, possibly, the mortality rate associated with influenza. The claim that commercially available NAIs are efficacious in the treatment of influenza infections has been based mainly on clinical trials involving otherwise healthy children, teens, and young to middle-aged adults [5]. Only limited studies have assessed the use of NAIs in immunocompromised patients with regard to the development of drug resistance in the context of prolonged viral shedding [1, 3]. Elsewhere, we recently have described influenza A variants with dual resistance to amantadine, via the S31N mutation in the M2 gene, and to oseltamivir, via the common NA-gene E119V mutation [1]. In the present report, we have described, in the same immunocompromised child, a novel genotype conferring (via a small NA-gene deletion) resistance to oseltamivir. It is noteworthy that this NA mutant was detected after cessation of oseltamivir, while the patient was receiving zanamivir. Because the virus was not resistant to zanamivir (the IC50 values were increased ≤3-fold), the most likely explanation is that this deletion mutant was selected as a minor quasispecies during oseltamivir therapy, as also was the case for the E119V mutant in other samples. Its transience may be due to decreased fitness compared with that of theWTstrain. The present report highlights the variety of mechanisms via which influenza viruses escape drug pressure and reinforces the need to develop novel antiviral strategies, such as combination therapy, that may be particularly beneficial in the treatment of severely immunocompromised patients.
Footnotes
-
Potential conflicts of interest: none reported.
-
Financial support: Canadian Institutes of Health Research (research grant 69559 to G.B.).
- Received May 21, 2008.
- Accepted July 25, 2008.
- © 2008 by the Infectious Diseases Society of America
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