Abstract
A zanamivir postapproval efficacy study was conducted in children (n = 279) in Japan during three influenza seasons. Pharyngeal swab specimens (n = 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors in in vitro assays or that were close to the receptor binding site. Their role in in vivo resistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.
INTRODUCTION
Influenza is a respiratory tract infection characterized by seasonal epidemics, widespread morbidity, and associated mortality, particularly in at-risk groups and during pandemics. Influenza pandemics are caused when a new strain of influenza A virus against which there is little or no existing immunity emerges in the human population and efficiently transmits from human to human.
The primary method for prevention of influenza is vaccination, but there is a role for treatment of infected individuals with antivirals. There are two classes of antivirals currently available for the treatment of influenza, adamantanes (adamantine and rimantadine) and neuraminidase (NA) inhibitors (NIs). There is widespread resistance to adamantanes, and therefore, treatment of influenza infection by this class of drugs is not currently recommended by the World Health Organization (WHO). There are four NA inhibitors currently licensed for treatment and prophylaxis of influenza infection, oseltamivir (Tamiflu), zanamivir (Relenza), peramivir (licensed for treatment in Japan and South Korea), and laninamivir (licensed for treatment in Japan and South Korea). Oseltamivir is administered orally, zanamivir and laninamivir are administered by oral inhalation, and peramivir is administered by injection. One of the factors that can impair the efficacy of NA inhibitors is the development of resistance. Zanamivir was designed to target the highly conserved active site of the influenza virus neuraminidase and is a close mimic of the natural substrate 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) (1). Zanamivir binds in the NA active site in a way similar to that of DANA, which appears to limit the potential for development of resistance to zanamivir (2–4). Resistance to zanamivir in immunocompetent patients is rare and has not been observed in more than 14,000 subjects participating in treatment and prophylaxis studies (5, 6). However, one resistant virus and three viruses with reduced susceptibility have been isolated from four immunocompromised patients treated with zanamivir. In one immunocompromised patient with influenza B virus infection treated for a prolonged period with inhaled zanamivir, amino acid substitutions in both hemagglutinin (HA) and NA were selected after 15 days of treatment (7). During the influenza pandemic of 2009-2010, there were three reports of the selection of viruses with reduced susceptibility to zanamivir in immunocompromised patients, two of whom were treated with an unlicensed formulation of intravenous zanamivir (as part of a named patient program) and one was treated with inhaled zanamivir (8–10). The variants harbored the I223R amino acid substitution in the NA, which conferred fold changes in susceptibility of 45 and 10 with oseltamivir and zanamivir, respectively.
In contrast, treatment with oseltamivir has resulted in widespread resistance. In Japan, studies have shown resistance in approximately 1% of adults and 4 to 18% of children treated with oseltamivir when infected with seasonal (A/H1N1, A/H3N2, and B) influenza virus (11, 12). Different oseltamivir resistance-associated amino acid substitutions have been identified in the different neuraminidase subtypes (N1, N2, and B). The most common oseltamivir resistance amino acid substitution in A/H1N1 viruses is H275Y (H274Y, N2 numbering), and such viruses were observed circulating within untreated subjects in many countries in the 2007-2008 influenza season and by the 2008-2009 Northern Hemisphere (NH) influenza season were at a level of 95% globally (13–15). Since the A/H1N1 pandemic started in 2009, there have been sporadic incidences of oseltamivir resistance in the pandemic H1N1 viruses (596 as of September 2011); all but one harbored the H275Y amino acid substitution (16). There have been clusters of infections with the resistant virus, and there have been four reported incidences of person-to-person transmission, but there was no sustained transmission between individuals, indicating that the variants in the pandemic H1N1 viruses are less fit than the wild-type virus (17–20). The global incidence of oseltamivir resistance in the pandemic H1N1 strain is approximately 1%; however, during the 2010-2011 Northern Hemisphere influenza season, there were several incidences of resistant virus infection in untreated patients, suggesting onward transmission of the resistant virus (21). Furthermore, during the 2011 Southern Hemisphere influenza season in Australia, there was a cluster of infections with virus with the H275Y amino acid substitution in untreated patients (22, 23). There is therefore cumulative evidence that the oseltamivir-resistant virus is becoming readily transmissible, and if the H275Y variant replaces all oseltamivir-sensitive viruses, as occurred with previous seasonal H1N1 virus, subsequent treatment options would be limited.
This study was carried out in order to ascertain if resistance to zanamivir was more readily selected in pediatric patients than adults. A postapproval study was conducted in Japan during three influenza seasons (2006 to 2009) to monitor for emergence of resistant influenza virus in pediatric patients treated with inhaled zanamivir. Efficacy analysis from this study has been reported elsewhere (24).
Analysis was carried out on pharyngeal swab specimens obtained at day 1 and during and after treatment. Susceptibility to zanamivir was determined using cultured virus. Population NA and HA sequencing and NA clonal analyses were carried out directly on swabs and/or cultured virus, to look for new or known resistance-associated amino acid substitutions.
MATERIALS AND METHODS
Compounds.
Zanamivir was provided by GlaxoSmithKline, Research and Development (Stevenage, United Kingdom).
Viruses and cell cultures.
A total of 279 pediatric patients in Japan (100 patients in 2006-2007; 79 patients in 2007-2008; 100 patients in 2008-2009) were enrolled in the study and were treated with 10 mg of inhaled zanamivir twice daily for 5 days. Viruses were isolated from pharyngeal swab specimens taken on day 1 (prior to treatment), during treatment (days 2 to 5), and at posttreatment time points (days 6 to 9), propagated in the absence of zanamivir in Madin-Darby canine kidney (MDCK) cells by standard techniques, and stored at −80°C prior to susceptibility analysis (25).
Study procedures.
The study was conducted in accordance with all applicable regulatory requirements, including the principles of the Declaration of Helsinki (1996). Before commencement of the study, all relevant study documentation was reviewed and approved by an ethics committee/institutional review board, and all subjects were provided with study information. Written consent was obtained from the legally authorized representative of all pediatric subjects.
NA activity inhibition assay.
Susceptibility to zanamivir was carried out on MDCK cell culture supernatants using an NA Star influenza neuraminidase inhibitor resistance detection kit as described by the manufacturer (Applied Biosystems). Viruses with a fold change in the 50% inhibitory concentration (IC50) that was 2-fold to 10-fold the IC50 for a sensitive reference strain were considered to have reduced susceptibility, and those with a fold change of >10 were considered resistant.
Virus gene sequence analysis.
Viral RNA was extracted from pharyngeal swabs using guanidinium isothiocyanate as described previously (25, 26). The NA and HA1 genes were amplified by reverse transcription-PCR. RNA was reverse transcribed using SuperScript II reverse transcriptase (Life Technologies Ltd., Paisley, United Kingdom) and gene-specific primers and amplified using two rounds of PCR with Pfx Platinum DNA polymerase (Life Technologies) and gene-specific primers. PCR products were sequenced using gene-specific primers. Primer sequences can be provided on request. Amino acid substitutions are shown in relation to the consensus sequence from the respective subtype obtained from the first season of this study. N2 numbering is used throughout, except where specified.
Clonal analysis.
PCR products were cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen) according to the manufacturer's protocol and sequenced with M13 forward and reverse primers. The mutation rate of the minority species was calculated by the following calculation: mutation rate of NA mutations = 1/[number of clones analyzed × (PCR1 + PCR2)], where PCR1 is the number of nucleotides amplified during the 1st-round PCR (influenza A/H1N1 virus = 1,408; influenza B virus = 1,396; influenza A/H3N2 virus = 1,424) × number of 1st-round PCR amplification cycles (n = 35), and PCR2 is the number of nucleotides amplified during the 2nd-round PCR (influenza A/H1N1 virus = 1,380; influenza B virus = 1,381; influenza A/H3N2 virus = 1398) × number of 2nd-round PCR amplification cycles.
Nucleotide sequence accession numbers.
The GenBank accession numbers of the NA and HA sequences from all viruses analyzed in this study are KC457353 to KC460206.
RESULTS
Samples analyzed.
The numbers of samples analyzed by susceptibility assays, NA sequencing, and HA sequencing are summarized in Table 1.
Table 1.
Number of swabs analyzed and results obtained for virus cultured for susceptibility using NA enzyme assay and genotyping directly from swabs for the NA and HA genes
| Parameter | No. of swabs analyzed/no. takena | No. of subjects with matched samplesb/total no. | No. of treatment swabs analyzed/no. taken | No. of swabs analyzed/no. taken on: |
No. of swabs with indicated virus/no. taken |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Day 1 | Day 2c | Day3c | Day 4c | Day 5c | Day 6/7d | Day 8/9d | Influenza A/H1N1 virus | Influenza A/H3N2 virus | Influenza B virus | ||||
| NA susceptibility | 371/714 | 108/255 | 119/437 | 252/277 | 39/68 | 49/123 | 19/70 | 7/38 | 5/95 | 0/38 | 166/391 | 105/174 | 100/149 |
| NA genotypes | 484/714 | 214/277 | 229/437 | 255/277 | 61/68 | 80/123 | 37/70 | 20/38 | 26/95 | 5/38 | 250/391 | 126/174 | 108/149 |
| HA genotypes | 413/714 | 135/277 | 175/437 | 238/277 | 56/68 | 63/123 | 25/70 | 15/38 | 14/95 | 2/38 | 186/391 | 120/174 | 107/149 |
Includes 5 untyped swab specimens from 2 subjects.
Matched samples are a baseline and a during-treatment or posttreatment sample from the same subject.
During treatment.
Posttreatment.
Virus susceptibility analysis.
Virus susceptibility to zanamivir was carried out on all cultured viruses. Samples from 24 subjects out of 279 could not be cultured and therefore were not analyzed phenotypically.
A total of 371 cultured viruses from 255 subjects (119 during/after treatment from 111 subjects) were analyzed (Table 1). IC50s were obtained for all samples (Table 2). For samples isolated in the 2006-2007 season, the type A/H1N1 isolates had a mean IC50 of 1.96 ± 5.27 nM, the type A/H3N2 isolates had a mean IC50 of 1.22 ± 0.65 nM, and the type B isolates had a mean IC50 of 3.95 ± 1.67 nM. One influenza A/H1N1 virus isolated at day 1 (before starting treatment) had fold changes in susceptibility to zanamivir and oseltamivir of 46 and 2.7, respectively. For samples isolated in the 2007-2008 season, the A/H1N1 isolates had a mean IC50 of 7.88 ± 44.06 nM and the B isolates had a mean IC50 of 4.8 ± 1.12 nM. Of the 371 viruses, 2 A/H1N1 viruses isolated on day 1 and day 2 (virus was not isolated after day 2) during the 2007-2008 influenza season were found to be resistant, with IC50s of 280.07 and 289.2 nM, and these are discussed in more detail below. The mean IC50 for A/H1N1 in this season, excluding these 2 resistant viruses, was 0.96 ± 1.41 nM. For samples isolated in the 2008-2009 season, the A/H1N1 isolates had a mean IC50 of 0.87 ± 0.78 nM, the A/H3N2 isolates had a mean IC50 of 1.07 ± 0.79 nM, and the B isolates had a mean IC50 of 3.92 ± 1.45 nM (Table 2).
Table 2.
Zanamivir susceptibilities determined using the NA Star assay for the different influenza virus subtypes isolated by cell culture
| Virus subtype | IC50 (nM) in the following influenza seasona: |
Total no. of isolates | |||||
|---|---|---|---|---|---|---|---|
| 2006-2007 |
2007-2008 |
2008-2009 |
|||||
| Range | Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | ||
| A/H1N1 | 0.21–19.49 | 1.96 ± 5.27 (13) | 0.08–289.2 | 7.88 ± 44.06 (82)b | 0.22–5.05 | 0.87 ± 0.78 (71) | 166 |
| A/H3N2 | 0.43–5.61 | 1.22 ± 0.65 (72) | NAc | NA (0) | 0.23–4.63 | 1.07 ± 0.79 (33) | 105 |
| B | 1.47–10.87 | 3.95 ± 1.67 (71) | 4.01–5.59 | 4.8 ± 1.12 (2) | 1.58–9.16 | 3.92 ± 1.45 (27) | 100 |
Data in parentheses represent numbers of isolates.
The mean IC50, excluding resistant viruses, was 0.96 ± 1.41 nM; see Table 3.
NA, not applicable.
NA gene sequencing.
Of the 279 subjects enrolled in the study, viruses from 2 subjects (5 swab specimens) were untyped and were not analyzed by genotypic analysis. A total of 484 NA sequences (250 A/H1N1; 126 A/H3N2; 108 B) were obtained from 714 swab specimens from 277 subjects (229 during/after treatment from 181 subjects) (Table 1). There were 214 subjects with matched day 1 and posttreatment swabs.
Results from this NA sequencing included those for the three viruses (A/H1N1) described above which showed reduced susceptibility to zanamivir at day 1 (before commencing treatment). The NA sequence from virus isolated from subject 1 (2006-2007) revealed an amino acid substitution at N74S (N70S by N1 numbering) and a 31- to 46-fold shift in susceptibility. The NA sequence for two viruses (day 1 and day 2) from subject 2 (2007/2008) revealed an amino acid substitution at Q136K (fold shifts, 292 and 301) (Table 3). The Q136K amino acid substitution was detected only in cultured virus and not in the swab, indicating that the amino acid substitution arose during in vitro passage (Table 3).
Table 3.
Genotypic (NA) and phenotypic (NA Star assay) analysis of resistant influenza A/H1N1 viruses
| Subject no. | Season | Visit (day) | Source | ZMV |
OSV |
Amino acid substitutions (N2 numbering)b | ||
|---|---|---|---|---|---|---|---|---|
| IC50 (nM) | FCa | IC50 (nM) | FC | |||||
| 1 | 2006-2007 | 1 | Swab | NAd | NA | NA | NA | N74S, P82S, K130R, I187 M, G248R, M266I, T286I, N347D, G357D, I370L, I397V, I454Tc |
| 1 | 2006-2007 | 1 | Culture | 19.5e | 46e | 1.8 | 2.7 | N74S, P82S, K130R, I187 M, G248R, M266I, T286I, N347D, G357D, I370L, I397V, I454Tc |
| 2 | 2007-2008 | 1 | Swab | NA | NA | NA | NA | A86V, V94I, N208K, Q216K, G331R, S452G |
| 2 | 2007-2008 | 2 | Swab | NA | NA | NA | NA | A86V, V94I, N208K, Q216K, G331R, S452G |
| 2 | 2007-2008 | 1 | Culture | 280.7 | 292 | 0.62 | 0.84 | A86V, V94I, Q136K, N208K, Q216K, G331R, S452G |
| 2 | 2007-2008 | 2 | Culture | 289.2 | 300 | 0.59 | 0.80 | A86V, V94I, Q136K, N208K, Q216K, G331R, S452G |
FC, fold change.
Resistance-associated amino acid substitutions are in bold.
Four viruses with the same sequence but without N74S did not show reduced susceptibility to zanamivir.
NA, not applicable.
On retesting, the IC50 was 12.9 nM and the fold change was 31.
Results from this NA sequencing for the remainder of the swabs showed that there were 12 subjects with treatment-emergent resistance-associated amino acid substitutions, in 1 of whom the substitution occurred close to the NA active site and in 3 of whom they were nonemergent resistance-associated amino acid substitutions present in day 1 samples (Table 4). Three published resistance-associated amino acid substitutions (11, 27) were detected within the NA before, during, or after treatment with zanamivir: G248R, N294K, and Y155H (N2 numbering; Table 4). The G248R amino acid substitution was present in all viruses isolated from 3 subjects (H1N1), including at day 1, but is reported to give rise to resistance only if present with the I266V substitution, which was not identified in virus from these subjects. Viruses from two subjects harboring the G248R amino acid substitution did not show elevated IC50s. One virus from subject 1 harbored the G248R along with the N74S amino acid substitution and had an elevated IC50, as discussed above. One virus analyzed in this study contained an amino acid substitution at position 294 (N294K) of NA (N2 numbering). The N294S amino acid substitution is a recognized resistance-associated amino acid substitution and has been detected in oseltamivir-treated patients infected with influenza A/H3N2 and A/H5N1 viruses (11, 28) and also prior to treatment in a patient infected with influenza B virus (29). The N294S amino acid substitution has been shown to give high-level resistance to oseltamivir in influenza A/H1N1, A/H1N1pdm2009, and A/H3N2 viruses, decreased susceptibility to oseltamivir in A/H5N1 and B viruses, and decreased susceptibility to zanamivir in A/H1N1, A/H3N2, and H5N1 viruses (11, 28, 30, 31). The amino acid substitution detected in this study was N294K in an H1N1 virus isolate recovered on day 7 after initiating treatment and was not present on day 1. Virus could not be cultured from this sample for susceptibility monitoring. The N294K substitution has never previously been identified as a zanamivir resistance-associated amino acid substitution; however, data presented here cannot preclude the possibility that this amino acid substitution confers resistance to zanamivir. Two influenza A H3N2 viruses recovered from two subjects on day 1 and day 3 possessed the Y155H amino acid substitution, which has previously been implicated in resistance to NIs in one influenza A/H1N1 virus isolate (27). The Y155H amino acid substitution has been shown to give rise to resistance to oseltamivir and zanamivir in one H1N1 virus isolate analyzed during surveillance studies but not in H3N2 viruses (27). In this study, a cultured H1N1 virus with the Y155H amino acid substitution did not have an elevated IC50 with zanamivir (0.61 nM). One day 6 virus had a deletion of 93 amino acids between positions 110 and 203 that was not present on day 1. This region of the NA encompasses part of the active site, and deletions in this region would affect the NA activity of the enzyme.
Table 4.
Emergent and resistance-associated amino acid substitutions detected in NA of influenza virus isolates
| Subject no. | Season | Virus type/subtype | IC50 (nM) | Incidence | Amino acid substitution(s) (N2 numbering) |
|---|---|---|---|---|---|
| 3, 4 | 2006-2007 | H1N1 | 0.5–1.0a | Nonemergent | G248Rc |
| 5 | 2006-2007 | B | 1.9–4.8b | Emergent, day 7 | L98L/F |
| 6 | 2007-2008 | H1N1 | NCe | Emergent, day 7 | T69T/I |
| 7 | 2007-2008 | H1N1 | NC | Emergent, day 3 | S367N |
| 8 | 2007-2008 | H1N1 | NC | Emergent, day 3 | S195Y |
| 9 | 2008-2009 | H1N1 | NC | Emergent, day 7 | N294Kd |
| 10 | 2008-2009 | H1N1 | NC | Emergent, day 6 | DEL110-202 |
| 11 | 2008-2009 | H1N1 | NC | Emergent, day 4 | P380N |
| 12 | 2008-2009 | H1N1 | NC | Emergent, day 7 | Y70C |
| 13 | 2008-2009 | H1N1 | 0.22 | Emergent, day 3 | V114I, E229G |
| 14 | 2008-2009 | H1N1 | NC | Emergent, day 2 | S105N |
| 15 | 2008-2009 | H1N1 | 1.7 | Emergent, day 6 | P389H |
| 16 | 2008-2009 | H3N2 | 0.61 | Nonemergent | Y155Hc |
| 17 | 2008-2009 | H3N2 | NC | Emergent, day 4 | G373R/G |
NA amino acid substitutions that were not present in the same subject's day 1 sample were detected following treatment with zanamivir at amino acid residues (N2 numbering) L98L/F in type B virus, amino acid residues T69T/I, Y70C, S105N, V114I, S195Y, E229G, N294K, S367N, P380N, and P389H in A/H1N1 virus, and amino acid residue G373R/G in A/H3N2 virus (Table 4). With the exception of N294K, none of the emergent amino acid substitutions were in the NA active site or have previously been observed in the clinic after treatment with NIs or during surveillance studies. The majority of the emergent variants could not be cultured, and therefore, no IC50s were determined. Two cultured viruses, one harboring the P389H amino acid substitution and one harboring both the V114I and E229G amino acid substitutions, had IC50s of 1.7 and 0.22 nM, respectively. One influenza B virus cultured on day 7 with a mixture of L98L/F had an IC50 range of 1.9 to 4.8 nM, which is within the normal range observed for influenza B viruses.
HA gene sequencing.
A total of 413 HA sequences (186 A/H1N1 sequences; 120 A/H3N2 sequences; 107 B sequences) were obtained from 714 swabs from 277 subjects (175 during/after treatment from 148 subjects) (Table 1). There were 166 subjects with matched day 1 and during/after treatment swab specimens. Seven HA amino acid substitutions identified in 18 subjects have previously been implicated in resistance to NIs (32) in in vitro assays (H3 numbering; E75K, A200V, and K222R in A/H1N1; G142R, S262N, and A304T in A/H3N2; N145S and N145I in B). In addition, two amino acid substitutions at positions 183 and 195 (H3 numbering) in the receptor binding site (RBS) of the HA were identified in separate viruses from two other subjects. HA amino acid substitutions from 15 of the subjects were present at day 1 and were therefore not selected as a result of drug pressure. A day 1 sample was not available for 5 subjects with HA amino acid substitutions, so it cannot be determined if they were treatment-emergent amino acid substitutions. Although the role of HA amino acid substitutions in resistance in vitro is well documented, their role in vivo is not well understood.
Clonal sequence analysis of neuraminidase gene.
Clonal sequence analysis of the neuraminidase gene was undertaken to determine if minority species of resistant viruses were present in samples taken on or after day 4 of treatment. A total of 1,682 clones from 91 swab specimens (90 subjects) were analyzed (Table 5). An additional 372 clones from control viruses isolated on or before day 3 from 19 subjects were analyzed (Table 5). Single clones from 12 subjects (from a total of 338 clones analyzed) were found to contain amino acid substitutions close to the NA active site on or after day 4 (Table 6).
Table 5.
Total number of samples analyzed by clonal analysis of the NA gene
| Season | Virus type | No. of swabs (no. of clones) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pretreatment (day 1) | During treatment |
Posttreatment |
Total analyzed | Total treated analyzed | Total controls analyzed | Total treated analyzed on day 4 and after | ||||||||
| Day 2 | Day 3 | Day 4 | Day 5 | Day 6 | Day 7 | Day 8 | Day 9 | |||||||
| 2006/07 | H1N1 | 2 (27) | 0 | 0 | 1 (27) | 1 (13) | 0 | 0 | 0 | 1 (24) | 5 (91) | 3 (64) | 2 (27) | 3 (64) |
| 2006/07 | H3N2 | 2 (47) | 2 (7) | 0 | 4 (13) | 6 (72) | 4 (17) | 0 | 0 | 0 | 18 (156) | 16 (109) | 4 (54) | 14 (102) |
| 2006/07 | B | 1 (30) | 0 | 1 (34) | 9 (90) | 4 (40) | 3 (33) | 1 (14) | 0 | 0 | 19 (241) | 18 (211) | 2 (64) | 17 (177) |
| 2007/08 | H1N1 | 7 (174) | 1 (0) | 0 | 9 (270) | 8 (209) | 3 (126) | 5 (146) | 5 (102) | 1 (31) | 39 (1,058) | 32 (884) | 7 (174) | 31 (884) |
| 2007/08 | B | 0 | 0 | 0 | 0 | 0 | 0 | 1 (0) | 0 | 0 | 1 (0) | 1 (0) | 0 | 1 (0) |
| 2008/09 | H1N1 | 2 (26) | 0 | 0 | 7 (150) | 0 | 4 (114) | 3 (60) | 1 (0) | 0 | 17 (350) | 15 (324) | 2 (26) | 15 (324) |
| 2008/09 | H3N2 | 0 | 0 | 0 | 3 (21) | 1 (1) | 2 (13) | 0 | 0 | 0 | 6 (35) | 6 (35) | 0 | 6 (35) |
| 2008/09 | B | 1 (27) | 0 | 0 | 4 (96) | 0 | 0 | 0 | 0 | 0 | 5 (123) | 4 (96) | 1 (27) | 5 (123) |
| Total | 15 (331) | 3 (7) | 1 (34) | 37 (667) | 20 (335) | 16 (303) | 10 (220) | 6 (102) | 2 (55) | 110 (2,054) | 95 (1,723) | 19 (372) | 91 (1,682) | |
Table 6.
Summary of potential NA resistance-associated amino acid substitutions identified by clonal analysis
| Season | Subject no. | Visit (day) | Virus subtype | No. of clones with mutation | Mutation rate | Amino acid substitution(s) (N2 numbering) |
|---|---|---|---|---|---|---|
| 2006-2007 | 18 | 1 | H1N1 | 0/17 | ||
| 2006-2007 | 18 | 4 | H1N1 | 1/27 | 0.4 × 10−6 | R152K |
| 2006-2007 | 19 | 1 | H3N2 | 0/26 | ||
| 2006-2007 | 19 | 5 | H3N2 | 1/14 | 0.8 × 10−6 | R224G |
| 2006-2007 | 20 | 1 | H3N2 | 0/21 | ||
| 2006-2007 | 20 | 5 | H3N2 | 1/16 | 0.7 × 10−6 | D198N |
| 2007-2008 | 21 | 1 | H1N1 | 0/27 | ||
| 2007-2008 | 21 | 6 | H1N1 | 1/33 | 0.3 × 10−6 | E227D |
| 2007-2008 | 21 | 6 | H1N1 | 1/33 | 0.3 × 10−6 | R224G |
| 2007-2008 | 22 | 1 | H1N1 | 0/23 | ||
| 2007-2008 | 22 | 8 | H1N1 | 1/33 | 0.3 × 10−6 | L134Q |
| 2007-2008 | 23 | 1 | H1N1 | 0/22 | ||
| 2007-2008 | 23 | 7 | H1N1 | 1/29 | 0.4 × 10−6 | E277G |
| 2007-2008 | 24 | 2 | H1N1 | NDa | ||
| 2007-2008 | 24 | 9 | H1N1 | 1/31 | 0.4 × 10−6 | R292G |
| 2007-2008 | 25 | 1 | H1N1 | 0/21 | ||
| 2007-2008 | 25 | 4 | H1N1 | 1/48 | 0.2 × 10−6 | R292S |
| 2007-2008 | 26 | 1 | H1N1 | 0/41 | ||
| 2007-2008 | 26 | 4 | H1N1 | 1/46 | 0.2 × 10−6 | L134P |
| 2008-2009 | 9 | 1 | H1N1 | 0/11 | ||
| 2008-2009 | 9 | 4 | H1N1 | 1/13 | 0.8 × 10−6 | L139P, K143N |
| 2008-2009 | 27 | 1 | H1N1 | 0/15 | ||
| 2008-2009 | 27 | 4 | H1N1 | 1/23 | 0.5 × 10−6 | Q136H |
| 2008-2009 | 28 | 1 | B | 0/27 | ||
| 2008-2009 | 28 | 4 | B | 1/25 | 0.4 × 10−6 | R224G |
ND, not done.
Two amino acid substitutions identified in clones from two subjects, R152K (A/H1N1) and D198N (A/H3N2), are known to be associated with reduced susceptibility and resistance in influenza B viruses (7, 33). Day 1 viruses from these 2 subjects were subjected to clonal analysis (17 and 22 clones, respectively) and did not possess the amino acid substitution detected in their respective during-treatment samples. Previous reverse genetics studies using an N2 background have indicated that the R152K amino acid substitution does not give rise to resistance (34) or gives low-level resistance but is unstable in N2 (35), but there are no data in an N1 background. It is not clear if the D198N substitution would produce resistance in influenza A virus strains, although the D198G substitution does reduce susceptibility to zanamivir by 6-fold in A/H1N1pdm2009 virus (31). Two clones from different subjects possessed amino acid substitutions at positions 136 and 143 of NA, which have been associated with resistance to NIs (27, 36, 37). The K143R amino acid substitution gives rise to resistance to oseltamivir, and the Q136K and Q136L amino acid substitutions give rise to resistance to zanamivir. It is not known if the amino acid substitutions identified in this study, Q136H and K143N, would give rise to resistance to NIs.
A further 9 clones from 9 subjects had amino acid substitutions in or close to the NA active site: L134Q, L134P, R224G, E227D, E277G, R292G, and R292S in type H1N1 virus, R224G in type H3N2 virus, and R224G in type B virus (N2 numbering). Clones from the day 1 isolate from the same subject did not contain the same amino acid substitution (Table 6). It is unclear whether these single amino acid substitutions could have been amplified as a result of drug pressure or represent chance mutations. The clinical implication of these amino acid substitutions is unclear because the majority of viruses with NA active-site amino acid substitutions have impaired fitness.
DISCUSSION
NIs are effective drugs for the treatment of influenza infections, but the development of resistance is a major factor that could reduce antiviral activity. Previous studies with oseltamivir have shown that the incidence of resistance may be higher in children than in adults. This study was conducted to investigate whether zanamivir-resistant viruses were selected in children during treatment. A study was conducted in Japan over three influenza seasons, and susceptibility analyses carried out on all isolated viruses. To date there have been no resistant isolates detected in immunocompetent patients treated with zanamivir. In this study, three resistant influenza A/H1N1 viruses were detected in samples from two subjects but were present at day 1 and therefore did not arise as a result of drug pressure. One pretreatment virus from one subject had reduced susceptibility with an IC50 of 19.49 nM (fold shift, 46) and harbored an N74S amino acid substitution (N70S by N1 numbering) in the NA which was outside the active site but gave rise to resistance to zanamivir. Two cultured viruses isolated on days 1 and 2 from another subject contained the amino acid substitution Q136K, which is close to the enzyme active site and confers high-level resistance to zanamivir, as observed previously (36, 38). This amino acid substitution was found only in cultured virus and not in the original clinical isolate and therefore was selected during in vitro passage rather than by drug pressure. This is consistent with findings from previous studies (36, 38). Although the Q136K amino acid substitution has not been detected in influenza A/H1N1 viruses from original clinical material, there has been one report of the amino acid substitution in a clinical specimen of an influenza A/H3N2 virus (39).
In this study, virus harboring the Q136K amino acid substitution was not observed in the swab but was found to be 100% mutant after just one passage in MDCK cells. This implies that the amino acid substitution may be present in the clinical isolate at a very low frequency and there is a strong selective pressure in favor of this amino acid substitution during growth in cell culture. However, the virus with the Q136K amino acid substitution selected in MDCK cells appeared to be unfit, as it did not grow to a high titer, in contrast to other isolates. Influenza A/H1N1 viruses with the Q136K amino acid substitution may be at a disadvantage as they have never been found in in vitro passage or directly from swabs from patients treated with zanamivir (40). In this study, there were an additional 15 viruses from 14 subjects that had NA sequences identical to those of the clinical isolates from which these 2 resistant viruses originated but for which resistant viruses were not selected in culture. Why only the two viruses from this subject identified here and not isolates with identical NA sequences from other patients underwent host cell selection is not clear, but the reason may be associated with other amino acid substitutions present in other genes. If there are compensatory amino acid substitutions that assist with the selection of Q136K, they do not appear to be in the NA. In previous studies, it was shown that viruses with the Q136K amino acid substitution can be transmitted between ferrets, and therefore, the possibility that the acquisition of compensatory amino acid substitutions with the Q136K substitution will give rise to a transmissible zanamivir-resistant virus in the future cannot be ruled out (36). In addition, the Q136L amino acid substitution was selected in vivo in zanamivir-treated ferrets infected with an influenza A/H5N1 virus (37). The virus harboring the Q136L amino acid substitution was resistant to oseltamivir and zanamivir, with fold changes in susceptibility of 350- and 26-fold, respectively. Structurally, it is not clear how the Q136K or Q136L amino acid substitution affects resistance to zanamivir, as position 136 is located at the base of the active site.
The susceptibilities of the zanamivir-sensitive isolates were comparable to data previously obtained for sensitive isolates and were in agreement with values obtained for the sensitive subtypes A/H1N1 (mean IC50, 0.76 nM), A/H3N2 (mean IC50, 1.82 nM), and B (mean IC50, 2.28 nM) (41).
NA sequences from samples from 12 subjects showed a difference between the day 1 sample and their posttreatment sample. Eleven out of 12 emergent amino acid substitutions identified here during zanamivir treatment have not been implicated in resistance to NIs and are not in the vicinity of the NA active site. The mutations were detected by PCR and sequencing, and viruses in only three of the samples could be cultured and were shown to be susceptible to zanamivir. The viruses in the majority of these samples could not be cultured, and therefore, the effect of these amino acid substitutions on susceptibility to zanamivir could not be determined. The clinical significance of the different amino acid substitutions identified is not clear, but most of these may have arisen due to natural variation.
One amino acid substitution, N294K, emerged during zanamivir treatment, and although N294S is a recognized resistance-associated amino acid substitution, it could not be determined if the N294K substitution affects susceptibility to zanamivir. Reverse genetics analysis will be carried out in the future to ascertain the effect, if any, of N294K on susceptibility.
It is noteworthy that all influenza A/H1N1 viruses isolated during the 2008-2009 influenza season harbored the H275Y NA amino acid substitution, which was expected from previous analyses of circulating oseltamivir-resistant viruses from that time (13–15). In contrast, all viruses isolated during the 2006-2007 and 2007-2008 influenza seasons analyzed in this study were wild type at position 275 of NA.
Sequencing of the HA gene showed that the majority of viruses analyzed did not contain amino acid substitutions in or near the RBS of the HA or amino acid substitutions in the HA that have previously been linked to resistance to zanamivir (32). Two amino acid substitutions at positions 179 and 191 in the RBS of the HA were present in day 1 viruses from different subjects and were therefore not selected as a result of drug pressure. HA amino acid substitutions located in the RBS have been shown to alter the affinity of the HA with the cellular receptor and can cause the virus to bypass the NA function completely in cell culture (42). Although HA amino acid substitutions are readily selected in vitro, the role of HA amino acid substitutions in resistance to NIs in vivo is not fully understood.
Although resistance to zanamivir is rare, resistance-associated amino acid substitutions may be present in clinical isolates as minority species. Population sequencing can identify minority species only down to 25% of the population, which is a major deficiency of this technique and may result in minority species not being identified. In studies with oseltamivir, resistant virus has generally been detected among viruses isolated on or after day 4 (43, 44). Clonal analysis of virus samples obtained on day 4 or beyond was therefore carried out in this study.
In this study, 13 single clones from during-treatment samples from 12 different subjects were found to contain an amino acid substitution in the NA active site or a previously identified resistance-associated amino acid substitution. All the amino acid substitutions are close to the NA active site and therefore may have the potential to give rise to zanamivir resistance. None of the amino acid substitutions were detected in the day 1 samples derived from the respective subjects. However, the fact that a single clone was detected in posttreatment samples does not mean that it was amplified by drug selection but means that it may have arisen by a single chance mutation.
The mutation rates of the clones are in the range 0.2 × 10−6 to 0.8 × 10−6 (Table 6). The error rate of platinum Pfx DNA polymerase used in the PCRs is 1.6 × 10−6 (45, 46). Therefore, the clonal mutation rate is comparable to the polymerase error rate. Consequently, it is possible that the mutations identified by clonal analysis arose as a result of polymerase error, although it cannot be completely ruled out that they arose during viral replication.
The clinical implication of the minority mutations is unclear because the majority of viruses with NA active-site amino acid substitutions have impaired viral fitness and the chance of an impaired virus producing a productive infection is limited (47). Minority species are important in the clinical outcome of patients with chronic infections, such as HIV infection. In a self-limiting acute disease such as influenza, the role of minority species is unclear, as the virus infection may become eradicated by immune clearance before an unfit virus subpopulation can become established. However, the presence of potential resistance-associated amino acid substitutions as minority species may be relevant in certain situations, for example, in the treatment of immunocompromised patients.
Further studies using reverse genetics in a known genetic background are warranted to analyze the phenotypes of some of the NA amino acid substitutions described in this paper.
ACKNOWLEDGMENTS
We thank GSK Japan and the clinical investigators for the design and coordination of the clinical study and the participating patients for their time and effort in providing samples for this study. Without all of their involvement, this study would not have been possible. We also acknowledge the work of Mitsubishi Chemical Japan for the culture of viruses from the swabs and for coordinating the transport of samples from Japan to the United Kingdom. In addition, we thank Sundip Modha, GlaxoSmithKline, Stevenage, United Kingdom, for carrying out purification of clonal plasmids using the Qiagen extraction robot.
Footnotes
Published ahead of print 18 January 2013
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