A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1

Journal of Virological Methods 153 (2008) 134–141

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A new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza A viruses of subtype H1N1, H3N2, and H5N1 Susanne Duwe, Brunhilde Schweiger ∗ National Reference Centre for Influenza, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

a b s t r a c t Article history: Received 17 October 2007 Received in revised form 18 July 2008 Accepted 23 July 2008 Keywords: Influenza A Neuraminidase Antiviral resistance A/H5N1 Pyrosequencing

The neuraminidase of influenza viruses is the target of the inhibitors oseltamivir and zanamivir. Recent reports on influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAI) are a cause for concern. Several amino acid substitutions, each as a consequence of one single nucleotide mutation, are known to confer resistance to NAI. An increase of NAI-resistant viruses appears to be likely as a result of a wider application of NAI for treatment and prophylaxis of seasonal influenza infections. Monitoring the occurrence and spread of resistant viruses is an important task. Therefore, RT-PCR assays were developed with subsequent pyrosequencing analysis (PSQ-PCR). These assays allow a rapid, high-throughput and cost-effective screening of subtype A/H1N1, A/H3N2, and A/H5N1 viruses. Various specimens such as respiratory swabs, allantoic fluid, or cell-propagated viruses can be used and results are available within hours. Several A/H1N1, A/H3N2, and A/H5N1 viruses isolated from human and avian specimens were tested to evaluate the method. Positive controls encoding resistance-associated mutations were created using site-directed mutagenesis. The results obtained with these controls showed that the assay can discriminate clearly the wild-type virus from a mutant virus. The detection limit of minor virus variants within the viral quasispecies amounts to 10%. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Influenza virus is a negative strand RNA virus belonging to the family Orthomyxoviridae, which is divided into three types A, B, and C. Every year influenza A and B viral infections are a major cause of morbidity and mortality worldwide. In 1999, the Food and Drug Administration (FDA) of the USA approved the neuraminidase inhibitors (NAIs) zanamivir (RelenzaTM ) and oseltamivir (TamifluTM ) for therapy and prophylaxis of influenza infections. The target of these anti-influenza drugs is the enzyme activity of the neuraminidase of influenza A and B viruses. The neuraminidase enzyme acts extracellularly and plays an essential role in the release and spread of progeny viruses by cleaving the terminal sialic acid from cellular receptors recognized by the hemagglutinin. Oseltamivir and zanamivir were developed by drug design strategies and interfere with the normal function of the influenza neuraminidase by mimicking the natural substrate sialic acid. Neuraminidase inhibitors are active against all subtypes of influenza A and type B viruses including H5N1 viruses (Govorkova et al., 2001; Yen et al., 2005). Clinical studies of NAIs

∗ Corresponding author. Tel.: +49 30 187542456; fax: +49 30 187542605. E-mail address: [email protected] (B. Schweiger). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.07.017

indicated their ability to reduce duration of symptoms and risk of complications (Hayden and Aoki, 2005; Moscona, 2005). The orally bio-available oseltamivir has become rapidly the principal drug of choice for treating influenza and for pandemic stockpiling. These antiviral drugs may play an important role in the early phase of a future pandemic, when vaccines against the new strain are not yet available or as long as the available vaccine is in short supply. Recent reports on the incidence of neuraminidase inhibitor resistant influenza A viruses of subtype H3N2, H1N1, and H5N1, are a cause for concern. Resistance to neuraminidase inhibitors occurs by amino acid substitutions at several positions, depending on the influenza subtype and the antiviral compound used (McKimm-Breschkin, 2000; Abed et al., 2006, Table 1). Each amino acid substitution is based on a single nucleotide mutation within the neuraminidase gene. There are several reports on oseltamivirresistant viruses of subtype A/H3N2 and A/H1N1 that developed in vivo and in vitro (Kiso et al., 2004; Whitley et al., 2001; Ison et al., 2006). Neuraminidase inhibitor resistant viruses of subtype A/H5N1 have been isolated from at least three patients treated with oseltamivir (Le et al., 2005; de Jong et al., 2005). Multidrug-resistant viruses have also occurred, although this may be a rare event, shown by a year-long shedding of such viruses by an immunocompromised child (Baz et al., 2006).

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Table 1 PSQ-PCR primers for amplification and pyrosequencing of neuraminidase gene fragments encoding for resistance-associated amino acid substitutions Influenza virus subtype amplified/sequenced gene fragment A/New Caledonia/20/99 H1N1 NA-N1 fragment A N1H1274s N1H1274Bas N1H1274SPs NA-N1 fragment B N1H1292Bs N1H1292as N1H1292SPas

Primer sequence

Fragment position

Fragment size

Resistance-associated amino acid substitution

798–1135

337

H274Y

835–913

78

7–488

481

E119V

550–892

342

R292K, N294S

644–864

220

H274Y

5 -ATA gAg TTg AAT gCA CCC AAT TTT 5 -Biotin-CAT TAg gAT CCC AAA TCA TCT CA 5 -TgA ATg CAC CCA ATT T R292K, N294S

5 -Biotin-CTg gCA CAg TgA TgT gTg TAT g 5 -ACA CCC AAg gTC gAT TTg AAC 5 -ggT CgA TTT gAA CCA T

A/Wisconsin/67/05 H3N2 NA-N2 fragment A N2-P-7s BN2-P-488as N2-S3 NA-N2 fragment B N2-P-550 BN2-P-892 N2-S5

5 -AgCAggAgTgAAgATgAATCC 5 -Biotin-TCg ATA Agg ggT CCT ATC AT 5 -ggg ACA TCT ggg TgA 5 ATggTCCAgCTCAAgTTgTCA 5 - Biotin-ATA TCT ACg ATg ggC CTA TTg gA 5 -CCT ggT gTC AgA TgT g

A/Thailand/676/2005 H5N1 NA-H5N1 N1H5274Bs N1H5274as N1H5Pas

5 -Biotin-ATg gCT CTT gCT TTA CTg TAA TgA 5 -TTg AAA gAT ACC CAT ggC CTA TT 5 -CCC ATg gCC TAT TTg Ag

Monitoring the emergence and spread of resistant human or avian influenza viruses should, therefore, be part of ongoing influenza surveillance. A new, high-throughput, and time-saving method using pyrosequencing was designed, which allows the screening of single nucleotide substitutions in the neuraminidase gene of subtype NA-N1 and subtype NA-N2 known to confer drug resistance. The assays include RT-PCR to amplify specific regions covering those specific positions within the neuraminidase gene in respiratory specimens. Subsequent pyrosequencing with specific sequencing primers detects resistance-associated substitutions in a specific, rapid, and cost-effective manner with access to the results in real time. The pyrosequencing-procedure (Ronaghi et al., 1996) has been used successfully, for instance, to identify and monitor resistance-associated mutations of HIV (O’Meara et al., 2001; Hoffmann et al., 2007). 2. Materials and methods 2.1. Virus strains and clinical specimens Influenza A virus reference strains H1N1 A/New Caledonia/20/99 and H3N2 A/Wisconsin/67/05 were obtained from the WHO Collaborating Centre, London (Dr. Alan Hay) and propagated in embryonated eggs at 37 ◦ C. Clinical samples (nasal swabs) from patients with respiratory disease symptoms were collected between October 2005 and April 2008 from the German National Reference Centre for Influenza in Berlin and were provided mainly by physicians working in the influenza surveillance scheme (Arbeitsgemeinschaft Influenza, Szecsenyi et al., 1995). The specimens were collected using Virocult swabs (Medical Wire & Equipment, Corsham, England) containing transport medium and

were sent by mail without any refrigeration. The time in transit was between 1 and 4 days. The swabs were vortexed with 5 ml of medium. For virus propagation, 200 ␮l of virus suspension were inoculated onto confluent Madin-Darby canine kidney (MDCK) cells. The cells were maintained in rolling tubes containing serum-free minimum essential medium (Gibco BRL, Life Technologies GmbH, Karlsruhe, Germany) supplemented with 1.25 ␮g of trypsin per ml (Gibco BRL). The cultures were incubated at 33 ◦ C and were examined every day for detectable cytopathic effect. The medium was tested continuously for hemagglutination of guinea pig red cells (0.5% vol/vol). Every hemagglutination-positive culture was identified by using the classical hemagglutination inhibition (HI) procedure (Chakraverty, 1971). Briefly, specific antisera raised in ferrets were treated with receptor-destroying enzyme. The HI tests were carried out by using four hemagglutination units of virus and 1.0% (vol/vol) guinea pig red blood cells. Influenza virus strains of subtype H5N1 were obtained from the Friedrich-Loeffler-Institute, Isle of Riems, Germany and from Prof. Klenk, Institute of Virology, Marburg, Germany, respectively. The viruses were propagated either in MDCK cell culture or in embryonated chicken eggs. 2.2. Neuraminidase inhibitor Oseltamivir carboxylate was kindly provided by HoffmannLa Roche Ltd. The neuraminidase inhibitor was resuspended and diluted in sterile dd H2 O. 100 ␮M stock solutions were generated and stored at −20 ◦ C. Serial dilutions for susceptibility assays were made in MES buffer [32.5 mM morpholineethanesulfonic acid (Sigma–Aldrich), pH 6.5, and 4 mM CaCl2 ].

Table 2 Primers for site-directed mutagenesis of influenza A neuraminidase genes Neuraminidase subtype

Primer

Primer sequence

Resistance-associated codon

H3N2 H3N2 H5N1

N2-119T N2-292A/294G N1H5274Ys

5 -CAT CTg ggT gAC AAg AGT ACC TTA TgT gTC 5 -CAg ATg TgT CTg CAA AgA CAg CTg gAA Agg CTC C 5 -gTg CAg ggA TAA TTg gTA Tgg CTC AAA TAg gCC ATg gg

119 292, 294 274

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2.3. RNA extraction and cDNA synthesis Viral RNA was extracted by using a commercial kit (Invitek, Germany). Briefly, 200 ␮l of clinical throat swab specimen, allantoic fluid, or tissue culture supernatant were mixed with an equal volume of lyses buffer, followed by 15 min incubation at 70 ◦ C, and subsequent application to a spin column. Unbound material was removed by several washing steps, and the RNA was eluted by using 60 ␮l of RNase-free water. The cDNA synthesis was carried out at 42 ◦ C for 10 min, afterwards at 37 ◦ C for 30 min followed by 5 min at 95 ◦ C by using 25 ␮l of RNA, 100 U of murine leukemia virus reverse transcriptase (Invitrogen), 10 mM dithiothreitol, 20 U of RNasin (Promega, Mannheim, Germany), and 0.25 ␮M random hexamer primers (TibMolBiol) in a final volume of 40 ␮l. 2.4. PSQ-PCR Amplification of the neuraminidase fragments encoding resistance-associated positions was performed by PCR in a 50 ␮l reaction volume with 10 ␮l cDNA, 2.5 U Platinum TaqPol polymerase (Invitrogen, Germany), 3 mM MgCl2 , 200 ␮M dNTP, and 0.25 ␮M of each specific primer (metabion GmbH, Martinsried). One primer of each primer pair was biotinylated. PCR was carried out in an ABI thermal cycler with following conditions: 10 min 95 ◦ C, 50 cycles of 94 ◦ C, and 55 ◦ C for 1 min each, and 2 min at 72 ◦ C, with a final extension of 10 min at 72 ◦ C. PCR products were analysed on a 1.5% agarose gel, stained with ethidium bromide under UV illumination, and stored at 4 ◦ C. 2.5. Pyrosequencing For the pyrosequencing reaction, 20 ␮l of the biotinylated PCR product was immobilized onto 4 ␮l Streptavidine SepharoseTM (Amersham Bioscience, Sweden) in 40 ␮l binding buffer with 16 ␮l H2 O for 10 min at room temperature with 5 min of shaking at 1000 rpm. Single-stranded DNA was prepared with the PyroMarkTM Vacuum Prep Workstation (Biotage, Sweden). The samples were washed in high purity water for approximately 20 s and the beads containing the immobilized template were captured subsequently on the filter probes. The Vacuum Prep Tool was moved into 70% ethanol for 5 s followed by washing with denaturation solution and washing buffer for 5 s each. The beads were released into a PSQ HS 96 plate prefilled with 0.625 ␮M sequencing primer in 39 ␮l annealing buffer per well. For primer annealing, the sample plate was heated at 80 ◦ C for 2 min and thereafter cooled to room temperature. Only the immobilized strand was used for pyrosequencing. Real-time pyrosequencing of the immobilized strand was performed with up to 96 samples in parallel at 28 ◦ C with the PyroMarkTM ID Instrument (Biotage, Sweden) using 96 PyroMarkTM enzyme and substrate mixture.

Great Britain) in a 377 DNA automated sequencer (Applied Biosystems). 2.7. Site-directed mutagenesis In order to generate positive controls for the pyrosequencing analysis (PSQ-PCR), resistance-associated mutations were inserted into cloned neuraminidase genes (Table 2). The wild-type clones were obtained from T. Wolff, Robert Koch-Institut Berlin and were composed of the pHW2000 plasmid and the neuraminidase of A/Panama/2007/99 (H3N2) and A/Vietnam/1194/04 (H5N1). Using the commercially available Quick ChangeTM Multi Site Directed Mutagenesis Kit (Stratagene) and oligonucleotides (30mer, Metabion) containing the resistance-associated mutations as primers, mutated neuraminidase strains were generated according to manufacture’s instructions. The plasmids were amplified in E. coli XL1 strains and prepared with the JetQuickTM Plasmid Preparation Kit (Genomed). The gene sequence of the mutated neuraminidase was confirmed by cycle sequencing. 2.8. Neuraminidase susceptibility assay The enzyme activity of neuraminidase was measured in a fluorometric enzyme assay with 2 -(4-methylumbelliferyl)-␣-d-Nacetylneuraminic acid (MUNANA; Sigma–Aldrich) at 100 ␮M used as substrate (Potier et al., 1979). Pretitration of the virus input was performed by serial dilution of virus stocks in MES buffer (32.5 mM morpholineethanesulfonic acid, pH 6.5) followed by incubation the virus suspensions with MUNANA substrate for 2 h at 37 ◦ C in black or white 96-well plates (Nunc, Denmark). For susceptibility testing, the virus isolates were adjusted to equivalent NA contents and preincubated with various concentrations of oseltamivir (4000 nM to 0 nM) for 1 h at 37 ◦ C. After addition of the substrate and incubation for 2 h at 37 ◦ C, the assay was stopped by adding stop solution (0.1 M glycine, 25% ethanol (99.7% stock), pH 10.7). Fluorescence values of the released 4-methylumbelliferone were measured using a spectrofluorometer (Tecan) at excitation and emission wavelengths of 355 nm and 460 nm, respectively. The 50% inhibitory concentration (IC50 ) for enzymatic activity of neuraminidase was determined from the dose–response curve, by using Excel software (Microsoft). 2.9. Neuraminidase activity assay Neuraminidase activity was determined by using MUNANA fluorogenic substrate as described above. The final concentration of the substrate ranged from 3 ␮M to 300 ␮M. Michaelis–Menten constants (Km ) were calculated by using the Lineweaver–Burk diagrams generated with Excel software (Microsoft). 3. Results

2.6. Sanger cycle sequencing A 1053-bp region (N2-A) and an 880-bp region (N2-B) of the NAN2 domain of the NA gene were amplified by using the primer pairs (A) H3-NA-7/H3-NA-1060 and (B) H3-NA-550/H3-NA-1430. The primer coordinates correspond to A/Wisconsin/67/05. For sequencing of the NA-N1, a 370-bp region (N1-A) H1-NA-414/H1-NA-44 and a 768-bp region (N1-B) with primers H1-NA-1162/H1-NA-394 corresponding to A/New Caledonia/20/99 were amplified (primer sequences on request). The generated amplicons were purified by using the PCR purification kit (QIAgen) and were directly sequenced with the Big Dye terminator cycle sequencing kit (Applied Biosystems, Warrington,

3.1. Design of primers for biotinylated PCR product and pyrosequencing Primer design was carried out using the PyroMarkTM Assay Design Software and the influenza A virus reference strains A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2). Two fragments of the neuraminidase gene were selected for each of the NA-N1 and NA-N2 subtype coding for the H274Y, R292K, and N294S resistant-associated mutations of the N1-NA and the E119V, R292K and N294S substitutions of the N2-NA, respectively (Table 1). Each fragment was amplified by using one biotinylated primer in antisense direction.

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The reference strain A/Thailand/676/2005 (H5N1) was used for designing of amplification and sequencing primers for neuraminidase gene fragments of subtype A/H5N1 encoding for the H274Y substitution. A 220 bp fragment was amplified with a biotinylated sense primer (Table 1). Specific primers were designed for pyrosequencing analysis of the resistanceassociated positions. These primers were located mainly adjacent to the relevant nucleotide. All primers used in this study, their nucleotide positions within the NA gene of the reference strains, and the sizes of the amplicons are summarized in Table 1. 3.2. Optimization and specificity of the PSQ-PCR Primer and template sequences were checked for complementary (primer-dimer) and self-complementary (hair-pin) formations. Controls were analysed for each amplicon with pyrosequencing for unspecific binding: (1) the PCR-no template control and one of the sequencing primer; (2) one sequencing primer only; (3) the biotinylated and one of the sequencing primer; (4) the biotinylated primer only; and (5) the template only. All these controls displayed negative results in pyrosequencing analysis (data not shown). The PCR conditions, especially the magnesium concentration and annealing temperature were optimized by using cDNA from egg-grown or MDCK cell propagated viruses as templates. The amplicons showed a clear, strong band without non-specific products or primer-dimers on an 1.5% agarose gel (Fig. 1).

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3.3. Sensitivity of the genotypic resistance assay 3.3.1. Sensitivity of the RT-PSQ-PCR The sensitivity of the A/H1N1 and the A/H3N2 PSQ-PCR was evaluated by serial dilution of the cell culture propagated reference strains A/New Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2). Tenfold serial dilutions of the reference virus stocks from 108 PFU/ml to 10−2 PFU/ml were used for evaluation of the assays including the first step of RNA extraction followed by cDNA synthesis and PSQ-PCR. The PCR products were analysed by gel electrophoresis and photometric quantification. Dilutions containing at least 10−1 PFU/ml showed positive results as distinct bands on agarose and, therefore, provided sufficient PCR product for pyrosequencing. To avoid non-specific high background within pyrosequencing it is necessary that all of the biotinylated primer is incorporated during the PCR. In that case, the amplification rate of the assay should leave the exponential phase and reach the plateau where no primer remains. To ensure that the assays described in this study attain the plateau phase, different template concentrations have been tested. The photometric quantification of the PCR products showed similar yields of amplified DNA, regardless of the template concentration used in the PSQ-PCR assays. 3.3.2. Detection of minor variants within the viral quasispecies To evaluate the pyrosequencing method and examine the detection level of minor virus variants within the viral quasispecies, positive controls with substitutions at resistance-associated

Fig. 1. Digital images of influenza A neuraminidase gene amplicons of subtype H3N2 (a), H1N1 (b), and H5N1 (c). The fragments were amplified with PSQ-PCR primers (Table 1) and subsequently analysed on a 1.5% agarose gel with ethidium bromide staining. The results showed a distinct band with expected fragment size without non-specific amplification. The marker ladder has a 100 bp gradient. The 600 bp band is indicated in bold face type and is marked with an arrow.

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Fig. 2. Typical pyrogram of PSQ-PCR analysis of the neuraminidase gene The pyrogram of A/H3N2 neuraminidase codon 119 was selected as reference for the presented PSQ-PCR assays. The cDNA sequence of the wild-type codon is GAA and the resistance-associated codon is GTA. The positions where different sequences of the two templates give rise to different signals are highlighted.

positions were required. These substitutions were placed into the neuraminidase of the A/Panama/2007/99 (H3N2) virus by using site-directed mutagenesis. As a result, neuraminidase genes of a subtype A/H3N2 virus encoding the E119V, R292K, and N294S amino acid substitutions were available. Mutant clones encoding the H274Y substitution of the neuraminidase of subtype A/H5N1 have been prepared by mutagenesis using the A/Vietnam/1194/04 (H5N1) neuraminidase gene as backbone. Subsequently performed PSQ-analysis showed that the assays discriminated clearly the wildtype from the mutant virus variants (Fig. 2). To analyse the sensitivity of the pyrosequencing method, wildtype clones and mutant clones were mixed in defined proportions from 100% of wild-type clone to 100% of mutant clone in 95:5, 90:10, 85:15, 80:20, 70:30, 50:50, 30:70, 20:80, 15:85, 10:90 (wildtype:mutant) mixtures. The PSQ-PCR analysis indicated that the detection level of minor variants within the viral quasispecies under these assay conditions is at least about 10% (Fig. 3). 3.4. Evaluation of the genotypic resistance assay In order to evaluate the genotypic assay, 37 clinical specimens were tested. Altogether 20 influenza A viruses of subtype H3N2 and 17 A/H1N1 viruses were analysed by the presented PSQ-PCR and Sanger cycle sequencing in parallel. Twelve avian A/H5N1 viruses isolated from wildfowl in Germany during the HPAIV outbreak last year and one virus isolated from a mute swan in 2007 were used to evaluate the PSQ-PCR for NA-N1 (A/H5N1). In addition one isolate from an A/H5N1 infected wild bird from south-east Asia and one avian A/H5N1 reference strain isolated during an HPAIV outbreak 1959 in Scotland was included (Table 3). The viruses represented the human and avian influenza viruses circulating in Germany dur-

Fig. 3. Detection limit of minor variants Defined mixtures of wild-type and mutant variants were analysed with PSQ-PCR assay. Analysis of A/H3N2 neuraminidase codon 119 is shown as reference. The peak levels are proportional to the percentage of each clone present in the mixture. The analysis has been carried out with mixtures of (a) 100% wild-type clone, (b) 10% mutant clone, (c) 20% mutant clone and (d) 30% mutant clone. The wild-type clone has the sequence GAA; the mutant clone has the sequence GTA. The positions where different sequences of the two templates give rise to different signals are highlighted.

ing the 2005/2006 and the 2006/2007 season, respectively. None resistance-associated mutation was detected neither by PSQ-PCR nor by Sanger cycle sequencing analysis. Therefore, comparable results were obtained by both methods, demonstrating the reliability of the new PSQ-assays presented here.

S. Duwe, B. Schweiger / Journal of Virological Methods 153 (2008) 134–141 Table 3 Isolates of avian influenza A/H5N1 viruses used to evaluate the resistance analysis assay A/whooper swan/Germany/R65/06 A/common buzzard/Berlin/06 A/canadagoose/Germany/R71/06 A/duck/Germany/R338/06 A/duck/Germany/R348/06 A/duck/Germany/R655/06 A/marten/Germany/R747/06 A/gull/Germany/R882/06 A/eagle owl/Germany/R1166/06 A/stork/Germany/R1239 A/falcon/Germany/R899/06 A/great crested grebe/Germany/R1226/06 A/mute swan/Germany/R1349/07 A/duck/Vietnam/Tg24-01/05 A/chicken/Scotland/59

3.5. Enzymatic activity and susceptibility of influenza A neuraminidase to the antiviral drug oseltamivir (TamifluTM ) To investigate the susceptibility of influenza A neuraminidase to the antiviral drug oseltamivir (TamifluTM ) recent clinical isolates of subtypes A/H1N1 (n = 16), A/H3N2 (n = 64), and A/H5N1 (n = 12) were examined in a neuraminidase enzymatic activity assay by using the fluorogenic substrate MUNANA. The calculated IC50 values and standard deviations amount to (0.20 ± 0.124) nM oseltamivir for A/H3N2 viruses, (0.97 ± 0.894) nM for A/H1N1 viruses, and (1.16 ± 0.498) nM for subtype A/H5N1 viruses. The results indicated that oseltamivir is a more potent inhibitor for neuraminidase of subtype N2 than of subtype N1 neuraminidase. Resistance-associated mutations within the neuraminidase lead to a reduced susceptibility to antiviral drugs (e.g. oseltamivir) in in vitro assays. Two clinical A/H1N1 virus isolates (A/BadenWürttemberg/124/08 and A/Bayern/40/08) carrying the 274Y neuraminidase substitution, known to confer reduced susceptibility to oseltamivir, were tested with the neuraminidase activity assay. Compared to two isolates carrying the sensitive wild-type sequence H274 (A/Berlin/45/08 and A/Niedersachsen/83/08) about 500-fold more oseltamivir was required to inhibit 50% of the enzyme activity (Table 4). Enzymatic activity analysis for resistant and sensitive neuraminidases was carried out by using the fluorogenic substrate MUNANA. The mutant neuraminidases were characterized by a more than twofold lower affinity to the substrate as the sensitive ones. This was shown by an increased value of the Michaelis–Menten constant (Km ) for viruses with the H274Y substitution in their neuraminidase (Table 4). 4. Discussion Influenza pandemic preparedness by various countries includes stockpiling of oseltamivir. The ongoing human A/H5N1 transmissions might lead to the next influenza pandemic caused by an A/H5N1 virus. At present (19 June 2008) 385 WHO confirmed human cases, 243 of them with fatal outcome, were reported

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(World Health Organisation, 2008a). Oseltamivir resistance of A/H5N1 viruses was detected in three immunocompetent patients during therapy. Resistant strains appeared after 5–9 days and seemed to be responsible for the fatal outcome (de Jong et al., 2005). In the case of a Vietnamese girl, oseltamivir-resistant A/H5N1 viruses occurred during prophylaxis and therapy (Le et al., 2005). Monitoring the emergence of resistant viruses with rapid access to results is, therefore, necessary. In this study, a rapid method for genotypic resistance testing of influenza A viruses of the subtypes A/H1N1, A/H3N2, and A/H5N1 is presented. The method includes all advantages and disadvantages of a genotypic assay. The advantages are quick and reproducible results. The RT-PCR with subsequent pyrosequencing can be carried out directly from original samples like nasal or throat swabs, without any need for virus propagation in eggs or in cell culture. This reduces not only the duration of the assay but might also be advantageous for detecting minor variants. Viruses generated by reverse genetics with resistance-associated mutations within the neuraminidase genes had reverted to wild-type neuraminidase in absence of the selection pressure due to oseltamivir (Zürcher et al., 2006). Thus, running the assay directly from original swabs excludes the risk of losing the resistant-associated mutations by reversion or overgrowing of the more replication-competent wildtype viruses. The presented analysis of enzymatic properties of the neuraminidase of reference and recently circulating influenza viruses revealed a significant higher susceptibility of subtype N2 neuraminidase compared to NA-N1. These findings confirm the data of other drug resistance surveillance programs (Monto et al., 2006; Hurt et al., 2004; Ferraris et al., 2005). Resistant A/H1N1 viruses isolated in Germany possessed a mutant neuraminidase with an approximately twofold decreased enzyme affinity to the substrate Munana. Comparable findings regarding the difference in enzyme affinity between sensitive and resistant viruses were reported previously (Wang et al., 2002; Rameix-Welti et al., 2006; Collins et al., 2008). Resistance to neuraminidase inhibitors was not only reported for A/H5N1 strains, but also for seasonal influenza viruses. The incidence of oseltamivir-resistant influenza A/H3N2 and A/H1N1 viruses seen in clinical trial samples of immunocompetent patients until July 2004 was 0.33% (4/1228) in adults (≥13 years) and 4% (17/421) in children (≤12 years), resulting in an overall incidence of 1.26% (Ward et al., 2005). An extremely high degree of A/H1N1 and A/H3N2 oseltamivir resistant viruses has been found within two clinical trials. During treatment about 16.3% (7/43) resistant A/H1N1 and 18% (9/59) resistant A/H3N2 viruses were detected (Ward et al., 2005; Kiso et al., 2004). In contrast to these findings, which may be connected to an insufficient dosage regime, the occurrence and spread of neuraminidase inhibitor resistant strains within seasonal influenza viruses seems to be rare. However, it is likely that the prevalence of influenza viruses with reduced susceptibility to antiviral drugs will arise as a consequence of wider application of neuraminidase inhibitors for therapy and prophylaxis of seasonal influenza infections. The PSQ-PCR assays described

Table 4 Enzymatic properties of mutant and wild-type of subtype N1 neuraminidase Km (␮M) ± S.D.

Isolate

WT/mutant

a

A/Berlin/45/08 A/Niedersachsen/83/08 A/Baden-Württemberg/124/08 A/Bayern/40/08

H274 sensitive H274 sensitive 274Y resistant 274Y resistant

49 29 89 84

± ± ± ±

9.1 2.4 13.0 2.6

IC50 nM Oseltamivir 1.3 0.6 500 600

WT, sensitive wild-type virus; H274, neuraminidase carried a histidine at amino acid position 274; 274Y, neuraminidase carried a tyrosine at amino acid position 274; Km , Michaelis–Menten constant; S.D., standard deviation. a Enzymatic activity assay was carried out in five independent duplicated experiments.

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above should be especially useful for monitoring the frequency of neuraminidase resistant influenza viruses within the seasonal influenza. It has to be stated that the presented PSQ-PCR assays can only detect mutations known to confer antiviral drug resistance. Mutations outside the active centre of the enzyme were not described so far as resistance-associated. Compensatory mutations within the hemagglutinin gene occurred during treatment and increased the phenotypic resistance (Ison et al., 2006; Abed et al., 2002). To detect resistance to neuraminidase inhibitors caused by such compensatory mutations phenotypic analysis is necessary. Resistance analysis is carried out broadly using the fluorometric neuraminidase assay that is regarded as the ‘gold standard’ for resistance analysis at present. However, this assay detects resistance that is caused by alterations of the neuraminidase structure. The detection of new resistance properties correlated also to other viral proteins requires assays for examination of the replication capacity in presence of inhibitor like plaque inhibition assays. Virus amplification by propagation in embryonated eggs or MDCK cells is necessary for phenotypic assays. As stated above, resistant neuraminidase has a lower substrate affinity indicating a reduced replication capacity of resistant viruses. Since cell cultures consisted both, resistant and sensitive viruses the phenotypic assays may not function as a result of increased replication of sensitive viruses or due to the failure of virus propagation. On the other hand, reversion of resistance-associated mutations to wild-type neuraminidase is known to occur (Zürcher et al., 2006). Genotypic analysis with sensitive and rapid assays as presented here is, therefore, the only possibility to detect antiviral resistance in an early state of occurrence. Follow up studies of neuraminidase inhibitor treated patients by complete sequencing of the neuraminidase and the hemagglutinin genes will also be important in identifying new mutations that might be associated to resistance. In this case the PSQ-PCR assays could be adapted rapidly to such new substitutions. Taken together, since mutations known to confer resistance outside the active centre of the neuraminidase are not described until now, the PSQ-PCR assays presented here can be a valuable tool for a rapid, high-throughput screening of respiratory samples. During the last influenza epidemic (2007/2008) an unexpected increase in the occurrence and spread of oseltamivir-resistant A/H1N1 viruses was observed in Europe, Asia and the United States of America (World Health Organisation, 2008b). All resistant viruses carried the neuraminidase mutation H274Y, known to confer high-level resistance to oseltamivir. By using the assays described above, a rapid and contemporary monitoring of the resistant viruses was carried out. Genotypic assays with subsequent pyrosequencing analysis were used successfully for monitoring the occurrence and spread of adamantine-resistant influenza viruses (Bright et al., 2006; Deyde et al., 2007). A rapid assay is not available for genotypic analysis of neuraminidase inhibitor resistant influenza viruses. The assays described complete the possibilities for genotypic analysis of resistant influenza viruses. The detection limit of resistant virus variants within the viral quasispecies is an important factor for monitoring the development of resistance during antiviral therapy. The prevalence of resistant viruses may vary from patient to patient. This has to be taken into account since it was shown that an A/H5N1 infection resulted in a mixed virus population. Of 10 viral clones picked from plaques, 6 showed the H274Y substitution causing a high resistance, 3 had the R294S mutation causing a slight resistance to oseltamivir, and one of the clones was sensitive (Le et al., 2005). The detection of minor resistant variants within the viral quasispecies of approximately 10% is possible using the presented assays. The method described above is not only faster,

but also more sensitive than the Sanger cycle sequencing procedure, which showed a detection limit of 25% (Larder et al., 1993). Rapid resistance analyses as well as the high sensitivity for detection of minor variants make the presented assays a valuable tool for seasonal resistance monitoring. Changing the antiviral medication is feasible by rapid detection of emerging resistant virus variants; towards a dosage increase or an additional administration of M2 proton channel blocker (Baz et al., 2006; Masihi et al., 2007). In conclusion, RT-PCR assays with subsequent PSQ-PCR were designed which allow rapid and sensitive detection of neuraminidase resistance-associated mutations within the neuraminidase genes of the circulating human and avian influenza A viruses of subtypes A/H1N1, A/H3N2, and A/H5N1.

Acknowledgements We would like to thank Manuela Friedrich for excellent technical assistance, Julia Tesch for cycle sequencing performance, and Ute Buwitt for helpful discussion. This study was funded by the German government programme “Forschung-Sofortprogramm Influenza (FSI)”.

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