Rapid and Specific Detection of Amantadine-Resistant Influenza AViruses with a Ser31Asn Mutation by the Cycling Probe Method�
Yasushi Suzuki,* Reiko Saito, Hassan Zaraket, Clyde Dapat, Isolde Caperig-Dapat, and Hiroshi SuzukiDepartment of Public Health, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
Received 6 April 2009/Returned for modification 17 June 2009/Accepted 28 October 2009
Amantadine is one of the antiviral agents used to treat influenza A virus infections, but resistant strains havewidely emerged worldwide. In the present study, we developed a novel method to detect amantadine-resistantstrains harboring the Ser31Asn mutation in the M2 gene based on the cycling probe method and real-timePCR. We also studied the rate of amantadine resistance in the 2007–2008 influenza season in Japan. Twodifferent primer and cycling probe sets were designed for A/H1N1 and A/H3N2 each to detect a singlenucleotide polymorphism corresponding to Ser/Asn at residue 31 of the M2 protein. By using nasopharyngealswabs from patients with influenza-like and other respiratory illnesses and virus isolates, the specificity andthe sensitivity of the cycling probe method were evaluated. High frequencies of amantadine resistance weredetected among the A/H1N1 (411/663, 62%) and A/H3N2 (56/56, 100%) virus isolates collected from sixprefectures in Japan in the 2007–2008 influenza season. We confirmed that the cycling probe method is suitablefor the screening of both nasopharyngeal swabs and influenza virus isolates for amantadine-resistant strainsand showed that the incidence of amantadine resistance among both A/H1N1 and A/H3N2 viruses remainedhigh in Japan during the 2007–2008 season.
Adamantanes (amantadine and rimantadine) have beenused for the prevention and treatment of influenza A virusinfections (25). The molecular basis of resistance has beenidentified as single nucleotide changes that lead to correspond-ing amino acid substitutions at one of four critical amino acidresidues (residues 26, 27, 30, and 31) in the transmembraneregion of the M2 ion channel protein (19, 26, 27). Recentstudies suggest that the rates of influenza virus A/H3N2 resis-tance to amantadine and rimantadine have been high globallysince 2005 (2, 6, 7, 9, 29, 30), while the rates of resistanceamong A/H1N1 viruses varied from country to country butincreased sharply from 2006 onwards (1, 9, 32). It should benoted that resistance in both subtypes was almost exclusivelyassociated with one amino acid substitution at residue 31 (Serto Asn) of the M2 ion channel protein after 2005 (1, 2, 6, 7, 9,29, 30, 32).
We have previously established methods for the detection ofamantadine susceptibility, such as the virus titration methodwith comparison of the 50% tissue culture infectious doses(TCID50s; TCID50/0.2 ml) in the presence and the absence ofamantadine (24) and PCR-restriction fragment length poly-morphism (PCR-RFLP) analysis (21, 31). Other methods forthe detection of resistant strains have also been reported, suchas enzyme-linked immunosorbent assay (ELISA) (4), plaquereduction assay (13), and DNA sequencing (24). In general,however, conventional methods are time-consuming. Recently,a high-throughput method of genetic analysis called pyrose-quencing was used as a rapid method for screening for aman-tadine and neuraminidase inhibitor resistance (6–9, 11); how-
ever, the cost of pyrosequencing is not always coverable forevery laboratories.
In the study described here, we developed a rapid assayusing a chimera probe-adapted real-time PCR, or the cyclingprobe method, to detect amantadine-resistant viruses withthe Ser31Asn substitution in the M2 ion channel protein.Furthermore, we report the frequency of amantadine resis-tance among influenza A viruses in six prefectures in Japanin the 2007–2008 influenza season.
MATERIALS AND METHODS
Design of cycling probes. The cycling probe technology is a unique nucleicacid-based method that detects single nucleic acid polymorphisms (SNPs) in atarget DNA sequence by using a probe-adapted real-time PCR (3, 10) (Fig. 1).The cycling probe method involves a reaction between a chimeric fluorescence-and quencher-labeled DNA/RNA oligonucleotide probe (cycling probe) andRNase (RNase H). This cycling probe is a short DNA fragment (normally 10- to20-mer) accommodating an RNA complementary to the nucleotide of interestthat undergoes degeneration by RNase H activity, once a DNA-RNA complex isformed during annealing. This degeneration leads to the emission of strongfluorescence (17), and by measuring the intensity of the fluorescence, the amountof amplified product can be quantified (Fig. 1a). For SNP typing, two cyclingprobes labeled with two different fluorescence dyes (6-carboxyfluorescein [FAM]or 6-carboxy-X-rhodamine [ROX]) are used, with each probe harboring RNAcorresponding to the wild-type nucleotide or the nucleotide with a mutation atthe SNP position (Fig. 1b).
RNA extraction and reverse transcription. Nasopharyngeal swab samples thatpreviously tested positive for influenza virus by virus isolation and for which theirgenetic substitution of interest was confirmed by sequencing were selected forevaluation of the assay’s specificity. These included 20 amantadine-sensitiveand 20 amantadine-resistant samples each of the influenza virus A/H1N1 andA/H3N2 subtypes. The viral RNA of influenza viruses A/H1N1 and A/H3N2 andother common respiratory viruses (respiratory syncytial virus, parainfluenza vi-rus, enterovirus, and rhinovirus) and the DNA of adenovirus were extracted from100 �l of the supernatants of the nasopharyngeal swabs or the virus culturesupernatant by using an Extragen II kit (Kainos, Tokyo, Japan), according to themanufacturer’s instructions. Reverse transcription was performed in a reactionseparate from the real-time PCR in order to obtain 25 �l of the first-strandcDNA of the influenza virus genome by using influenza A virus universal primerUni12, as reported elsewhere (16). The RNA of the other respiratory viruses
* Corresponding author. Mailing address: Department of PublicHealth, Niigata University, Graduate School of Medical and DentalSciences, 1-757, Asahimachi-Dori, Niigata City, Niigata Prefecture951-8510, Japan. Phone: 81-25-227-2129. Fax: 81-25-227-0765. E-mail:[email protected].
used to check for cross-reactions was reverse transcribed by using random prim-ers (Invitrogen Corp., Carlsbad, CA).
Primers, probes, and PCR conditions. PCR primers (sets of forward andreverse primers for H1N1 and H3N2) were designed to specifically amplify theM2 gene transmembrane region of influenza viruses A/H1N1 and A/H3N2; thePCR product sizes were 155 bp and 98 bp, respectively. The chimera probesH1N1-AS (where AS indicates amantadine sensitive), H1N1-AR (where ARindicates amantadine resistant), H3N2-AS, and H3N2-AR were created to detectSer31 (AGT) and Asn31(AAT) in the M2 gene, respectively (the underscoresindicate the nucleotide replaced by RNA) (TaKaRa Bio Inc.) (Table 1). ACycleavePCRCore kit (TaKaRa Bio Inc.) was used for the PCR and the simul-taneous cleavage of RNase H. The amplification was carried out in a totalvolume of 25 �l. The reaction mixture contained (final concentrations are given)1� CycleavePCR buffer, 3 mM Mg2�, 0.3 mM each deoxynucleoside triphos-phates, 5 pmol of each PCR primer (forward and reverse), 5 pmol of each probe(the FAM-labeled probe and the ROX-labeled probe), 100 U of Tli RNase HII,and 1.25 U of Ex Taq HS (TaKaRa Bio Inc.). One microliter of cDNA was addedto the reaction mixture as the DNA template. To detect A/H1N1 viruses, a set ofprimers and probes consisting of the H1N1 forward primer, the H1N1 reverseprimer, and the H1N1-AS and H1N1-AR probes was used; and to detectA/H3N2 viruses, a relevant set of H3N2 primers and probes was employed(Table 1). PCR amplification and fluorescence detection were performed with a
TP800 thermal cycler Dice real-time PCR system (TaKaRa Bio Inc.). The con-ditions of the PCR cycles were as follows: a hold at 95°C for 10 s, followed by 40cycles of denaturation at 95°C for 5 s, primer annealing at 57°C for 10 s, andextension and emission of fluorescence at 72°C for 15 s. Duplicate wells wereused for each sample, and amantadine-sensitive (Ser31) and -resistant (Asn31)control plasmids were included in each run in a 96-well plate.
Control plasmids. Four positive control plasmids, H1N1-AS, H1N1-AR,H3N2-AS, and H3N2-AR, were made from the PCR product of each subtypeamplified with the same PCR primers used in this study. Both the H1N1-AS andH3N2-AS controls contained sequences that code for serine at position 31(amantadine sensitive), while the H1N1-AR and H3N2-AR controls possessedsequences with mutations that code for asparagine at position 31. The purifiedPCR product was ligated and cloned into a pMD20-T vector (TaKaRa Bio Inc.)and was transformed into JM109 competent cells (TaKaRa Bio Inc.) with aMighty TA cloning kit (TaKaRa Bio Inc.), according to the manufacturer’sinstructions. Positive clones were selected by the blue-white colony pickupmethod and were then cultured in Luria-Bertani broth and incubated overnightat 37°C in a shaking incubator. The bacterial culture was pelleted by centrifu-gation, and the plasmids were extracted with a Wizard Plus SV Minipreps DNApurification system (Promega, Madison, WI), according to the manufacturer’sinstructions. Sequencing was performed with an ABI Prism BigDye Terminator(version 3.1) cycle sequencing kit (Applied Biosystems, Foster City, CA), ac-
FIG. 1. Illustration of cycling probe technology using real-time PCR for SNP typing. F and Q represent fluorescence and quencher, respectively.(a) I, denaturation of target DNA; II, primer annealing and prove hybridization and then cleavage by RNase H; III, extension of target DNA byTaq polymerase and detection of fluorescence. (b) Each probe detects SNP in the matched sequence; IV, FAM-labeled probe matches target DNAand is cleaved to release the FAM fluorescence by RNase H; V, ROX-labeled probe matched target DNA and is cleaved by RNase H to releasethe ROX fluorescence.
cording to the manufacturer’s instructions, and the products were sequencedwith an ABI 3100 automatic sequencer (Applied Biosystems) to confirm thepresence of the insert. The sequences were aligned and compiled by usingBioEdit software (version 7.0.7) (14).
After the DNA concentrations of the plasmids with the wild-type and mutantsequences were measured with a spectrophotometer (GeneQuant 1300; GEHealthcare UK Ltd., Buckinghamshire, England), serial 10-fold dilutions weremade to determine the detection sensitivities of this method for the wild-type andmutant sequences of each subtype. In addition, mixtures of plasmids with thewild-type and mutant sequences were tested at ratios of 1:1, 1:10, 1:100, and1:1,000 to examine the detection sensitivity of the method with a mixed popu-lation.
Prevalence of amantadine resistance in clinical samples. Clinical sampleswere collected from patients with influenza-like illnesses at 14 outpatient clinicsand hospitals from October 2007 to April 2008 in six prefectures (Hokkaido,Niigata, Gunma, Kyoto, Hyogo, and Nagasaki) in Japan. Briefly, after written ororal informed consent was obtained, nasopharyngeal swab specimens were col-lected from the patients with influenza-like illnesses, and the medication used inthe clinic for the treatment of influenza (amantadine, oseltamivir, or zanamivir)was recorded. The swabs were stored in viral transport medium and were kept at4°C until transportation to the Department of Public Health, Niigata University,for virus isolation within 1 week. Then, 100-�l aliquots of supernatants of thenasopharyngeal swab specimens were inoculated into Madin-Darby canine kid-ney cells (MDCK), prepared in 48-well plates. The plates were incubated at 34°Cunder a 5% CO2 atmosphere for up to 10 days to assess the samples for thepresence of cytopathic effects (CPE). Fifty-microliter aliquots of the superna-tants of CPE-positive samples were then passaged twice to obtain a sufficientvirus titer for virus identification. The influenza virus isolates were typed andsubtyped by a hemagglutination inhibition (HAI) assay with commercially avail-able antisera to the influenza virus vaccine strain for the 2007–2008 season inJapan (Denka Seiken Co., Ltd., Tokyo, Japan), namely, A/Solomon Islands/3/2006 (A/H1N1), A/Hiroshima/52/2005 (A/H3N2), and B/Malaysia/2506/2004,and with guinea pig red blood cells. RNA extraction, cDNA synthesis, and thecycling probe real-time PCR were employed with the nasopharyngeal swabspecimens and virus isolates as described above to examine whether they pos-sessed the S31N substitution.
RESULTS
Establishment of cycling probe method. A FAM-labeledcycling probe was designed to detect the sequence for aman-tadine sensitivity (AGT) at amino acid position 31 in the M2protein by replacing guanine DNA with RNA, while the ROXprobe replaced the adenine DNA with RNA corresponding tothe sequence for amantadine resistance (AAT) (Table 1).If the sample in question had a sequence conferring sensitivity,the fluorescent emission of FAM was detected after the hy-bridized RNA and DNA complex at the guanine position wasdegenerated by RNase H and the dye was subsequently liber-ated from the quencher. The same reaction occurred with the
sequence conferring amantadine resistance, which harboredadenine at the identical position (position 31) and which isreported by the detection of ROX.
The cycling probes were first tested with DNA plasmids withthe known wild-type and mutant sequences in the M2 proteinfor both subtype A/H1N1 and subtype A/H3N2, which servedas controls for our two-step cycling probe real-time PCR. Flu-orescence intensities with threshold cycle (CT) values of be-tween 16 and 20 for 2.9 � 107 copies for A/H1N1 and 2.95 �107 copies for A/H3N2 were detected for the plasmids with thewild-type sequence and the plasmids with the mutant se-quence (Fig. 2a and b). The detection limits for the controlswere 2.9 � 102 copies for the A/H1N1 plasmid and 2.95 �102 copies for the A/H3N2 plasmid, giving CT values ofabout 38 (Fig. 2c and d).
The study of mixtures of control plasmids with the wild-typeand mutant sequences showed that the cycling probes gave CT
values with each dye in a plasmid concentration-dependentmanner. A ratio of 1:1 gave similar CT values for both thewild-type and the mutant plasmids, and the CT values graduallyincreased as the proportion of the wild-type or mutant plas-mids became smaller (data not shown). The detection limit wasa ratio of 1:100 for the mutant and wild-type plasmids for bothsubtype H1N1 and subtype H3N2.
We next evaluated the sensitivity and the specificity of themethod for the detection of strains previously determined tobe amantadine sensitive and resistant using clinical influenzaisolates (high template concentration) and nasopharyngealswab specimens (low template concentration). The H1N1-ASprobe successfully detected the amantadine-sensitive virus(Fig. 3a) and the H1N1-AR probe successfully detected theamantadine-resistant virus (Fig. 3b) from both clinical isolatesand nasopharyngeal swab specimens. Similar results for thedifferentiation (specific detection) of sensitive and resistantstrains were obtained with A/H3N2 probes and clinical samplesof A/H3N2 (Fig. 3c and d). The genetic sequencing resultsmatched the cycling probe results. The average CT value ob-served for the isolates was low (15 to 20 cycles) compared withthat for the nasopharyngeal swab specimens (25 to 35 cycles).A reaction was considered positive only when the CT value didnot exceed 38 cycles after a 40-cycle PCR run.
We evaluated the specificities of the probes for human in-fluenza virus and other common respiratory viruses (Table 2).
TABLE 1. Primers and probes used for real-time PCR
Subtype Primers and probes Sequence (5�–3�) Locationa
H1N1 H1N1 forward primer 5�-GCTCTAGCACTGGTCTGAAA-3� 696–715H1N1 reverse primer 5�-AGGCGATCAATAATCCACAA-3� 831–850H1N1-AS probeb 5�-(FAMc)-TGCCGCAAGTA-(Eclipsed)-3� 797–807H1N1-AR probeb 5�-(ROXc)-TGCCGCAAATA-(Eclipsed)-3� 797–807
H3N2 H3N2 forward primer 5�-AGACCTATCAGAAACGAATG-3� 738–757H3N2 reverse primer 5�-CACAGTATCAAGTGCAAG-3� 818–835H3N2-AS probeb 5�-(FAMc)-TGCTGCGAGTA-(Eclipsed)-3� 796–807H3N2-AR probeb 5�-(ROXc)-TTGCTGCGAATA-(Eclipsed)-3� 796–807
a Location of primers and probes in the M gene (total length, 1,027 bp), segment 7, of influenza A virus.b Fluorescent dye- and quencher-labeled DNA-RNA chimeric probe. The boldface italic letters in the sequences of these probes indicate the nucleotide replaced
by RNA.c Fluorescent molecules.d Quenching molecule.
VOL. 48, 2010 DETECTION OF AMANTADINE-RESISTANT INFLUENZA A VIRUS 59
The H1N1 probes reacted only with amantadine-sensitive and-resistant A/H1N1 strains and did not react with human influ-enza virus A/H3N2, whereas the H3N2 probes reacted onlywith amantadine-sensitive and -resistant A/H3N2 strains anddid not react with human influenza virus A/H1N1. Neitherprobe reacted with samples containing influenza B virus, otherrespiratory virus-positive samples, or negative (no-template)samples. Furthermore, 99 swab specimens, including 85 isola-tion-positive samples and 14 isolation-negative samples, weredirectly tested with the cycling probe. Forty isolates each ofH1N1 and H3N2 were identified, and 20 isolates each of aman-tadine-sensitive and isolation-positive H1N1 and H3N2 strainswere also identified by these methods. Neither probe reactedwith influenza B viruses, respiratory syncytial virus-positivesamples, or isolation-negative samples.
Prevalence of amantadine-resistant influenza viruses in the2007–2008 season in Japan. A total of 1,027 primary clinicalsamples were collected from clinicians, and eventually, 756(73.4%) influenza viruses were isolated by the use of MDCKcells. Of these, 672 (88.9%) isolates were influenza virusA/H1N1, 62 (8.2%) were influenza virus A/H3N2, and 22(2.9%) were influenza B virus by HAI testing. The cycling
probe assay successfully identified 663 (98.7%) of the 672A/H1N1 viruses and 56 (90.0%) of the 62 A/H3N2 viruses. Inthis assay, 62.0% (411 of 663) of the A/H1N1 isolates and100% (56 of 56) of the A/H3N2 isolates were amantadineresistant (Table 3). None of the patients received amantadinebefore sampling or after diagnosis.
DISCUSSION
We developed a rapid and high-throughput real-time PCRassay for the detection of the Ser31Asn mutation in theM2 gene transmembrane region in both the influenza virusA/H1N1 and the influenza virus A/H3N2 subtypes using spe-cific florescent-labeled chimeric probes. The assay is called thecycling probe technology (3, 10). We demonstrated in the studydescribed here that the method is highly specific for the de-tection of the SNP for amantadine resistance in the M2 geneand could successfully differentiate the two influenza A virussubtypes. Eventually, we showed a high frequency of occur-rence of amantadine-resistant strains of both subtypes duringthe 2007–2008 season in Japan.
The results of the cycling probe assay described in this paper
FIG. 2. Testing of serial dilutions of control plasmids with inserts of the M2 gene sequences of influenza A viruses by the cycling probe method.FAM fluorescence was detected with the sequence for amantadine sensitivity (AS; Ser31), and ROX fluorescence was seen with the sequence foramantadine resistance (AR; Asn31). Control plasmids with the A/H1N1 sequences (H1N1-AS and H1N1-AR) reacted with probes H1N1-AS andH1N1-AR, respectively (a and b). Control plasmids with the A/H3N2 sequences (H3N2-AS and H3N2-AR) specifically reacted with the respectiveA/H3N2 probes (c and d).
demonstrated agreement with the results of gene sequencing.Virus detection was successful by the use of both nasopharyn-geal swabs and virus isolates from clinical samples, despite thedifferences in the virus concentration between the two types ofsamples. In addition, the cycling probe sets used to detect bothsubtypes did not show false-positive reactions with the otherinfluenza A virus subtypes, influenza B virus, or other respira-tory viruses. The sensitivity of the method for the detection ofinfluenza virus A/H3N2, based on virus isolation, was lowerthan that for the detection of A/H1N1, which was due tosporadic nucleotide mismatches in the primers and/or probesfor A/H3N2. At present, the proportions of viruses with amismatch is not sufficiently significant to require a change tothe sequence of the primers or the probes. Thus, our methodis highly specific and is suitable for the subtyping of humaninfluenza A viruses, along with the identification of amanta-dine-resistant viruses. In addition, our method successfully de-tected mixed populations of plasmids with mutant and wild-type sequences at a ratio as low as 1:100. This method can beused to quantify the virus loads in both clinical samples andsamples tested in vitro containing a mixed mutant and wild-type virus population.
Most protocols currently employed are based on one-step
reverse transcription real-time PCR (22, 33), and thus, it iscommon to use as the control a known concentration of RNA.However, in the cycling probe method, it is not possible toadopt a one-step real-time PCR, because it includes RNase Hand the template could not achieve a sufficient length of cDNAduring the reverse transcription. Other common controls arecDNA (two-step method), but plasmid preparations havehigher degrees of stability and reproducibility (20, 23, 28).
Various methods has been used to examine virus isolates fortheir amantadine susceptibilities, such as ELISA (4), plaquereduction assay (13), the TCID50/0.2-ml method (24), PCR-RFLP analysis (21, 31), and DNA sequencing (24). However,the time to the retrieval of results by these methods may befrom several hours to a few days. The advantage of our ap-proach is that we can detect amantadine-resistant strains di-rectly from patients’ nasopharyngeal swab specimens in only3 h: 1.5 h for RNA extraction and cDNA synthesis and 1.5 h forthe real-time PCR run. Our method can be also used for dualsubtyping and resistance detection if the two subtype-specificreactions are performed in parallel in a 96-well plate for sam-ple sizes of �45. Pyrosequencing (6–9, 11) and DNA microar-ray analysis (34) were recently developed for the detectionof amantadine-resistant strains. These methods have high
FIG. 3. Detection of amantadine-resistant influenza A virus strains with a mutation at position 31 in the M2 gene by the use of clinical isolatesand nasopharyngeal swab specimens. A/H1N1 amantadine-sensitive (a) and amantadine-resistant (b) strains reacted with each of the FAM andthe ROX probes. A/H3N2 amantadine-sensitive (c) and amantadine-resistant (d) strains reacted with the specific corresponding probes.
VOL. 48, 2010 DETECTION OF AMANTADINE-RESISTANT INFLUENZA A VIRUS 61
throughputs and are rapid, just as our cycling probe techniqueis, but unlike our method, they have the disadvantage of thecosts for the machine and the reagents, which are too high fortheir routine use in laboratories. Thus, our method is perhapsone of the quickest and most affordable with respect to therunning cost (which is equivalent to that of the TaqMan probemethod) among the methods currently available for the iden-tification of amantadine resistance, and it is ideal for large-scale screening for resistant mutants and subtyping even withspecimens with low template concentrations, such as nasopha-ryngeal swab specimens. Of note, the TaqMan probe real-timePCR was applied for the detection of oseltamivir-resistantinfluenza virus A/H1N1 possessing His274Tyr (N2 numbering)in the neuraminidase gene and used for monitoring for resis-tant viruses (5). We are currently developing new cycling probesets to detect other amantadine resistance mutations in the M2gene and the oseltamivir resistance mutation (His274Tyr) inthe neuraminidase gene.
None of the patients in our study were known to have re-ceived amantadine. However, we detected amantadine-resis-
tant viruses among both A/H1N1 (62.0%) and A/H3N2(100%) viruses at a high frequency during the 2007–2008 in-fluenza season. The high prevalence of amantadine-resistantA/H1N1 strains detected in Japan was one season before the2006–2007 season, and that of the A/H3N2 strains was theprevious two seasons (2005–2006 and 2006–2007) (29, 30, 32).The source of the international spread of amantadine-resistantstrains is speculated to be Southeast and East Asia (6). Inparticular, in China, information from the NonprescriptionMedicines Association shows that amantadine is available inover-the-counter formulations and is included in various coldremedies for which humans do not need prescriptions, andchicken farmers frequently add amantadine to chicken food orwater for the treatment and prophylaxis of avian influenzavirus and other viral diseases with low levels of pathogenicity(15). In addition, amantadine-resistant strains that could beefficiently transmitted without drug pressure were eventuallygenerated by genetic reassortment (18).
Recently, a high rate of resistance to oseltamivir was detectedamong A/H1N1 viruses in several countries in different regions ofthe world in 2007 and 2008 (35), and combined treatment withadamantane and neuraminidase inhibitors should be consideredfor high-risk patients when the subtype is unknown (12). How-ever, monitoring of influenza viruses for resistance to both drugsis crucial when combination treatment is used.
In conclusion, this study shows that the cycling probemethod can specifically and rapidly detect amantadine-resis-tant influenza A viruses with the Ser31Asn mutation in the M2gene directly from nasopharyngeal swab specimens and is quiteuseful for monitoring for drug-resistant strains to elicit thejudicial use of amantadine for chemoprophylaxis and the treat-ment of influenza.
TABLE 2. Probe reaction performance with various virus samples
Sample type Virusa Subtypeb Susceptibility toamantadinec
No. ofsamples
No. of samples positive with probe set:
H1N1 H3N2
FAMprobe
ROXprobe
FAMprobe
ROXprobe
Clinical samples Influenza A virus H1N1 Sensitive 4 4 0 0 0Influenza A virus H1N1 Resistant 4 0 4 0 0Influenza A virus H3N2 Sensitive 4 0 0 4 0Influenza A virus H3N2 Resistant 4 0 0 0 4Influenza B virus NAd NA 4 0 0 0 0Respiratory syncytial virus NA NA 1 0 0 0 0Parainfluenza virus NA NA 1 0 0 0 0Enterovirus NA NA 1 0 0 0 0Rhinovirus NA NA 2 0 0 0 0Adenovirus NA NA 1 0 0 0 0
Nasopharyngeal swab Influenza A virus H1N1 Sensitive 20 20 0 0 0Influenza A virus H1N1 Resistant 20 0 20 0 0Influenza A virus H3N2 Sensitive 20 0 0 20 0Influenza A virus H3N2 Resistant 20 0 0 0 20Influenza B virus NA NA 5 0 0 0 0Negative sample NA NA 10 0 0 0 0Respiratory syncytial virus NA NA 4 0 0 0 0
a Viruses were initially detected by virus isolation and PCR with specific primers.b Typed and subtyped by hemagglutinin inhibition assay with vaccine strain antisera.c Resistant strains of both subtypes had a serine-to-asparagine change in residue 31 of the M2 ion channel protein.d NA, not addressed.
TABLE 3. Rate of amantadine resistance among influenza A virusisolates in the 2007–2008 season in Japana
Subtype
No. of isolatesRate (%) of amantadine-
resistant virusesInfluenza Avirus
Amantadine-resistantvirusb
H1N1 663 411 62.0H3N2 56 56 100.0
a Samples were collected from individuals in the Hokkaido, Gunma, Niigata,Kyoto, Hyogo, and Nagasaki Prefectures in Japan.
b Amantadine resistance was conferred by the Ser31Asn mutation in the M2gene.
This study was supported by a research grant from the KurozumiMedical Foundation.
We thank Junko Yamamoto, Kazuhide Okazawa, and KentaroMoro of Takara Bio Inc. for technical assistance in developing thecycling probe assay. We thank clinical doctors Rika Sugai in HokkaidoPrefecture; Takashi Kawashima in Gunma Prefecture;, Isamu Sato inNiigata Prefecture; Shigeyoshi Hibi, Satoshi Ikushima, Fumitomo Fu-jiwara, and Kentaro Tsunamoto in Kyoto Prefecture; Tetsuo Hashidain Hyogo Prefecture; and Hironori Masaki, Yutaka Shirahige, Hide-humi Ishikawa, Satoshi Degawa, Noritika Asou, and Hironobu Ka-geura in Nagasaki Prefecture. We are grateful to Akinori Miyashitaand Ryozo Kuwano in the Department of Molecular Genetics, Biore-source Science Branch, Center for Bioresources, Brain Research In-stitute, Niigata University, for utilization of their DNA sequencer. Wethank Akemi Watanabe for technical assistance with virus isolationand Yoshiko Kato for intensive secretarial work.
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