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  • Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibitors

    Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibitors

    Hatakeyama, MD, PhD; <NOBR>Norio Sugaya, MD</NOBR>; <NOBR>Mutsumi Ito, DVM</NOBR>; <NOBR>Masahiko Yamazaki, MD</NOBR>; <NOBR>Masataka Ichikawa, MD</NOBR>; <NOBR>Kazuhiro Kimura, MD, PhD</NOBR>; <NOBR>Maki Kiso, DVM</NOBR>; <NOBR>Hideaki Shimizu</NOBR>; <NOBR>Chiharu Kawakami</NOBR>; <NOBR>Kazuhiko Koike, MD, PhD</NOBR>; <NOBR>Keiko Mitamura, MD</NOBR>; <NOBR>Yoshihiro Kawaoka, DVM, PhD</NOBR>


    JAMA. 2007;297:1435-1442.
    <!-- null -->ABSTRACT <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <!--startindex-->Context Very little is known about the frequency of generation<SUP> </SUP>and transmissibility of influenza B viruses with reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors. Furthermore, transmission of resistant<SUP> </SUP>virus, whether influenza A or B, has not been recognized to<SUP> </SUP>date.<SUP> </SUP>
    Objective To assess the prevalence and transmissibility<SUP> </SUP>of influenza B viruses with reduced sensitivity to neuraminidase<SUP> </SUP>inhibitors.<SUP> </SUP>
    Design, Setting, and Patients Investigation of the neuraminidase<SUP> </SUP>inhibitor sensitivity of influenza B isolates from 74 children<SUP> </SUP>before and after oseltamivir therapy and from 348 untreated<SUP> </SUP>patients with influenza (including 66 adults) seen at 4 community<SUP> </SUP>hospitals in Japan during the 2004-2005 influenza season. Four<SUP> </SUP>hundred twenty-two viruses from untreated patients and 74 samples<SUP> </SUP>from patients after oseltamivir therapy were analyzed.<SUP> </SUP>
    Main Outcome Measure Sialidase inhibition assay was used<SUP> </SUP>to test the drug sensitivities of influenza B viruses. The neuraminidase<SUP> </SUP>and hemagglutinin genes of viruses showing reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors were sequenced to identify mutations<SUP> </SUP>that have the potential to confer reduced sensitivity to these<SUP> </SUP>drugs.<SUP> </SUP>
    Results In 1 (1.4%) of the 74 children who had received<SUP> </SUP>oseltamivir, we identified a variant with reduced drug sensitivity<SUP> </SUP>possessing a Gly402Ser neuraminidase substitution. We also identified<SUP> </SUP>variants with reduced sensitivity carrying an Asp198Asn, Ile222Thr,<SUP> </SUP>or Ser250Gly mutation in 7 (1.7%) of the 422 viruses from untreated<SUP> </SUP>patients. Review of the clinical and viral genetic information<SUP> </SUP>available on these 7 patients indicated that 4 were likely infected<SUP> </SUP>in a community setting, while the remaining 3 were probably<SUP> </SUP>infected through contact with siblings shedding the mutant viruses.<SUP> </SUP>
    Conclusions In this population, influenza B viruses with<SUP> </SUP>reduced sensitivity to neuraminidase inhibitors do not arise<SUP> </SUP>as frequently as resistant influenza A viruses. However, they<SUP> </SUP>appear to be transmitted within communities and families, requiring<SUP> </SUP>continued close monitoring.<SUP> </SUP>

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    INTRODUCTION <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    Clinical use of any antiviral drug can lead to the development<SUP> </SUP>of drug-resistant viruses.<SUP>1-2</SUP> Two neuraminidase inhibitors,<SUP> </SUP>zanamivir and oseltamivir, are effective against influenza and<SUP> </SUP>are used extensively for treatment of this infection, especially<SUP> </SUP>in Japan.<SUP>3-4</SUP> We and others have documented the emergence of<SUP> </SUP>oseltamivir-resistant type A viruses, including H5N1 subtypes,<SUP>3,<SUP> </SUP>5-7</SUP> but similar information on influenza B viruses is limited.<SUP> </SUP>Although influenza B viruses usually cause smaller epidemics<SUP> </SUP>than type A viruses, they are nonetheless associated with annual<SUP> </SUP>outbreaks of illness and excess mortality rates worldwide.<SUP>8</SUP><SUP> </SUP>
    Of the 2 type B viruses with reduced sensitivity that have been<SUP> </SUP>reported, 1 carried an Arg152Lys mutation (amino acid numbering<SUP> </SUP>system adapted for an N2 neuraminidase<SUP>9</SUP>; N2 numbering is used<SUP> </SUP>throughout this article) in its neuraminidase and was isolated<SUP> </SUP>from an immunocompromised child treated with zanamivir.<SUP>10</SUP> The<SUP> </SUP>other virus had an Asp198Asn neuraminidase mutation and was<SUP> </SUP>isolated from an immunocompromised child treated with oseltamivir.<SUP>11</SUP><SUP> </SUP>The known neuraminidase substitutions identified in drug-resistant<SUP> </SUP>viruses from humans tend to be type- or subtype-specific: Glu119Val,<SUP> </SUP>Arg292Lys, and Asn294Ser in the neuraminidase of the N2 subtype;<SUP> </SUP>His274Tyr in that of the N1 subtype (including not only H1N1<SUP> </SUP>viruses but also H5N1 viruses)<SUP>6-7</SUP>; and Arg152Lys and Asp198Asn<SUP> </SUP>in that of the type B virus.<SUP>10-11</SUP> All of these substitutions<SUP> </SUP>have been identified at catalytic or framework residues in the<SUP> </SUP>sialidase active sites of the neuraminidase protein,<SUP>9</SUP> which<SUP> </SUP>are relatively conserved in all type A and type B neuraminidase<SUP> </SUP>molecules and are the targets of neuraminidase inhibitors.<SUP> </SUP>
    The results of cell culture experiments in which multiple passages<SUP> </SUP>were required for the generation of viruses resistant to neuraminidase<SUP> </SUP>inhibitors<SUP>12</SUP> have suggested that resistance to these agents<SUP> </SUP>arises infrequently. It is thus reasonable that a low frequency<SUP> </SUP>of oseltamivir resistance?5.5% for children aged 1 to<SUP> </SUP>12 years infected with type A viruses and 0% in children infected<SUP> </SUP>with type B viruses?was observed in a clinical trial.<SUP>13</SUP><SUP> </SUP>More recent studies, however, have demonstrated a higher-than-expected<SUP> </SUP>rate of drug-resistant influenza A virus generation in oseltamivir-treated<SUP> </SUP>children: 18% of children with H3N2 virus infection<SUP>5</SUP> and 16%<SUP> </SUP>of those with H1N1 virus infection<SUP>3</SUP> harbored resistant variants<SUP> </SUP>with neuraminidase mutations after drug treatment.<SUP> </SUP>
    The biological fitness of viruses resistant to neuraminidase<SUP> </SUP>inhibitors differs depending on the type of mutations in the<SUP> </SUP>neuraminidase. In mouse or ferret studies, the infectivity and<SUP> </SUP>transmissibility of clinical isolates of human influenza A viruses<SUP> </SUP>carrying the Arg292Lys or the His274Tyr mutation in their neuraminidases<SUP> </SUP>were compromised<SUP>14-17</SUP>; a similar result was reported for a mutant<SUP> </SUP>type B virus with the Arg152Lys mutation in ferrets.<SUP>10</SUP> By contrast,<SUP> </SUP>a resistant virus with the Glu119Val mutation infected ferrets<SUP> </SUP>and was transmitted among these animals as efficiently as the<SUP> </SUP>wild-type virus.<SUP>17</SUP> Also, influenza B virus carrying the Asp198Asn<SUP> </SUP>substitution grows as well as the wild-type virus in this animal<SUP> </SUP>model.<SUP>18</SUP> Nonetheless, the pathogenicity and transmissibility<SUP> </SUP>of neuraminidase inhibitor?resistant viruses remain unanswered<SUP> </SUP>questions with significance for predicting pandemic strains.<SUP> </SUP>
    In Japan, the neuraminidase inhibitors zanamivir and oseltamivir<SUP> </SUP>were approved for clinical use in 2000 and 2001, respectively,<SUP> </SUP>and are now used more extensively there than anywhere else in<SUP> </SUP>the world.<SUP>3-4</SUP> In the winter of 2004-2005, an influenza B virus<SUP> </SUP>caused a widespread epidemic in Japan, creating opportunities<SUP> </SUP>to assess in a natural setting the prevalence and transmissibility<SUP> </SUP>of influenza B viruses with reduced sensitivity to neuraminidase<SUP> </SUP>inhibitors. The results reported here suggest a low but appreciable<SUP> </SUP>rate of emergence of type B viruses with reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors and their person-to-person transmission,<SUP> </SUP>both in the community and within single families.<SUP> </SUP>
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    METHODS <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    Study Population and Settings
    Pharyngeal or nasal swabs for influenza B virus analysis were<SUP> </SUP>obtained from patients who visited the pediatric services at<SUP> </SUP>4 community hospitals in Japan during the 2004-2005 influenza<SUP> </SUP>season. To identify the frequency of developing neuraminidase<SUP> </SUP>inhibitor?resistant influenza B viruses after oseltamivir<SUP> </SUP>therapy, we analyzed paired specimens from each patient, one<SUP> </SUP>taken at the initial hospital visit (pretreatment samples) and<SUP> </SUP>the other during treatment with oseltamivir (posttreatment samples).<SUP> </SUP>Patients diagnosed with influenza B virus infection by a rapid<SUP> </SUP>diagnostic kit who received oseltamivir therapy, and from whom<SUP> </SUP>we could obtain at least 2 sequential samples for virus isolation,<SUP> </SUP>were enrolled in the first series of studies (Figure 1). In<SUP> </SUP>the second series, we attempted to assess in a community setting<SUP> </SUP>the prevalence of influenza B viruses with reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors. To this end, we obtained samples<SUP> </SUP>before oseltamivir treatment from patients who visited the 4<SUP> </SUP>community hospitals. The influenza B viruses isolated from these<SUP> </SUP>samples and the viruses from the pretreatment samples from the<SUP> </SUP>first series of studies were combined and analyzed (Figure 1).<SUP> </SUP>Because these studies included patients who visited community<SUP> </SUP>hospitals, several family members sought consultation at the<SUP> </SUP>same facility.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=1 width="100%" bgColor=#6a90aa border=0><TBODY><TR><TD align=middle><TABLE cellSpacing=0 cellPadding=0 width="100%" align=center bgColor=#6a90aa border=0><TBODY><TR><TD vAlign=top align=left bgColor=#dce4ee><TABLE cellSpacing=0 cellPadding=10 width=250 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center align=middle bgColor=#dce4ee>
    View larger version (32K):
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    </TD></TR></TBODY></TABLE></TD><TD width=1 bgColor=#6a90aa> </TD><TD vAlign=top width=1000 bgColor=#ffffff><TABLE cellSpacing=0 cellPadding=10 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top align=left bgColor=#ffffff>Figure 1. Flowchart of Participants
    IC<SUB>50</SUB> indicates concentration of neuraminidase inhibitor required to inhibit 50% of the sialidase activity of influenza B viruses.

    </TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    <SUP></SUP>
    Oral informed consent was obtained from the parents of all patients.<SUP> </SUP>This study was conducted with the approval of the ethics committees<SUP> </SUP>of 3 of the 4 hospitals; in the case of the single hospital<SUP> </SUP>in which an ethics committee did not exist, the activities of<SUP> </SUP>the study were undertaken under the auspices of the informed<SUP> </SUP>consent.<SUP> </SUP>
    Clinical Specimens and Viruses
    Pharyngeal or nasal swabs for influenza B virus isolation were<SUP> </SUP>obtained by attending physicians after informed consent was<SUP> </SUP>obtained. The viruses isolated were stored at ?80?C<SUP> </SUP>until used. The viral isolates were used as mixed populations<SUP> </SUP>without plaque purification. Madin-Darby canine kidney cells<SUP> </SUP>overexpressing the -galactoside 2,6-sialyltransferase I (ST6Gal<SUP> </SUP>I) gene<SUP>19</SUP> were used for viral isolation and plaque assay. These<SUP> </SUP>cells support the growth of clinical isolates of human influenza<SUP> </SUP>viruses better than unmanipulated Madin-Darby canine kidney<SUP> </SUP>cells.<SUP>19</SUP> To assess the sensitivity of the influenza B viruses<SUP> </SUP>to neuraminidase inhibitors, the concentration of neuraminidase<SUP> </SUP>inhibitor required to inhibit 50% of the sialidase activity<SUP> </SUP>of the viruses (IC<SUB>50</SUB>) was determined with pretreatment and posttreatment<SUP> </SUP>influenza B isolates using a sialidase inhibition assay.<SUP>19-20</SUP><SUP> </SUP>The IC<SUB>50</SUB> values demonstrated in this study were assessed for<SUP> </SUP>viruses present in culture supernatant fluids, without plaque<SUP> </SUP>purification of the isolates. For strains demonstrating reduced<SUP> </SUP>susceptibility to the inhibitors, we sequenced their neuraminidase<SUP> </SUP>and hemagglutinin genes.<SUP> </SUP>
    Sialidase Sensitivity to Neuraminidase Inhibitors
    Sialidase sensitivities of influenza B viruses to neuraminidase<SUP> </SUP>inhibitors were evaluated with a sialidase inhibition assay<SUP> </SUP>as described previously.<SUP>19-20</SUP> Briefly, 2'-(4-methylumbelliferyl)--D-N-acetylneuraminic<SUP> </SUP>acid (MUNANA; Sigma, St Louis, Mo) at a final concentration<SUP> </SUP>of 0.1 mmol/L was used as a substrate. Ten microliters of the<SUP> </SUP>virus dilution (predetermined to contain sialidase activity<SUP> </SUP>in the range of 800-1200 fluorescence units in this assay) and<SUP> </SUP>10 ?L of the neuraminidase inhibitor (0.01 nmol/L to 10<SUP> </SUP>?mol/L) in calcium-MES buffer (33 mmol/L 2-[N-morpholino]ethanesulfonic<SUP> </SUP>acid, 4 mmol/L CaCl<SUB>2</SUB>, pH 6.0) were mixed and incubated at 37?C<SUP> </SUP>for 30 minutes, followed by the addition of 30 ?L of the<SUP> </SUP>substrate. The mixtures were further incubated at 37?C for<SUP> </SUP>60 minutes, and the reaction was stopped by adding 150 ?L<SUP> </SUP>of 0.1 mol/L sodium hydroxide in 80% ethanol (pH 10.0). We quantified<SUP> </SUP>fluorescence at an excitation wavelength of 360 nm and an emission<SUP> </SUP>wavelength of 465 nm. The IC<SUB>50</SUB> value was determined by extrapolation<SUP> </SUP>of the relation between the concentration of inhibitor and the<SUP> </SUP>proportion of fluorescence inhibition. Results are reported<SUP> </SUP>as the mean of duplicate IC<SUB>50</SUB> values. Oseltamivir carboxylate<SUP> </SUP>(GS4071; Roche Products, Basel, Switzerland), the active metabolite<SUP> </SUP>of the ethyl ester prodrug oseltamivir phosphate, and zanamivir<SUP> </SUP>(Relenza; GlaxoSmithKline, Brentford, UK) were used as neuraminidase<SUP> </SUP>inhibitors.<SUP> </SUP>
    Sequence Analyses of the Neuraminidase and Hemagglutinin Genes
    Viral RNA was extracted from virus in cell-culture supernatant<SUP> </SUP>fluid with an RNA extraction kit (ISOGEN-LS; Nippon Gene, Tokyo,<SUP> </SUP>Japan), without prior plaque purification of the virus. Reverse<SUP> </SUP>transcription was performed with reverse transcriptase (SUPERSCRIPT<SUP> </SUP>III; Invitrogen, Carlsbad, Calif) and a primer complementary<SUP> </SUP>to the 3' end of the viral RNA (5'-AGCAGAAGCAGAGCA-3'). The<SUP> </SUP>cDNA products were then used to amplify the viral neuraminidase<SUP> </SUP>and hemagglutinin genes by a standard polymerase chain reaction<SUP> </SUP>method (Pfu Ultra DNA Polymerase; Stratagene, La Jolla, Calif)<SUP> </SUP>(primer sequences and amplification conditions available from<SUP> </SUP>the authors on request). We cloned these products into the pCRBlunt<SUP> </SUP>II-TOPO vector (Invitrogen) and transformed them into TOP10<SUP> </SUP>chemically competent cells (Invitrogen). Transformed cells were<SUP> </SUP>grown on Luria broth agar containing 50 mg/L of kanamycin, after<SUP> </SUP>which the kanamycin-resistant colonies were selected and incubated<SUP> </SUP>in Luria broth at 37?C overnight in a shaking incubator.<SUP> </SUP>Plasmid DNA was extracted with the MagExtractor-plasmid system<SUP> </SUP>(TOYOBO, Osaka, Japan). We determined complete sequences of<SUP> </SUP>the neuraminidase and hemagglutinin genes by cycle sequencing<SUP> </SUP>with dye-terminator chemistry (Big Dye; Applied Biosystems,<SUP> </SUP>Foster City, Calif) on the Applied Biosystems 3100 or 3130Xl<SUP> </SUP>auto sequencer using M13F-20, neuraminidase-specific, or hemagglutinin-specific<SUP> </SUP>primers. For each sample, 5 to 8 cDNA clones of the neuraminidase<SUP> </SUP>and hemagglutinin genes were analyzed.<SUP> </SUP>
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    RESULTS <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    Study Population
    A total of 75 pairs of pretreatment and posttreatment samples<SUP> </SUP>were obtained from pediatric patients. One pair was excluded<SUP> </SUP>because influenza virus was not isolated from either the pretreatment<SUP> </SUP>or posttreatment sample. Thus, 74 patients aged 0 to 15 years<SUP> </SUP>(median, 3 years) with influenza B virus infection were enrolled<SUP> </SUP>in the study (Figure 1). All were treated with oseltamivir for<SUP> </SUP>5 days. Eighteen children received 2 mg/kg of body weight twice<SUP> </SUP>daily, while the remaining 56 children received weight-based<SUP> </SUP>unit doses<SUP>21</SUP> (body weight 15 kg, 30 mg twice daily; >15-23<SUP> </SUP>kg, 45 mg twice daily; >23-40 kg, 60 mg twice daily; and<SUP> </SUP>>40 kg, 75 mg twice daily).<SUP> </SUP>
    In the second series of experiments, we analyzed a total of<SUP> </SUP>442 influenza B viruses isolated from patients prior to treatment<SUP> </SUP>(348 patients plus the above-mentioned 74 patients) during the<SUP> </SUP>2004-2005 influenza season (Figure 1). Of the 422 patients,<SUP> </SUP>356 were children aged 0 to 15 years (median, 5 years); the<SUP> </SUP>remaining 66 were adults 16 years or older (range, 17 to 61<SUP> </SUP>years; median, 34 years).<SUP> </SUP>
    Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibitors After Oseltamivir Treatment
    Viruses were recovered from all pretreatment samples and from<SUP> </SUP>65 posttreatment samples collected from the 74 children who<SUP> </SUP>had received a full course of oseltamivir. In 1 child (1.4%),<SUP> </SUP>the IC<SUB>50</SUB> value of the posttreatment isolate tested against zanamivir<SUP> </SUP>and oseltamivir increased by 7.1-fold and 3.9-fold, respectively,<SUP> </SUP>compared with results for the virus isolated before treatment<SUP> </SUP>(Table, patient 1). This child was an immunocompetent 7-year-old<SUP> </SUP>boy who had received oseltamivir immediately after diagnosis.<SUP> </SUP>The virus with reduced sensitivity to the neuraminidase inhibitors<SUP> </SUP>was isolated from a pharyngeal swab collected on day 3 after<SUP> </SUP>initiation of oseltamivir therapy. To understand the molecular<SUP> </SUP>basis of the observed reduced sensitivity to the drugs, we molecularly<SUP> </SUP>cloned the neuraminidase gene from the virus exhibiting this<SUP> </SUP>property. The sequence analysis revealed an amino acid substitution,<SUP> </SUP>Gly402Ser, in 7 of the 8 cDNA clones of the neuraminidase gene.<SUP> </SUP>No other difference was observed in the amino acid sequence<SUP> </SUP>of the neuraminidase and hemagglutinin proteins between the<SUP> </SUP>wild-type parent and the posttreatment mutant virus. The neuraminidase<SUP> </SUP>mutation Gly402Ser was located near the sialidase enzymatic<SUP> </SUP>center.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=1 width="100%" bgColor=#6a90aa border=0><TBODY><TR><TD align=middle><TABLE cellSpacing=0 cellPadding=0 width="100%" align=center bgColor=#6a90aa border=0><TBODY><TR><TD vAlign=top align=left bgColor=#dce4ee><TABLE cellSpacing=0 cellPadding=10 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center align=middle bgColor=#dce4ee>View this table:
    <NOBR>[in this window]
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    </TD></TR></TBODY></TABLE></TD><TD width=1 bgColor=#6a90aa> </TD><TD vAlign=top width=1000 bgColor=#ffffff><TABLE cellSpacing=0 cellPadding=10 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top align=left bgColor=#ffffff>Table. Influenza B Isolates With Reduced Sensitivity to Neuraminidase Inhibitors Before or After Antiviral Treatment
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    <SUP></SUP>
    Influenza B Viruses With Reduced Susceptibility to Neuraminidase Inhibitors Detected in Patients Prior to Treatment
    The median (interquartile range) IC<SUB>50</SUB> values for influenza B<SUP> </SUP>viruses isolated from 422 untreated patients during the 2004-2005<SUP> </SUP>influenza season and tested against both oseltamivir and zanamivir<SUP> </SUP>with the sialidase inhibition assay were 70.5 (55.8-85.1) nmol/L<SUP> </SUP>and 10.1 (7.0-15.8) nmol/L, respectively (Figure 2). Considering<SUP> </SUP>the level of increase in the IC<SUB>50</SUB> value of the virus from the<SUP> </SUP>posttreatment sample as compared with that of the original virus<SUP> </SUP>obtained before oseltamivir therapy from patient 1, we regarded<SUP> </SUP>viruses whose IC<SUB>50</SUB> values were higher than 1.5 times interquartile<SUP> </SUP>range above the third quartile as drug-resistant (Figure 2).<SUP> </SUP>Using this criterion, 7 (1.7%) of the 422 influenza B viruses<SUP> </SUP>isolated from untreated patients (Table, patients 2-8) were<SUP> </SUP>found to have reduced sensitivity to zanamivir, oseltamivir,<SUP> </SUP>or both. Each of the 7 isolates with reduced sensitivity contained<SUP> </SUP>amino acid substitutions in the neuraminidase at the sialidase<SUP> </SUP>active center, by comparison with the consensus type B neuraminidase<SUP> </SUP>sequence: 3 had Asp198Asn mutations, 3 had Ile222Thr mutations,<SUP> </SUP>and 1 had a Ser250Gly mutation (Table). None of these patients<SUP> </SUP>had an underlying disease and none had received immunosuppressive<SUP> </SUP>drugs.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=1 width="100%" bgColor=#6a90aa border=0><TBODY><TR><TD align=middle><TABLE cellSpacing=0 cellPadding=0 width="100%" align=center bgColor=#6a90aa border=0><TBODY><TR><TD vAlign=top align=left bgColor=#dce4ee><TABLE cellSpacing=0 cellPadding=10 width=250 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center align=middle bgColor=#dce4ee>
    View larger version (17K):
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    </TD></TR></TBODY></TABLE></TD><TD width=1 bgColor=#6a90aa> </TD><TD vAlign=top width=1000 bgColor=#ffffff><TABLE cellSpacing=0 cellPadding=10 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top align=left bgColor=#ffffff>Figure 2. Distribution of IC<SUB>50</SUB> Values for Influenza B Viruses Isolated From 422 Patients Prior to Treatment, Tested Against Oseltamivir Carboxylate and Zanamivir
    IC<SUB>50</SUB> indicates concentration of neuraminidase inhibitor required to inhibit 50% of the sialidase activity of influenza B viruses. The IC<SUB>50</SUB> values were determined by the sialidase inhibition assay.<SUP>19-20</SUP> Boxes indicate interquartile ranges (IQRs); vertical lines, median values; and bars to the left and right of boxes, 1.5 times IQR. The median (IQR) IC<SUB>50</SUB> values of the 422 type B viruses from patients before treatment were 70.5 (55.8-85.1) nmol/L for oseltamivir and 10.1 (7.0-15.8) nmol/L for zanamivir. Patient numbers in blue indicate those untreated patients with influenza B viral isolates found to have reduced sensitivity to oseltamivir (left) and/or zanamivir (right).

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    <SUP></SUP>
    An 8-year-old boy (patient 2) was diagnosed with influenza B<SUP> </SUP>virus infection 6 days before the onset of influenza B infection<SUP> </SUP>in his 1-year-old sister (patient 3). The IC<SUB>50</SUB> values for the<SUP> </SUP>pretreatment isolate from patient 2 (47.4 nmol/L for zanamivir<SUP> </SUP>and 237.3 nmol/L for oseltamivir) indicated reduced sensitivity<SUP> </SUP>of the isolate to these compounds. We identified a neuraminidase<SUP> </SUP>mutation at position 198 (Asp198Asn) in all of the 8 cDNA clones<SUP> </SUP>of the neuraminidase gene of this isolate. The virus isolated<SUP> </SUP>from patient 3 also showed reduced sensitivity to zanamivir<SUP> </SUP>and oseltamivir (Table). Sequence analyses of the neuraminidase<SUP> </SUP>and hemagglutinin genes were identical between viruses isolated<SUP> </SUP>from patients 2 and 3, including the presence of an Asp198Asn<SUP> </SUP>mutation in the neuraminidase protein (in all of the 8 cDNA<SUP> </SUP>clones of the neuraminidase gene of the isolate from patient<SUP> </SUP>3). Thus, it may be possible that patient 2 was infected with<SUP> </SUP>an influenza B virus having reduced sensitivity to neuraminidase<SUP> </SUP>inhibitors and then transmitted the virus to his sister, patient<SUP> </SUP>3.<SUP> </SUP>
    Another influenza B virus possessing the Asp198Asn mutation<SUP> </SUP>in the neuraminidase was isolated from patient 4 (aged 6 years,<SUP> </SUP>female) on February 9, 2005, before oseltamivir treatment (Table).<SUP> </SUP>This neuraminidase mutation was observed in all of the 7 cDNA<SUP> </SUP>clones of this isolate. Her 4-year-old sister, from whom a wild-type<SUP> </SUP>influenza B virus had been isolated on January 31, 2005, had<SUP> </SUP>received oseltamivir from January 31 to February 4 (Table).<SUP> </SUP>The sequences of both the neuraminidase and hemagglutinin genes<SUP> </SUP>from the 2 patients were identical, with the exception of a<SUP> </SUP>neuraminidase substitution at amino acid position 198. Thus,<SUP> </SUP>it is possible that a drug-resistant virus might have arisen<SUP> </SUP>in the 4-year-old sister during oseltamivir therapy and been<SUP> </SUP>transmitted to patient 4. However, because we were unable to<SUP> </SUP>obtain samples after oseltamivir therapy from the 4-year-old<SUP> </SUP>sister, we cannot prove that this was indeed the case.<SUP> </SUP>
    The IC<SUB>50</SUB> values for the Asp198Asn mutants ranged from 42 to<SUP> </SUP>62 nmol/L (zanamivir) and from 204 to 255 nmol/L (oseltamivir),<SUP> </SUP>indicating that the mutation was associated with approximately<SUP> </SUP>3- to 4-fold and 4- to 6-fold reductions in drug sensitivity,<SUP> </SUP>respectively, compared with the corresponding median IC<SUB>50</SUB> values<SUP> </SUP>for the entire group of type B viruses. The variant with reduced<SUP> </SUP>sensitivity to oseltamivir and with the Asp198Asn mutation was<SUP> </SUP>recently identified by Gubareva<SUP>11</SUP> and by Mishin et al<SUP>18</SUP> in a<SUP> </SUP>posttreatment sample from an immunocompromised child with influenza<SUP> </SUP>B virus, further supporting the notion that this mutation was<SUP> </SUP>introduced during oseltamivir therapy and that it reduced sensitivity<SUP> </SUP>to the neuraminidase inhibitors.<SUP> </SUP>
    Several type B viruses carrying other neuraminidase mutations<SUP> </SUP>with reduced sensitivity were also identified in other patients.<SUP> </SUP>Viruses carrying an Ile222Thr mutation were isolated from pretreatment<SUP> </SUP>samples of 3 patients: patients 5 and 6 (siblings) and patient<SUP> </SUP>7 (Table). The nucleotide sequences of the neuraminidase and<SUP> </SUP>hemagglutinin genes of isolates from these patients were identical,<SUP> </SUP>and the neuraminidase Ile222Thr mutation was observed in all<SUP> </SUP>of the cDNA clones of each viral NA gene. The IC<SUB>50</SUB> values for<SUP> </SUP>viruses carrying the Ile222Thr mutation ranged from 443 to 514<SUP> </SUP>nmol/L (oseltamivir), representing a 6- to 7-fold reduction<SUP> </SUP>in sensitivity compared with the median IC<SUB>50</SUB> values for type<SUP> </SUP>B viruses (Table). This mutation appeared to lack strong effect<SUP> </SUP>on viral sensitivity to zanamivir. An influenza B virus with<SUP> </SUP>reduced sensitivity to the neuraminidase inhibitors was also<SUP> </SUP>isolated from patient 8, a 22-year-old woman (Table). The isolate<SUP> </SUP>from patient 8 possessed a Ser250Gly mutation in all of the<SUP> </SUP>7 cDNA clones of the neuraminidase gene. The Ser250Gly mutation<SUP> </SUP>conferred an approximately 19-fold resistance to zanamivir (when<SUP> </SUP>compared with the median type B virus IC<SUB>50</SUB> value) but did not<SUP> </SUP>reduce sensitivity to oseltamivir.<SUP> </SUP>
    None of the family members of patients 2, 5, 7, and 8 were affected<SUP> </SUP>by influenza B virus before onset of their symptoms, suggesting<SUP> </SUP>that they were possibly infected with mutants with reduced drug<SUP> </SUP>sensitivity circulating in the community. These results suggest<SUP> </SUP>that influenza B viruses with reduced sensitivity to neuraminidase<SUP> </SUP>inhibitors might possibly be transmitted from person to person,<SUP> </SUP>not only within single families but also among members of the<SUP> </SUP>same community.<SUP> </SUP>
    Finally, we did not observe any appreciable differences in the<SUP> </SUP>clinical course of viral infection between patients infected<SUP> </SUP>with wild-type viruses or those with reduced sensitivity to<SUP> </SUP>neuraminidase inhibitors. Mean durations of fever after antiviral<SUP> </SUP>therapy were 2.4, 2.6, and 2.0 days in patients infected with<SUP> </SUP>wild-type viruses (n = 32), those infected with reduced<SUP> </SUP>sensitivity to neuraminidase inhibitors (patient 2 [3 days],<SUP> </SUP>patient 3 [5.5 days], patient 7 [1 day], patient 8 [1 day]),<SUP> </SUP>and the patient with the variant that developed during therapy<SUP> </SUP>(patient 1), respectively. Similarly, we did not find an appreciable<SUP> </SUP>difference in the extent of virus shedding (duration and titer)<SUP> </SUP>between patients infected with a drug-resistant virus and those<SUP> </SUP>infected with a drug-sensitive virus. However, the number of<SUP> </SUP>patients infected with viruses with reduced drug sensitivity<SUP> </SUP>is too small to assess the statistical significance of the effect<SUP> </SUP>of drug resistance on virus shedding.<SUP> </SUP>
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    COMMENT <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    We demonstrated that influenza B viruses with reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors can emerge during routine therapy<SUP> </SUP>and that such mutants appear to be transmitted from person to<SUP> </SUP>person, not only within the same family but possibly through<SUP> </SUP>community contacts as well. The rate of generation of influenza<SUP> </SUP>B viruses with reduced drug sensitivity during oseltamivir treatment<SUP> </SUP>in this study, 1.4%, is lower than that among influenza A viruses<SUP> </SUP>(5.5%-18%).<SUP>3, 5, 13</SUP> This discrepancy could reflect the higher<SUP> </SUP>doses of oseltamivir used in our study (76% of the patients<SUP> </SUP>received weight-based unit doses of the drug, in contrast to<SUP> </SUP>the twice-daily 2 mg/kg dose uniformly administered in previous<SUP> </SUP>Japanese studies<SUP>3, 5</SUP>).<SUP> </SUP>
    Four mutations in the type B neuraminidase reduced sensitivity<SUP> </SUP>to neuraminidase inhibitors: Asp198Asn, Ile222Thr, Ser250Gly,<SUP> </SUP>and Gly402Ser substitutions. Residues 198, 222, and 250 are<SUP> </SUP>located in the framework of the neuraminidase active site, which<SUP> </SUP>interacts with the catalytic residues by hydrogen bonds or salt<SUP> </SUP>bridges (Figure 3).<SUP>9, 22</SUP> The framework residues Asp198 and Ser<SUP> </SUP>250 (the corresponding residue in the type A neuraminidase is<SUP> </SUP>Ala) interact with the catalytic residues Arg152 and Arg224,<SUP> </SUP>respectively, and Ile222 forms a hydrophobic pocket into which<SUP> </SUP>the substrate fits.<SUP>22</SUP> The novel substitution detected in the<SUP> </SUP>neuraminidase of a virus recovered from an oseltamivir-treated<SUP> </SUP>patient in this study occurred at residue 402. Although Gly402<SUP> </SUP>is not a catalytic or framework residue, it is located near<SUP> </SUP>the sialidase enzymatic center (Figure 3). Therefore, Gly402Ser<SUP> </SUP>substitution may alter the interaction of the enzymatic center<SUP> </SUP>and the neuraminidase inhibitors, resulting in reduced drug<SUP> </SUP>sensitivity. Further analysis is needed to understand the interaction<SUP> </SUP>of this residue with sialic acid.<SUP> </SUP>
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    <TABLE cellSpacing=0 cellPadding=1 width="100%" bgColor=#6a90aa border=0><TBODY><TR><TD align=middle><TABLE cellSpacing=0 cellPadding=0 width="100%" align=center bgColor=#6a90aa border=0><TBODY><TR><TD vAlign=top align=left bgColor=#dce4ee><TABLE cellSpacing=0 cellPadding=10 width=250 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center align=middle bgColor=#dce4ee>
    View larger version (86K):
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    </TD></TR></TBODY></TABLE></TD><TD width=1 bgColor=#6a90aa> </TD><TD vAlign=top width=1000 bgColor=#ffffff><TABLE cellSpacing=0 cellPadding=10 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top align=left bgColor=#ffffff>Figure 3. Locations of Mutations on the 3-Dimensional Structure of Neuraminidase
    Three-dimensional structure of the complex between influenza virus B/Beijing/1/87 neuraminidase and zanamivir (Molecular Modeling DataBase Identifier: 10147; Protein DataBank Identifier: 1A4G). The locations of the neuraminidase mutations* identified in the present study (aspartic acid 198 [Asp198], isoleucine 222 [Ile222], serine 250 [Ser250], and glycine 402 [Gly402]) that are associated with decreased drug sensitivity are marked in yellow. These mutations are located at or near the sialidase active site, where neuraminidase inhibitors bind. (See online interactive neuraminidase model at http://jama.com/cgi/content/full/297/13/1435/DC1.) <SUP></SUP>
    *Amino acids are numbered according to the N2 NA numbering system. Corresponding influenza virus B/Beijing/1/87 positions are Asp196, Ile220, Ser248, and Gly406; for currently circulating type B viruses, Asp197, Ile221, Ser249, and Gly407, respectively.

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    <SUP></SUP>
    The framework mutations we identified appear to reduce oseltamivir<SUP> </SUP>sensitivity at a moderate level as compared with the catalytic<SUP> </SUP>Arg292Lys mutation. When tested against oseltamivir, the IC<SUB>50</SUB><SUP> </SUP>values for H3N2 viruses with the framework mutation Glu119Val<SUP> </SUP>or Asn294Ser were 239 nmol/L or 106 nmol/L,<SUP>5</SUP> respectively, while<SUP> </SUP>that for an H5N1 strain with the framework mutation His274Tyr<SUP> </SUP>was 763 nmol/L.<SUP>6</SUP> On the other hand, the catalytic Arg292Lys<SUP> </SUP>mutation in N2 viruses conferred a high level of resistance<SUP> </SUP>to oseltamivir (IC<SUB>50</SUB> >10 000 nmol/L).<SUP>5</SUP> Viruses with<SUP> </SUP>framework mutations might have the ability to be transmitted<SUP> </SUP>among experimental animals, as has been shown with type A variants<SUP> </SUP>with a framework mutation at position 119 or 274.<SUP>17</SUP> These results<SUP> </SUP>suggest that influenza viruses with a framework mutation in<SUP> </SUP>the neuraminidase might be of clinical concern, even though<SUP> </SUP>their IC<SUB>50</SUB> values are lower than those of viruses with mutations<SUP> </SUP>in the catalytic domain. Thus, recent reports of oseltamivir<SUP> </SUP>resistance in H5N1 influenza A viruses harboring the framework<SUP> </SUP>His274Tyr mutation<SUP>6-7</SUP> warrant particular attention and careful<SUP> </SUP>monitoring for the spreading of resistant variants.<SUP> </SUP>
    The question remains as to whether the variants isolated from<SUP> </SUP>untreated patients demonstrate person-to-person transmissibility<SUP> </SUP>in a community or the spontaneous emergence of mutants with<SUP> </SUP>reduced drug sensitivity. We favor the first possibility because<SUP> </SUP>the global Neuraminidase Inhibitor Susceptibility Network did<SUP> </SUP>not identify influenza viruses with resistance to neuraminidase<SUP> </SUP>inhibitors before these drugs were introduced into clinical<SUP> </SUP>use.<SUP>23-24</SUP> However, in the first 3 influenza seasons (1999-2002)<SUP> </SUP>following the introduction of neuraminidase inhibitors to the<SUP> </SUP>market, the network detected a small number (8 [0.35%] of 2287<SUP> </SUP>isolates) of influenza viruses, isolated from untreated patients,<SUP> </SUP>with decreased susceptibility to neuraminidase inhibitors.<SUP>25</SUP><SUP> </SUP>Of those, 2 possessed neuraminidase mutations previously identified<SUP> </SUP>in neuraminidase inhibitor?resistant viruses. Moreover,<SUP> </SUP>in the 2003-2004 influenza season, the network identified 3<SUP> </SUP>H3N2 viruses in 1180 samples collected in Japan that contained<SUP> </SUP>neuraminidase mutations conferring resistance to neuraminidase<SUP> </SUP>inhibitors, although it was not possible to determine with certainty<SUP> </SUP>whether these patients had been exposed to neuraminidase inhibitors<SUP> </SUP>or to neuraminidase inhibitor?treated individuals.<SUP>26</SUP> Our<SUP> </SUP>findings are consistent with these surveillance data, which<SUP> </SUP>imply a possible transmission of neuraminidase inhibitor?resistant<SUP> </SUP>viruses from person to person.<SUP> </SUP>
    When healthy children were given oseltamivir at 2 mg/kg of body<SUP> </SUP>weight, the mean peak plasma concentration of oseltamivir carboxylate,<SUP> </SUP>the active metabolite of the drug, was 630 nmol/L among children<SUP> </SUP>aged 3 to 5 years and 426 nmol/L among children aged 1 to 2<SUP> </SUP>years.<SUP>27</SUP> This indicates that the IC<SUB>50</SUB> values for influenza B<SUP> </SUP>viruses tested against oseltamivir in our study were close to<SUP> </SUP>the plasma drug concentration, suggesting that this drug may<SUP> </SUP>not be as effective against influenza B virus as against influenza<SUP> </SUP>A virus. By contrast, the topical concentration of zanamivir<SUP> </SUP>in the human respiratory tract is estimated to be more than<SUP> </SUP>10 000 nmol/L when healthy adults inhale 10 mg zanamivir,<SUP>28</SUP><SUP> </SUP>well above the influenza B virus IC<SUB>50</SUB> values.<SUP> </SUP>
    In Japan, prescriptions for oseltamivir were estimated to be<SUP> </SUP>90 times more common than those for zanamivir during the 2004-2005<SUP> </SUP>influenza season (official notice from the Ministry of Health,<SUP> </SUP>Labor and Welfare of Japan). It is therefore possible that the<SUP> </SUP>mutants with reduced drug sensitivity found in communities in<SUP> </SUP>this study had been generated by widespread use of oseltamivir.<SUP> </SUP>Continued surveillance for the emergence or spread of neuraminidase<SUP> </SUP>inhibitor?resistant influenza viruses is critically important.<SUP> </SUP>
    Finally, the clinical course of influenza B virus infection<SUP> </SUP>in this study did not appear to be affected by the sensitivity<SUP> </SUP>of the virus to neuraminidase inhibitors, although larger numbers<SUP> </SUP>of cases will need to be studied to confirm this impression.<SUP> </SUP>Nonetheless, the symptoms of patients infected with viruses<SUP> </SUP>with reduced sensitivities to neuraminidase inhibitors were<SUP> </SUP>similar to those of patients infected with wild-type viruses,<SUP> </SUP>indicating that these mutant viruses, at least those carrying<SUP> </SUP>the framework mutation, do not lose virulence even though they<SUP> </SUP>have evolved to a status that is less sensitive to the drug.<SUP> </SUP>Further evaluation of the biological properties of neuraminidase<SUP> </SUP>inhibitor?resistant influenza viruses is needed to fully<SUP> </SUP>assess their pathogenicity in humans.<SUP> </SUP>
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    AUTHOR INFORMATION <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE>
    <TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    Corresponding Author: Yoshihiro Kawaoka, DVM, PhD, Division<SUP> </SUP>of Virology, Department of Microbiology and Immunology, Institute<SUP> </SUP>of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,<SUP> </SUP>Minato-ku, Tokyo 108-8639, Japan (kawaoka@ims.u-tokyo.ac.jp<SCRIPT type=text/javascript><!-- var u = "kawaoka", d = "ims.u-tokyo.ac.jp"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></SCRIPT> ).<SUP> </SUP>
    Author Contributions: Drs Hatakeyama and Kawaoka had full access<SUP> </SUP>to all of the data in the study and take responsibility for<SUP> </SUP>the integrity of the data and the accuracy of the data analysis.<SUP> </SUP>
    Study concept and design: Hatakeyama, Sugaya, Kawaoka.<SUP> </SUP>
    Acquisition of data: Hatakeyama, Ito, Yamazaki, Ichikawa, Kimura,<SUP> </SUP>Kiso, Shimizu, Kawakami, Mitamura.<SUP> </SUP>
    Analysis and interpretation of data: Hatakeyama, Koike, Kawaoka.<SUP> </SUP>
    Drafting of the manuscript: Hatakeyama.<SUP> </SUP>
    Critical revision of the manuscript for important intellectual<SUP> </SUP>content: Sugaya, Ito, Yamazaki, Ichikawa, Kimura, Kiso, Shimizu,<SUP> </SUP>Kawakami, Koike, Mitamura, Kawaoka.<SUP> </SUP>
    Statistical analysis: Hatakeyama.<SUP> </SUP>
    Obtained funding: Kawaoka.<SUP> </SUP>
    Administrative, technical, or material support: Sugaya.<SUP> </SUP>
    Study supervision: Kawaoka.<SUP> </SUP>
    Financial Disclosures: Dr Kawaoka reports receiving speakers'<SUP> </SUP>honoraria from Chugai Pharmaceuticals, Novartis, Sankyo, Toyama<SUP> </SUP>Chemical, and Wyeth, and grant support from Chugai Pharmaceuticals,<SUP> </SUP>Daiichi Pharmaceutical, Sankyo, and Toyama Chemical. Dr Sugaya<SUP> </SUP>reports receiving speakers' honoraria from Chugai Pharmaceuticals,<SUP> </SUP>Sankyo Pharmaceuticals, and Takeda. Dr Koike reports receiving<SUP> </SUP>grant support from Chugai Pharmaceuticals. The information described<SUP> </SUP>in this article has been included in a patent application for<SUP> </SUP>novel amino acid substitutions found to affect sensitivity to<SUP> </SUP>neuraminidase inhibitors, compositions containing these substitutions,<SUP> </SUP>and methods for detecting them. No other disclosures were reported.<SUP> </SUP>
    Funding/Support: This work was supported by grants from Core<SUP> </SUP>Research for Evolutional Science and Technology (CREST), Japan;<SUP> </SUP>grants from the Japan Science and Technology Agency (JST); grants-in-aid<SUP> </SUP>from the Ministry of Education, Culture, Sports, Science and<SUP> </SUP>Technology and the Ministry of Health, Labor and Welfare, Japan;<SUP> </SUP>and grants from the National Institute of Allergy and Infectious<SUP> </SUP>Diseases, National Institutes of Health. The oseltamivir carboxylate<SUP> </SUP>used in the study was provided by Roche Products.<SUP> </SUP>
    Role of the Sponsors: None of the funding sources had any role<SUP> </SUP>in the design or conduct of the study; the collection, management,<SUP> </SUP>analysis, or interpretation of the data; or the preparation,<SUP> </SUP>review, or approval of the manuscript.<SUP> </SUP>
    Acknowledgment:We thank biostatistician Hiroshi Ohtsu, MS (Department<SUP> </SUP>of Clinical Bioinformatics, University of Tokyo), for his advice<SUP> </SUP>on the statistical analyses. We thank Larisa Gubareva, PhD (Department<SUP> </SUP>of Internal Medicine, University of Virginia Health Sciences<SUP> </SUP>Center), for providing us with a protocol for the sialidase<SUP> </SUP>inhibition assay, John Gilbert, BS (self-employed), for editing<SUP> </SUP>the manuscript, and Krisna Wells, BS, RN (University of Wisconsin-Madison),<SUP> </SUP>for technical assistance. We also thank Hideo Cho, MD (Department<SUP> </SUP>of Pediatrics, Kawasaki Municipal Hospital, Kanagawa, Japan),<SUP> </SUP>for his supervision. None of those acknowledged received compensation<SUP> </SUP>with the exception of John Gilbert, who is contracted by and<SUP> </SUP>receives a monthly salary from Dr Kawaoka to provide editorial<SUP> </SUP>services.<SUP> </SUP>
    <!--stopindex--><!--null-->Author Affiliations: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science (Drs Hatakeyama, Ito, Kiso, and Kawaoka), Department of Infectious Diseases, Graduate School of Medicine (Drs Hatakeyama and Koike), and International Research Center for Infectious Diseases, Institute of Medical Science (Dr Kawaoka), University of Tokyo, Tokyo, Japan; Department of Pediatrics, Keiyu Hospital, Kanagawa, Japan (Dr Sugaya); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama (Drs Ito, Kiso, and Kawaoka); Zama Children's Clinic, Kanagawa (Dr Yamazaki); Department of Pediatrics, Isehara Kyodo Hospital, Kanagawa (Drs Ichikawa and Kimura); Kawasaki City Institute of Public Health, Kanagawa (Mr Shimizu); Yokohama City Institute of Health, Kanagawa (Ms Kawakami); Department of Pediatrics, Eiju General Hospital, Tokyo (Dr Mitamura); and Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison (Dr Kawaoka).
    <!-- null -->
    REFERENCES <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD><TD></TD></TR></TBODY></TABLE><TABLE cellSpacing=0 cellPadding=0 align=right border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=top noWrap><TABLE cellSpacing=0 cellPadding=0 width=140 border=0 marginheight="0" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD align=left width=1 bgColor=#ffffff rowSpan=2></TD><TD align=left width=1 bgColor=#b1bdbf rowSpan=2></TD><TD align=left width=138><TABLE cellSpacing=0 cellPadding=1 width=138 align=right bgColor=#ffffff border=0 marginheight="0" valign="top" marginwidth="0" topmargin="0" leftmargin="0"><TBODY><TR><TD vAlign=center bgColor=#b1bdbf colSpan=2 height=20> Jump to Section</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Top</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Introduction</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Methods</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Results</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Comment</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>Author information</TD></TR><TR><TD vAlign=top width=5><NOBR> ?</NOBR></TD><TD vAlign=top>References</TD></TR><TR><TD colSpan=2 height=5></TD></TR></TBODY></TABLE></TD><TD align=right width=1 bgColor=#b1bdbf rowSpan=2></TD></TR><TR><TD width=138 bgColor=#b1bdbf height=1></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
    <!-- null -->1. Pillay D, Zambon M. Antiviral drug resistance. BMJ. 1998;317:660-662.<!-- HIGHWIRE ID="297:13:1435:1" --> FREE FULL TEXT <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->2. De Clercq E. Antiviral drugs in current clinical use. J Clin Virol. 2004;30:115-133.<!-- HIGHWIRE ID="297:13:1435:2" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->3. Ward P, Small I, Smith J, Suter P, Dutkowski R. Oseltamivir (Tamiflu) and its potential for use in the event of an influenza pandemic. J Antimicrob Chemother. 2005;55(suppl 1):i5-i21.<!-- HIGHWIRE ID="297:13:1435:3" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->4. Roche. Factsheet Tamiflu. December 2005. http://www.roche.com/med_mbtamiflu05e.pdf. Accessed July 1, 2006.<!-- HIGHWIRE ID="297:13:1435:4" --><!-- /HIGHWIRE --> <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->5. Kiso M, Mitamura K, Sakai-Tagawa Y, et al. Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet. 2004;364:759-765.<!-- HIGHWIRE ID="297:13:1435:5" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->6. Le QM, Kiso M, Someya K, et al. Avian flu: isolation of drug-resistant H5N1 virus [published correction appears in Nature. 2005;438:754]. Nature. 2005;437:1108.<!-- HIGHWIRE ID="297:13:1435:6" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->7. de Jong MD, Tran TT, Truong HK, et al. Oseltamivir resistance during treatment of influenza A (H5N1) infection. N Engl J Med. 2005;353:2667-2672.<!-- HIGHWIRE ID="297:13:1435:7" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->8. Treanor JJ. Influenza virus. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases. 6th ed. Philadelphia, Pa: Elsevier/Churchill Livingstone; 2005:2060-2085.<!-- HIGHWIRE ID="297:13:1435:8" --><!-- /HIGHWIRE --> <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->9. Colman PM, Hoyne PA, Lawrence MC. Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase. J Virol. 1993;67:2972-2980.<!-- HIGHWIRE ID="297:13:1435:9" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->10. Gubareva LV, Matrosovich MN, Brenner MK, Bethell RC, Webster RG. Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus. J Infect Dis. 1998;178:1257-1262.<!-- HIGHWIRE ID="297:13:1435:10" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->11. Gubareva LV. Molecular mechanisms of influenza virus resistance to neuraminidase inhibitors. Virus Res. 2004;103:199-203.<!-- HIGHWIRE ID="297:13:1435:11" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->12. McKimm-Breschkin JL. Resistance of influenza viruses to neuraminidase inhibitors?a review. Antiviral Res. 2000;47:1-17.<!-- HIGHWIRE ID="297:13:1435:12" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->13. Whitley RJ, Hayden FG, Reisinger KS, et al. Oral oseltamivir treatment of influenza in children. Pediatr Infect Dis J. 2001;20:127-133.<!-- HIGHWIRE ID="297:13:1435:13" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->14. Carr J, Ives J, Kelly L, et al. Influenza virus carrying neuraminidase with reduced sensitivity to oseltamivir carboxylate has altered properties in vitro and is compromised for infectivity and replicative ability in vivo. Antiviral Res. 2002;54:79-88.<!-- HIGHWIRE ID="297:13:1435:14" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->15. Herlocher ML, Carr J, Ives J, et al. Influenza virus carrying an R292K mutation in the neuraminidase gene is not transmitted in ferrets. Antiviral Res. 2002;54:99-111.<!-- HIGHWIRE ID="297:13:1435:15" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->16. Ives JA, Carr JA, Mendel DB, et al. The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antiviral Res. 2002;55:307-317.<!-- HIGHWIRE ID="297:13:1435:16" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->17. Herlocher ML, Truscon R, Elias S, et al. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J Infect Dis. 2004;190:1627-1630.<!-- HIGHWIRE ID="297:13:1435:17" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->18. Mishin VP, Hayden FG, Gubareva LV. Susceptibilities of antiviral-resistant influenza viruses to novel neuraminidase inhibitors. Antimicrob Agents Chemother. 2005;49:4515-4520.<!-- HIGHWIRE ID="297:13:1435:18" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->19. Hatakeyama S, Sakai-Tagawa Y, Kiso M, et al. Enhanced expression of an 2,6-linked sialic acid on MDCK cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor. J Clin Microbiol. 2005;43:4139-4146.<!-- HIGHWIRE ID="297:13:1435:19" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->20. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, Hayden FG. Selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir. J Infect Dis. 2001;183:523-531.<!-- HIGHWIRE ID="297:13:1435:20" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->21. Harper SA, Fukuda K, Uyeki TM, Cox NJ, Bridges CB, Advisory Committee on Immunization Practices, Centers for Disease Control and Prevention (CDC). Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices [published correction appears in MMWR Morb Mortal Wkly Rep. 2005; 54:750]. MMWR Recomm Rep. 2005;54;(RR-8):1-40.<!-- HIGHWIRE ID="297:13:1435:21" --> PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->22. Burmeister WP, Ruigrok RW, Cusack S. The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 1992;11:49-56.<!-- HIGHWIRE ID="297:13:1435:22" --> ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->23. McKimm-Breschkin J, Trivedi T, Hampson A, et al. Neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir. Antimicrob Agents Chemother. 2003;47:2264-2272.<!-- HIGHWIRE ID="297:13:1435:23" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->24. Moscona A. Neuraminidase inhibitors for influenza. N Engl J Med. 2005;353:1363-1373.<!-- HIGHWIRE ID="297:13:1435:24" --> FREE FULL TEXT <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->25. Monto AS, McKimm-Breschkin JL, Macken C, et al. Detection of influenza viruses resistant to neuraminidase inhibitors in global surveillance during the first 3 years of their use. Antimicrob Agents Chemother. 2006;50:2395-2402.<!-- HIGHWIRE ID="297:13:1435:25" --> <NOBR>FREE FULL TEXT</NOBR> <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->26. World Health Organization. Use of influenza antivirals during 2003-2004 and monitoring of neuraminidase inhibitor resistance. Wkly Epidemiol Rec. 2005;80:156.<!-- HIGHWIRE ID="297:13:1435:26" --> PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->27. Oo C, Hill G, Dorr A, Liu B, Boellner S, Ward P. Pharmacokinetics of anti-influenza prodrug oseltamivir in children aged 1-5 years. Eur J Clin Pharmacol. 2003;59:411-415.<!-- HIGHWIRE ID="297:13:1435:27" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE><!-- null -->28. Cass LM, Brown J, Pickford M, et al. Pharmacoscintigraphic evaluation of lung deposition of inhaled zanamivir in healthy volunteers. Clin Pharmacokinet. 1999;36(suppl 1):21-31.<!-- HIGHWIRE ID="297:13:1435:28" --> FULL TEXT | ISI | PUBMED <!-- /HIGHWIRE --><TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD></TD></TR></TBODY></TABLE>

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    THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES <TABLE cellSpacing=0 cellPadding=0 width="100%" border=0><TBODY><TR><TD width="100%" bgColor=#6a90aa></TD></TR></TBODY></TABLE>
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  • #2
    Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

    does this mean that influenza B is becoming drug resitant to certain anti virals,dr niman.?

    Comment


    • #3
      Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

      Originally posted by russell family View Post
      does this mean that influenza B is becoming drug resitant to certain anti virals,dr niman.?
      Yes, and resistance includes Tamiflu.

      Comment


      • #4
        Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

        Study finds drug-resistant flu virus in Japan

        Tue Apr 3, 2007 4:27PM EDT
        CHICAGO (Reuters) - Type B flu viruses, which usually cause smaller epidemics than type A, developed partial resistance to two front-line anti-viral drugs used to combat seasonal influenza, according to a study published on Tuesday.
        While there has been evidence of some type A flu viruses developing a resistance to anti-viral drugs, researchers at the University of Tokyo said until their study there has been only limited information involving type B.
        The research involved Tamiflu, an antiviral drug made by Roche and Gilead Sciences, and Relenza, made by GlaxoSmithKline and Biota Holdings, and known generically as zanamivir.
        Tamiflu, also known as oseltamivir, is the first choice against both seasonal flu and the H5N1 avian influenza.
        The findings come from Japan where both drugs to prevent and treat seasonal flu are used more extensively than anywhere else in the world. The study involved an influenza B virus outbreak in the winter of 2004-2005 that caused a widespread epidemic.
        Writing in this week's journal of the American Medical Association, researchers said they collected flu B virus samples from 74 children before and after Tamiflu use and from 348 influenza patients -- also mostly children -- who were not treated with the drug.
        They said they found a virus with reduced drug sensitivity in one of the 74 children who had received Tamiflu. In addition, seven of the 422 influenza B viruses isolated from untreated patients were found to have reduced sensitivity to Relenza, Tamiflu, or both.
        Drug resistant flu viruses present a risk because the mutations can make current drugs ineffective for both seasonal flu and a potentially devastating bird flu epidemic.
        An editorial in the same issue commenting on the study said it raised more questions than it answered.
        But, it said, "some facts are strikingly clear. Influenza B mutants with reduced sensitivity to (anti-viral drugs) are circulating, and these viruses can cause infections with no difference in duration of symptoms...
        "Contrary to what had been hoped until now, some resistant variants are vigorous pathogens. Whether these viruses arise by spontaneous mutation ... or whether they are transmitted within families or acquired from the community, the resistant variants may be here to stay," the editorial added.

        Comment


        • #5
          Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

          You're all full of good news today, Dr. Niman.

          Seriously, I wanted to ask you about the conference you spoke to in Kullifornia, as the Governator would say. In the bio for the head of NAMRU-3, it is mentioned that one field test (ELISA) is only 36% effective/accurate, while the PCR test is 92% accurate.

          When we read about tests initially showing negative but awaiting blood test confirmation, which test is usually used in the field? The 36% or the 92%?

          Thanks as always.

          Comment


          • #6
            Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

            Originally posted by scottmcpherson View Post
            You're all full of good news today, Dr. Niman.

            Seriously, I wanted to ask you about the conference you spoke to in Kullifornia, as the Governator would say. In the bio for the head of NAMRU-3, it is mentioned that one field test (ELISA) is only 36% effective/accurate, while the PCR test is 92% accurate.

            When we read about tests initially showing negative but awaiting blood test confirmation, which test is usually used in the field? The 36% or the 92%?

            Thanks as always.
            Many of the rapid tests (results in minutes, not hours) have a high false negative rate.

            Comment


            • #7
              Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

              I'm not so much concerned about what happens in Influenza-B.
              Influenza B never went pandemic and mutates much slower.
              Only two sorts of Influenza B, they can be covered by vaccine.

              I'm concerned that it took more than 2 years that they decided to tell us about it and that people have already accepted these delays,
              no one had noticed and commented.

              They probably have much more data about influenza-A resistance
              as well and are still thinking whether to publish them or not...
              Last edited by gsgs; April 3, 2007, 07:03 PM.
              I'm interested in expert panflu damage estimates
              my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

              Comment


              • #8
                Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                Good point, gsgs.

                Comment


                • #9
                  Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                  What is interesting, though, from what I have read about B over the past two flu seasons:

                  First, there was a B not too long ago with some increased mortality. I think it was 2004 season. It was a very, very nasty B.

                  Second, for the past two years, WHO has apparently missed the B target in its trivalent vaccine. The last statistics I saw (from December or January) stated that only 35&#37; of the B flu typed in the US actually matched the vaccine target. Fully 65% of the typed B was another strain, or else the B flu in the vaccine drifted pretty far prior to manufacture.

                  Third, Dr. Niman has stated that H5N1 has acquired a polymorphism that exists in A/H1N1, A/H3N2 and B strains. I think it is in the PB1 strand but could be wrong and it could be in HA.

                  I sometimes think science ignores B too much, the same way agriculture ignores low-path A viruses. Whatever; we need to follow the Japanese very carefully, as they clearly have blazed a Tamiflu trail that we need to tread carefully upon.

                  Comment


                  • #10
                    Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                    Second, for the past two years, WHO has apparently missed the B target in its trivalent vaccine. The last statistics I saw (from December or January) stated that only 35&#37; of the B flu typed in the US actually matched the vaccine target. Fully 65% of the typed B was another strain, or else the B flu in the vaccine drifted pretty far prior to manufacture.
                    According to the official report in 2005 there was only a < 20 % match of Influenza B in Germany (vaccine strain= B/Jiangsu/10/03 (Yamagata line); circulating = B/Victoria like strains)

                    Comment


                    • #11
                      Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                      Wow, less than 20% in flu season 2005-06. That is telling. I wonder why they are missing the B target so badly?

                      Comment


                      • #12
                        Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                        all available complete aligned HA-flu-A protein sequences in genbank
                        and lanl have less than 0&#37;,1%,2%,...,differences from at least
                        one member in a set of

                        3810,951,478,302,210,144,108,81,65,52,43,35,30,27, 23,21,18,18,17,16,16,
                        15,14,14,13,12,12,12,12,12,12,12,12,12,11,10,9,8,7 ,7,6,6,5,5,5,5,5,5,5,
                        5,5,4,4,3,2,2,2,2,2,1
                        sequences


                        the same for flu-B gives:
                        253,45,14,9,5,3,3,1
                        sequences.

                        You can see, how much more diversity there is for flu-A with its 16 HAs.
                        So, there is a flu-B sequence (e.g. B/Memphis/3/1989) such that any
                        other complete flu-B-HA-sequence
                        is less than 7% different from it at protein-level.



                        If I had to distinguish the flu-A HAs, I'd make 12 classes instead of 16
                        and group H5 with H2,H14 with H4,H15 with H7 and H16 with H13.
                        So, maybe H2 should be H2a and H5 should be H2b or such.

                        With a coarser filter you have 18 HAs , H3 and H7 splitting into 2 groups.


                        For the vaccine not the whole HA is critical but only the antigenic sites
                        and if the mutations specially target these, then the vaccine could be missed
                        although the HAs are very similar elsewhere.
                        I'm interested in expert panflu damage estimates
                        my current links: http://bit.ly/hFI7H ILI-charts: http://bit.ly/CcRgT

                        Comment


                        • #13
                          Re: Emergence of Influenza B Viruses With Reduced Sensitivity to Neuraminidase Inhibi

                          The mismatch in 2005/06 was not due to mutation but false prediction - as it happens frequently . It was simply not the strain expected to become dominant which made the race.

                          Comment

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