Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Peter Doherty Institute for Infection and Immunity

The E protein of most flaviviruses is modified by Asn-linked glycosylation at residue 153/154 and in the case of the four dengue virus (DENV) serotypes by a second glycan at residue 67. However, the absence of E protein glycosylation among numerous natural isolates of different flaviviruses suggests that the glycan, per se, is not critically important in the virus life cycle. Consistent with this notion, we show that ablation of both glycans from the DENV-2 E protein reduces but does not prevent growth of the variant in mammalian and mosquito cells. We found a pronounced and opposing effect of glycan ablation on two stages of the virus growth cycle: infectivity and release. Loss of either of the two DENV E protein glycans markedly enhanced infectivity of variants for mosquito cells at the expense of efficient virion release. The variants also displayed reduced release in mammalian cells, which was more prominent for viruses lacking the Asn 67-linked glycan than for those lacking the Asn 153-linked glycan, without a marked change in infectivity. Mutations, which compensated for the defect in virus morphogenesis associated with ablation of the Asn 67-linked glycan in mammalian cells but interestingly not in mosquito cells, were identified at the glycosylation acceptor motif and a second site in E protein domain II. The dueling influences of infectivity and release on virus growth affected by the glycans may explain the plasticity in E protein glycosylation among the flaviviruses.

Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release.

Analysis of mutations I117V and I117M in the neuraminidase of influenza A pandemic (H1N1) 2009 viruses showed that I117V confers a mild reduction in oseltamivir sensitivity and has a synergistic effect of further increasing resistance when combined with H275Y. Contrary to recent reports, the I117M mutation does not alter oseltamivir sensitivity.

Mutations I117V and I117M and oseltamivir sensitivity of pandemic (H1N1) 2009 viruses.

&#x5C3D;&#x7BA1;&#x5927;&#x4E8E; 99%&#x7684;&#x7532;&#x578B;&#x6D41;&#x611F;&#x75C5;&#x6BD2;&#x5728;&#x4E9A;&#x6D32;-&#x592A;&#x5E73;&#x6D0B;&#x5730;&#x533A;&#x88AB;&#x8010;&#x5BF9;&#x91D1;&#x521A;&#x70F7;&#x6297;&#x75C5;&#x6BD2;&#x836F;&#x7269;&#x5728; 2011 &#x5E74;&#xFF0C;&#x5927;&#x591A;&#x6570;&#x7532;&#x578B;&#x6D41;&#x611F;&#x7684;&#x5FAA;&#x73AF; (&gt; 97%&#xFF09; &#x548C; B &#x682A; (&sim;99%) &#x4ECD;&#x7136;&#x5BB9;&#x6613;&#x53D7;&#x5230;&#x7684;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x6291;&#x5236;&#x5242;&#x5965;&#x53F8;&#x4ED6;&#x97E6;&#x548C;&#x624E;&#x3002;&#x4E0D;&#x8FC7;&#xFF0C;&#x76F8;&#x6BD4; 2009&#x5E74;&#x7684;&#x5965;&#x53F8;&#x4ED6;&#x97E6;&#x8010;&#x7684;&#x5927;&#x6D41;&#x884C;&#xFF0C;&#x6848;&#x4EF6;&#x7684;&#x7B2C;&#x4E00;&#x5E74;&#x589E;&#x52A0;&#x5728; 2011 &#x5E74;&#xFF0C;&#x4E3B;&#x8981;&#x662F;&#x7531;&#x4E8E;&#x66B4;&#x53D1;&#x53D1;&#x751F;&#x5728;&#x7EBD;&#x5361;&#x65AF;&#x5C14;&#xFF0C;&#x6B63;&#x5982;&#x62A5;&#x544A;&#x4E2D;&#x4F24;&#x5BB3;&#x7ECD;&#x5174;&#x6587;&#x7406;&#x5B66;&#x9662; 2012a 2011c&#xFF09;&#xFF0C;&#x90A3;&#x91CC;&#x7684;&#x6297;&#x75C5;&#x6BD2;&#x7684;&#x591A;&#x6570;&#x662F;&#x4ECE;&#x793E;&#x533A;&#x7684;&#x5965;&#x53F8;&#x4ED6;&#x97E6;&#x6297;&#x75C5;&#x6BD2;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176; H275Y &#x7A81;&#x53D8;&#x4E0E; A (H1N1) pdm09 &#x75C5;&#x6BD2;&#x4E0D;&#x88AB;&#x5965;&#x53F8;&#x4ED6;&#x97E6;&#x6CBB;&#x7597;&#x7684;&#x60A3;&#x8005;&#x3002;&#x6B64;&#x5916;&#x53D1;&#x73B0;&#x4E86;&#x5C11;&#x91CF;&#x7684;&#x6D41;&#x611F; B &#x75C5;&#x6BD2;&#x7684;&#x51CF;&#x5C11;&#x5965;&#x53F8;&#x4ED6;&#x97E6;&#x6216;&#x624B;&#x5E15;&#x6613;&#x611F;&#x6027;&#x3002;&#x589E;&#x52A0;&#x7684;&#x68C0;&#x6D4B;&#x7684;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x6291;&#x5236;&#x5242;&#x8010;&#x836F;&#x682A;&#x5728;&#x793E;&#x533A;&#x548C;&#x65B0;&#x578B;&#x53D8;&#x79CD;&#x7684;&#x68C0;&#x6D4B;&#x4E0E;&#x654F;&#x611F;&#x6027;&#x964D;&#x4F4E;&#x5FAA;&#x73AF;&#x662F;&#x8981;&#x63D0;&#x9192;&#x4EBA;&#x4EEC;&#x7684;&#x6D41;&#x611F;&#x75C5;&#x6BD2;&#x76D1;&#x6D4B;&#x662F;&#x786E;&#x4FDD;&#x9002;&#x5F53;&#x7684;&#x6297;&#x75C5;&#x6BD2;&#x6CBB;&#x7597;&#x6307;&#x5BFC;&#x65B9;&#x9488;&#x4ECD;&#x7136;&#x5F88;&#x91CD;&#x8981;&#x3002;

&#x6D41;&#x611F;&#x6297;&#x75C5;&#x6BD2;&#x6297;&#x4E9A;&#x6D32;-&#x592A;&#x5E73;&#x6D0B;&#x5730;&#x533A; 2011&#x5E74;&#x671F;&#x95F4;&#x3002;

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (âˆ¼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Influenza antiviral resistance in the Asia-Pacific region during 2011.

Malgr&eacute; plu de 99 % des virus de l&#39;influenza en circulation dans la r&eacute;gion Asie-Pacifique &eacute;tant r&eacute;sistante aux m&eacute;dicaments antiviraux adamantane en 2011, la grande majorit&eacute; de l&#39;influenza A (&amp;gt; 97 %) et les souches B (&sim;99 %) sont rest&eacute;s sensibles au zanamivir et l&#39;oseltamivir inhibiteurs de la neuraminidase. Cependant, par rapport &agrave; la premi&egrave;re ann&eacute;e des cas pand&eacute;mie 2009 de r&eacute;sistant &agrave; l&#39;oseltamivir une virus a (H1N1) pdm09 avec la mutation de la neuraminidase H275Y a augment&eacute; en 2011, principalement &agrave; cause d&#39;une &eacute;pid&eacute;mie de virus r&eacute;sistants &agrave; l&#39;oseltamivir qui ont eu lieu &agrave; Newcastle, tel que rapport&eacute; dans Hurt et coll. (2011c, 2012a), o&ugrave; la majorit&eacute; des virus r&eacute;sistants provenaient de communaut&eacute; patients non trait&eacute;s par l&#39;oseltamivir. Un petit nombre de virus de la grippe B avec r&eacute;duit l&#39;oseltamivir ou le zanamivir susceptibilit&eacute; ont aussi &eacute;t&eacute; d&eacute;tect&eacute;. La d&eacute;tection d&#39;une augmentation de souches r&eacute;sistant aux inhibiteurs de la neuraminidase circulant dans la communaut&eacute; et la d&eacute;tection de nouvelles variantes pr&eacute;sentant une sensibilit&eacute; r&eacute;duite nous rappellent que la surveillance des virus grippaux est important de s&#39;assurer que les lignes directrices restent un traitement antiviral ouvre.

R&eacute;sistance antivirale de grippe dans la r&eacute;gion Asie-Pacifique au cours de 2011.

Trotz mehr als 99 % des Influenza-A-Viren im Umlauf in der Asien-Pazifik-Region werden resistent gegen das Adamantan Virostatika 2011, die gro&szlig;e Mehrheit der Influenza-A (&gt; 97 %) und B-St&auml;mme (&sim;99 %) blieb die Neuraminidase-Hemmer Oseltamivir und Zanamivir anf&auml;llig. Jedoch im Vergleich zum ersten Jahr 2009 Pandemie, F&auml;lle von Oseltamivir-resistente A (H1N1)-pdm09-Viren mit der H275Y-Neuraminidase-Mutation, erh&ouml;hte sich im Jahr 2011, vor allem durch einen Ausbruch von Oseltamivir-resistenten Viren, die in Newcastle, aufgetreten sind, wie berichtet in Hurt Et Al. (2011c, 2012a), wo die Mehrheit der resistenten Viren aus der Gemeinschaft waren wird nicht mit Oseltamivir behandelten Patienten. Eine kleine Anzahl von Influenza-B-Viren mit reduzierten Oseltamivir oder Zanamivir Anf&auml;lligkeit wurden ebenfalls entdeckt. Der erh&ouml;hte Nachweis von Neuraminidase-Hemmer resistente St&auml;mme in der Gemeinschaft und die Erkennung von neue Varianten mit einer geringeren Anf&auml;lligkeit in Umlauf sind Erinnerungen, die &Uuml;berwachung von Influenza-Viren ist darauf zu achten, dass gegebenenfalls antivirale Therapie, die Richtlinien zu bleiben.

Grippe antiviral Widerstand in der Region Asien-Pazifik w&auml;hrend 2011.

Nonostante maggiore del 99% dei virus influenzali A che circolano nella regione Asia-Pacifico essere resistente ai farmaci antivirali adamantano nel 2011, la grande maggioranza di influenza A (&amp;gt; 97%) e ceppi B (% &sim;99) rimasero suscettibili al neuraminidasi inibitori oseltamivir e zanamivir. Tuttavia, rispetto al primo anno del 2009 pandemia, casi di oseltamivir-resistenti A (H1N1) pdm09 virus con la mutazione di neuraminidasi H275Y aumentato nel 2011, principalmente a causa di un&#39;epidemia di virus oseltamivir-resistenti che si sono verificati a Newcastle, come riportato nel male et al (2011c, 2012a), dove la maggior parte dei virus resistenti era da comunit&agrave; pazienti non trattati con oseltamivir. Sono stato individuato anche un piccolo numero di virus dell&#39;influenza B con ridotta oseltamivir o zanamivir suscettibilit&agrave;. La rilevazione aumentata di neuraminidasi inibitore ceppi circolanti nella Comunit&agrave; e il rilevamento di nuove varianti con suscettibilit&agrave; ridotta sono i ricordi che monitoraggio dei virus dell&#39;influenza &egrave; importante assicurare che trattamento antivirale rimangono orientamenti appropriati.

Antivirale resistenza influenza nella regione Asia-Pacifico nel corso del 2011.

&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x306B;&#x304F;&#x3044;&#x306B;&#x30A2;&#x30C0;&#x30DE;&#x30F3;&#x30BF;&#x30F3;&#x6297;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x85AC;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6;&#x306E;&#x5927;&#x591A;&#x6570;&#x3067;&#x3001;2011 &#x5E74;&#x306E;&#x30A2;&#x30B8;&#x30A2;&#x592A;&#x5E73;&#x6D0B;&#x5730;&#x57DF;&#x3067;&#x5FAA;&#x74B0;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E; 99 &#xFF05; &#x3092;&#x8D85;&#x3048;&#x308B; &#xFF08;&#x76EE; 97%) &#x3068; B &#x682A; (&sim;99%) &#x6B8B;&#x3063;&#x305F;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x963B;&#x5BB3;&#x85AC;&#x30AA;&#x30BB;&#x30EB;&#x30BF;&#x30DF;&#x30D3;&#x30EB;&#x53CA;&#x3073;&#x30B6;&#x30CA;&#x30DF;&#x30D3;&#x30EB;&#x306B;&#x5BFE;&#x3057;&#x3066;&#x5F71;&#x97FF;&#x3092;&#x53D7;&#x3051;&#x3084;&#x3059;&#x304B;&#x3063;&#x305F;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x30AA;&#x30BB;&#x30EB;&#x30BF;&#x30DF;&#x30D3;&#x30EB;&#x8010;&#x6027;&#x306E; 2009&#x5E74;&#x30D1;&#x30F3;&#x30C7;&#x30DF;&#x30C3;&#x30AF;&#x3001;&#x4F8B;&#x306E;&#x6700;&#x521D;&#x306E;&#x5E74;&#x306B; (H1N1) pdm09 &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x3067;&#x50B7;&#x8010;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x5927;&#x90E8;&#x5206;&#x304C;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30C6;&#x30A3;&#x304B;&#x3089;&#x3089; (2011 c, 2012a) &#x3092;&#x5831;&#x544A;&#x3057;&#x305F;&#x30CB;&#x30E5;&#x30FC;&#x30AB;&#x30C3;&#x30B9;&#x30EB;&#x3067;&#x304C;&#x767A;&#x751F;&#x3057;&#x305F;&#x30AA;&#x30BB;&#x30EB;&#x30BF;&#x30DF;&#x30D3;&#x30EB;&#x8010;&#x6027;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x767A;&#x751F;&#x306B;&#x3088;&#x308A;&#x4E3B;&#x306B; 2011 &#x5E74;&#x306B;&#x5897;&#x52A0; H275Y &#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x5909;&#x7570;&#x3092;&#x6BD4;&#x8F03;&#x30AA;&#x30BB;&#x30EB;&#x30BF;&#x30DF;&#x30D3;&#x30EB;&#x306E;&#x6CBB;&#x7642;&#x3092;&#x53D7;&#x3051;&#x3066;&#x3044;&#x306A;&#x3044;&#x60A3;&#x8005;&#x3002;&#x524A;&#x6E1B;&#x30AA;&#x30BB;&#x30EB;&#x30BF;&#x30DF;&#x30D3;&#x30EB;&#x307E;&#x305F;&#x306F;&#x30B6;&#x30CA;&#x30DF;&#x30D3;&#x30EB;&#x611F;&#x53D7;&#x6027;&#x3068;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6; B &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x5C11;&#x6570;&#x3082;&#x691C;&#x51FA;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B3;&#x30DF;&#x30E5;&#x30CB;&#x30C6;&#x30A3;&#x3068;&#x65B0;&#x898F;&#x30D0;&#x30EA;&#x30A2;&#x30F3;&#x30C8;&#x306E;&#x691C;&#x51FA;&#x306E;&#x611F;&#x53D7;&#x6027;&#x306E;&#x4F4E;&#x4E0B;&#x3068;&#x5FAA;&#x74B0;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x963B;&#x5BB3;&#x5264;&#x8010;&#x6027;&#x83CC;&#x306E;&#x767A;&#x898B;&#x306E;&#x5897;&#x52A0;&#x306F;&#x3001;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306E;&#x76E3;&#x8996;&#x306E;&#x30AC;&#x30A4;&#x30C9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x7DAD;&#x6301;&#x6297;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x6CBB;&#x7642;&#x3092;&#x9069;&#x5207;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x901A;&#x77E5;&#x3067;&#x3059;&#x3002;

2011 &#x5E74;&#x30A2;&#x30B8;&#x30A2;&#x592A;&#x5E73;&#x6D0B;&#x5730;&#x57DF;&#x306B;&#x304A;&#x3051;&#x308B;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6;&#x6297;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x62B5;&#x6297;&#x3002;

&#xB3C4; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xC800;&#xD56D; adamantane &#xD56D; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC57D;&#xBB3C; 2011 &#xB144; &#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; A&#xC758; &#xB300;&#xB2E4;&#xC218;&#xB97C; &#xC544;&#xC2DC;&#xC544;-&#xD0DC;&#xD3C9;&#xC591; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C; &#xC21C;&#xD658; &#xD558;&#xB294; &#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; A &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; 99% &#xBCF4;&#xB2E4; &#xD070; (&amp;gt; 97%)&#xC640; B &#xBCC0;&#xC885; (&sim;99%) &#xC720;&#xC9C0; &#xC751;&#xC9D1; &#xC5B5;&#xC81C;&#xC81C; oseltamivir&#xC640; zanamivir. &#xADF8;&#xB7EC;&#xB098;, &#xC8FC;&#xB85C;&#xC5D0; &#xC0C1;&#xCC98; &#xC678;. (2011c, 2012a) &#xC5B4;&#xB514; &#xB0B4;&#xC131; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xB300;&#xBD80;&#xBD84;&#xC740; &#xC9C0;&#xC5ED; &#xC0AC;&#xD68C;&#xC5D0;&#xC11C; &#xC788;&#xC5C8;&#xB2E4; &#xBCF4;&#xACE0; &#xB274;&#xCE90;&#xC2AC;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD55C; oseltamivir &#xB0B4;&#xC131; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xD655;&#xC0B0;&#xC73C;&#xB85C; &#xC778;&#xD574; 2011 &#xB144;&#xC5D0; &#xC99D;&#xAC00; H275Y &#xC751;&#xC9D1; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774;&#xC640; (H1N1) pdm09 &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; oseltamivir &#xB0B4;&#xC131;&#xC758; 2009 &#xC720;&#xD589; &#xC131;, &#xACBD;&#xC6B0; &#xCCAB; &#xD574;&#xC5D0; &#xBE44;&#xD574; &#xD658;&#xC790; oseltamivir &#xD568;&#xAED8; &#xCE58;&#xB8CC; &#xD558;&#xC9C0;. &#xC18C;&#xC218; &#xAC10;&#xC18C; oseltamivir &#xB610;&#xB294; zanamivir &#xBBFC;&#xAC10;&#xC131; &#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; B &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xB3C4; &#xAC80;&#xCD9C; &#xB410;&#xB2E4;. &#xC751;&#xC9D1; &#xC5B5;&#xC81C;&#xC81C; &#xB0B4;&#xC131; &#xBCC0;&#xC885; &#xAC10;&#xC18C; &#xBBFC;&#xAC10;&#xC131;&#xACFC; &#xC9C0;&#xC5ED; &#xC0AC;&#xD68C;&#xC640; &#xC0C8;&#xB85C;&#xC6B4; &#xBCC0;&#xC885; &#xD0D0;&#xC9C0; &#xC21C;&#xD658;&#xC758; &#xC99D;&#xAC00; &#xAC80;&#xCD9C;&#xC740; &#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; &#xBC14;&#xC774;&#xB7EC;&#xC2A4;&#xC758; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;&#xC774; &#xC911;&#xC694; &#xC9C0;&#xCE68;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xB294; &#xD56D; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xCE58;&#xB8CC;&#xC5D0; &#xC801;&#xC808; &#xD55C; &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xC54C;&#xB9BC;.

&#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; 2011 &#xC911; &#xC544;&#xC2DC;&#xC544;-&#xD0DC;&#xD3C9;&#xC591; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C; &#xD56D; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xC800;&#xD56D;

A pesar de m&aacute;s de 99% de los virus de la gripe A circulan en la regi&oacute;n de Asia y el Pac&iacute;fico ser resistente a los medicamentos antivirales adamantano en 2011, la gran mayor&iacute;a de la gripe A (&gt; 97%) y las cepas B (&sim;99%) segu&iacute;a siendo susceptibles a los inhibidores de neuraminidasa oseltamivir y zanamivir. Sin embargo, comparado con el primer a&ntilde;o de los casos de pandemia, 2009 de resistentes al oseltamivir un virus de pdm09 (H1N1) con la mutaci&oacute;n de neuraminidasa H275Y aument&oacute; en 2011, principalmente debido a un brote de virus resistentes al oseltamivir que hubo en Newcastle, como en da&ntilde;o et al (2011c, 2012a), donde la mayor&iacute;a de los virus resistentes proced&iacute;an de comunidad pacientes no tratados con oseltamivir. Tambi&eacute;n se detectaron una peque&ntilde;a cantidad de virus de la influenza B con reducida oseltamivir o zanamivir susceptibilidad. El aumento de la detecci&oacute;n de cepas resistentes a los inhibidores de la neuraminidasa circulando en la comunidad y la detecci&oacute;n de nuevas variantes con susceptibilidad reducida son recordatorios que monitoreo de virus de la influenza es importante para asegurar que siguen las pautas de tratamiento antiviral apropiado.

Resistencia antiviral de la gripe en la regi&oacute;n de Asia y el Pac&iacute;fico durante el 2011.

A minor viral population of oseltamivir-resistant A(H3N2) viruses (E119V neuraminidase mutation) was selected and maintained in a continually infected immunocompromised child following initial oseltamivir treatment. A subsequent course of oseltamivir given 7 weeks later rapidly selected for the E119V variant resulting in a near-pure population of the resistant virus. The study highlights the challenges of oseltamivir treatment of immunocompromised patients that are continually shedding virus and demonstrates the ability of the E119V oseltamivir-resistant virus to be maintained for prolonged periods even in the absence of drug-selective pressure.

Progressive emergence of an oseltamivir-resistant A(H3N2) virus over two courses of oseltamivir treatment in an immunocompromised paediatric patient.

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

Estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant A(H1N1)pdm09 influenza viruses.

Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.

Peramivir and laninamivir susceptibility of circulating influenza A and B viruses.

The emergence of the pandemic influenza A(H1N1)pdm09 virus in 2009 saw a significant increase in the therapeutic and prophylactic use of neuraminidase inhibitors (NAIs) to mitigate the impact of this highly transmissible virus. Prior to the pandemic, many countries stockpiled NAIs and developed pandemic plans for the use of antiviral drugs, based on either treatment of high-risk individuals and/or prophylaxis of contacts. However, to date there has been a lack of in vivo models to test the efficacy of treatment or prophylaxis with NAIs, for influenza-infected individuals or exposed contacts, in a household setting.

Evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact.

Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 10,641 viruses collected by WHO-recognized National Influenza Centres between May 2013 and May 2014 to determine 50% inhibitory concentration (IC50) data for neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, peramivir and laninamivir. In addition, neuraminidase (NA) sequence data, available from the WHO CCs and from sequence databases (n=3206), were screened for amino acid substitutions associated with reduced NAI susceptibility. Ninety-five per cent of the viruses tested by the WHO CCs were from three WHO regions: Western Pacific, the Americas and Europe. Approximately 2% (n=172) showed highly reduced inhibition (HRI) against at least one of the four NAIs, commonly oseltamivir, while 0.3% (n=32) showed reduced inhibition (RI). Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=169), A(H3N2) with NA E119V (n=1), B/Victoria-lineage with NA E117G (n=1) and B/Yamagata-lineage with NA H273Y (n=1); amino acid position numbering is A subtype and B type specific. Although approximately 98% of circulating viruses tested during the 2013-2014 period were sensitive to all four NAIs, a large community cluster of A(H1N1)pdm09 viruses with the NA H275Y substitution from patients with no previous exposure to antivirals was detected in Hokkaido, Japan. Significant numbers of A(H1N1)pdm09 NA H275Y viruses were also detected in China and the United States: phylogenetic analyses showed that the Chinese viruses were similar to those from Japan, while the United States viruses clustered separately from those of the Hokkaido outbreak, indicative of multiple resistance-emergence events. Consequently, global surveillance of influenza antiviral susceptibility should be continued from a public health perspective.

Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2013-2014.

The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like).

Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility.

Rett syndrome (RTT) is a neurodevelopmental disorder associated with mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2) and consequent dysregulation of brain maturation. Patients suffer from a range of debilitating physical symptoms, however, behavioral and emotional symptoms also severely affect their quality of life. Here, we present previously unreported and clinically relevant affective dysfunction in the female heterozygous Mecp2(tm1Tam) mouse model of RTT (129sv and C57BL6 mixed background). The affective dysfunction and aberrant anxiety-related behavior of the Mecp2(+/-) mice were found to be reversible with environmental enrichment (EE) from 4 weeks of age. The effect of exercise alone (via wheel running) was also explored, providing the first evidence that increased voluntary physical activity in an animal model of RTT is beneficial for some phenotypes. Mecp2(+/-) mutants displayed elevated corticosterone despite decreased Crh expression, demonstrating hypothalamic-pituitary-adrenal axis dysregulation. EE of Mecp2(+/-) mice normalized basal serum corticosterone and hippocampal BDNF protein levels. The enrichment-induced rescue appears independent of the transcriptional regulation of the MeCP2 targets Bdnf exon 4 and Crh. These findings provide new insight into the neurodevelopmental role of MeCP2 and pathogenesis of RTT, in particular the affective dysfunction. The positive outcomes of environmental stimulation and physical exercise have implications for the development of therapies targeting the affective symptoms, as well as behavioral and cognitive dimensions, of this devastating neurodevelopmental disorder.

Affective dysfunction in a mouse model of Rett syndrome: Therapeutic effects of environmental stimulation and physical activity.

Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue Q136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.

Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring.

Sook-Kwan Leang

WHO Collaborating Centre for Reference and Research on Influenza

Peter Doherty Institute for Infection and Immunity

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

Sook-Kwan Leang

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Peter Doherty Institute for Infection and Immunity

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

WHO Collaborating Centre for Reference and Research on Influenza