Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

The Francis Crick Institute

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors.

&#xC870;&#xB958; &#xC778;&#xD50C;&#xB8E8;&#xC5D4;&#xC790; &#xC751;&#xC9D1; &#xC5B5;&#xC81C;&#xC81C; (NAIs) &#xAC10; &#xADF8;&#xB9AC;&#xACE0; &#xC751;&#xC9D1; (NA)&#xC758; &#xD65C;&#xB3D9;&#xC5D0; &#xC751;&#xC9D1; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; h 5 n 1 I117V, I314V &#xBC0F; I117V + i314v&#xC758; &#xC601;&#xD5A5;&#xC744; &#xACF5;&#xBD80; &#xD569;&#xB2C8;&#xB2E4;.

[&#xC870;&#xB958; &#xB3C5;&#xAC10; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; h 5 n 1 &#xC751;&#xC9D1; &#xB3CC;&#xC5F0;&#xBCC0;&#xC774; &#xC751;&#xC9D1;&#xACFC; &#xC751;&#xC9D1; &#xC5B5;&#xC81C;&#xC81C;&#xB97C; &#xAC10;&#xC131; &#xD65C;&#xB3D9;&#xC5D0;&#xC758; &#xC601;&#xD5A5;].

Para estudiar el impacto de la gripe aviar virus H5N1 neuraminidasa mutaciones I117V, I314V y I117V + I314V en la sensibilidad de los inhibidores de la neuraminidasa (NAIs) y la actividad de neuraminidasa (NA).

[Impacto de mutaciones de neuraminidasa virus H5N1 de gripe aviar en la actividad de la neuraminidasa y la sensibilidad a los inhibidores de la neuraminidasa].

&#x8981;&#x7814;&#x7A76;&#x79BD;&#x6D41;&#x611F; H5N1 &#x75C5;&#x6BD2;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x7A81;&#x53D8; I117V&#x3001; I314V &#x548C; I117V + I314V &#x611F;&#x6027;&#x7684;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x6291;&#x5236;&#x5242; (NAIs) &#x548C;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176; (NA) &#x7684;&#x6D3B;&#x52A8;&#x7684;&#x5F71;&#x54CD;&#x3002;

[&#x79BD;&#x6D41;&#x611F; H5N1 &#x75C5;&#x6BD2;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x7A81;&#x53D8;&#x6D3B;&#x6027;&#x7684;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x548C;&#x795E;&#x7ECF;&#x6C28;&#x9178;&#x9176;&#x6291;&#x5236;&#x5242;&#x654F;&#x611F;&#x6027;&#x7684;&#x5F71;&#x54CD;]&#x3002;

To study the impact of avian influenza virus H5N1 neuraminidase mutations I117V, I314V and I117V + I314V on the sensibility of neuraminidase inhibitors (NAIs) and the activity of neuraminidase (NA).

[Impact of avian influenza virus H5N1 neuraminidase mutations on the activity of neuraminidase and the sensibility to neuraminidase inhibitors].

Pour &eacute;tudier l&#39;impact des mutations de la neuraminidase de virus H5N1 grippe aviaire I117V, I314V et I117V + I314V sur la sensibilit&eacute; des inhibiteurs de la neuraminidase (SNIA) et l&#39;activit&eacute; de la neuraminidase (NA).

[L&#39;impact de la grippe aviaire virus H5N1 la neuraminidase mutations sur l&#39;activit&eacute; de la neuraminidase et la sensibilit&eacute; aux inhibiteurs de la neuraminidase].

Die Auswirkungen des Vogelgrippe-Virus H5N1 Neuraminidase Mutationen I117V, I314V und I117V + I314V auf die Sensibilit&auml;t der Neuraminidase-Hemmer (NAIs) und die Neuraminidase (NA)-Aktivit&auml;t zu untersuchen.

[Auswirkungen der Gefl&uuml;gelpest H5N1-Virus Neuraminidase Mutationen auf die Aktivit&auml;t der Neuraminidase und die Sensibilit&auml;t auf Neuraminidase-Inhibitoren].

Per studiare l&#39;impatto dell&#39;influenza aviaria virus H5N1 neuraminidasi mutazioni I117V, I314V e I117V + I314V la sensibilit&agrave; degli inibitori della neuraminidasi (NAIs) e nell&#39;attivit&agrave; di neuraminidasi (NA).

[Impatto dell&#39;influenza aviaria virus H5N1 neuraminidasi mutazioni sull&#39;attivit&agrave; della neuraminidasi e la sensibilit&agrave; di inibitori della neuraminidasi].

&#x9CE5;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6; &#x30A6;&#x30A4;&#x30EB;&#x30B9; h5n1 &#x578B;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x5909;&#x7570; I117V&#x3001;I314V&#x3001;I117V + I314V &#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x963B;&#x5BB3;&#x5264; &#xFF08;NAIs&#xFF09; &#x306E;&#x611F;&#x6027;&#x3068;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC; &#xFF08;NA&#xFF09; &#x306E;&#x6D3B;&#x52D5;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x7814;&#x7A76;&#x3059;&#x308B;&#x306B;&#x306F;&#x3002;

[&#x9CE5;&#x30A4;&#x30F3;&#x30D5;&#x30EB;&#x30A8;&#x30F3;&#x30B6; &#x30A6;&#x30A4;&#x30EB;&#x30B9; H5N1 &#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x5909;&#x7570;&#x6D3B;&#x6027;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x3068;&#x611F;&#x6027;&#x306B;&#x30CE;&#x30A4;&#x30E9;&#x30DF;&#x30CB;&#x30C0;&#x30FC;&#x30BC;&#x963B;&#x5BB3;&#x5264;&#x306E;&#x5F71;&#x97FF;]&#x3002;

The identification of antigenic variants and the selection of influenza viruses for vaccine production are based largely on antigenic characterisation of the haemagglutinin (HA) of circulating viruses using the haemagglutination inhibition (HI) assay. However, additional to evolution related to escape from host immunity, variants emerging as a result of propagation in different cell substrates can complicate interpretation of HI results.

Optimization of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses.

Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

Yipu Lin

Mill Hill Laboratory

The Francis Crick Institute

Optimization of a Quantitative Micro-neutralization Assay

Yipu Lin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Mill Hill Laboratory

The Francis Crick Institute

Optimization of a Quantitative Micro-neutralization Assay

Mill Hill Laboratory