Dataset Information

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

ABSTRACT: The rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so the emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time consuming and mid-throughput. To facilitate virological surveillance and epidemiological studies, we developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of viruses of the H3 clades 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a, and 3C.3b is based on the interrogation of five single nucleotide polymorphisms (SNPs) within three neighboring HA regions, namely 412 to 431, 465 to 481, and 559 to 571. Two bioinformatics tools, IdentiFire (Qiagen) and FireComb (developed in-house), were utilized to expedite pyrosequencing data analysis. The assay's analytical sensitivity was 10 focus forming units, and respiratory specimens with threshold cycle (CT) values of <34 typically produced good quality pyrograms. When applied to 120 A(H3N2) virus isolates and 27 respiratory specimens, the assay displayed 100% agreement with clades determined by HA sequencing coupled with phylogenetics. The multi-SNP analysis described here was readily adopted by another laboratory with pyrosequencing capabilities. The implementation of this approach enhanced the findings from virological surveillance and epidemiological studies between 2013 and 2016, which examined more than 3,000 A(H3N2) viruses.

SUBMITTER: Mishin VP

PROVIDER: S-EPMC5228225 | biostudies-other | 2017 Jan

REPOSITORIES: biostudies-other

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A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

Mishin Vasiliy P VP Baranovich Tatiana T Garten Rebecca R Chesnokov Anton A Abd Elal Anwar I AI Adamczyk Michelle M LaPlante Jennifer J St George Kirsten K Fry Alicia M AM Barnes John J Chester Stephanie C SC Xu Xiyan X Katz Jacqueline M JM Wentworth David E DE Gubareva Larisa V LV

Journal of clinical microbiology 20161228 1

The rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so the emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time consuming and mid-throughput. To facilitat ...[more]

PMID: 27795346

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Project description:BACKGROUND: In pandemic and epidemic forms, avian and human influenza viruses often cause significant damage to human society and economics. Gradually accumulated mutations on hemagglutinin (HA) cause immunologically distinct circulating strains, which lead to the antigenic drift (named as antigenic variants). The "antigenic variants" often requires a new vaccine to be formulated before each annual epidemic. Mapping the genetic evolution to the antigenic drift of influenza viruses is an emergent issue to public health and vaccine development RESULTS: We developed a method for identifying antigenic critical amino acid positions, rules, and co-mutated positions for antigenic variants. The information gain (IG) and the entropy are used to measure the score of an amino acid position on hemagglutinin (HA) for discriminating between antigenic variants and similar viruses. A position with high IG and entropy implied that this position is highly correlated to an antigenic drift. Nineteen positions with high IG and high genetic diversity are identified as antigenic critical positions on the HA proteins. Most of these antigenic critical positions are located on five epitopes or on the surface based on the HA structure. Based on IG values and entropies of these 19 positions on the HA, the decision tree was applied to create a rule-based model and to identify rules for predicting antigenic variants of a given two HA sequences which are often a vaccine strain and a circulating strain. The predicting accuracies of this model on two sets, which consist of a training set (181 hemagglutination inhibition (HI) assays) and an independent test set (31,878 HI assays), are 91.2% and 96.2% respectively. CONCLUSION: Our method is able to identify critical positions, rules, and co-mutated positions on HA for predicting the antigenic variants. The information gains and the entropies of HA positions provide insight to the antigenic drift and co-evolution positions for influenza seasons. We believe that our method is robust and is potential useful for studying influenza virus evolution and vaccine development.

Project description:H3N2 strains of influenza A virus emerged in humans in 1968 and have continued to circulate, evolving in response to human immune pressure. During this process of "antigenic drift," viruses have progressively lost the ability to agglutinate erythrocytes of various species and to replicate efficiently under the established conditions for amplifying clinical isolates and generating vaccine candidates. We have determined the glycome profiles of chicken and guinea pig erythrocytes to gain insights into reduced agglutination properties displayed by drifted strains and show that both chicken and guinea pig erythrocytes contain complex sialylated N-glycans but that they differ with respect to the extent of branching, core fucosylation, and the abundance of poly-N-acetyllactosamine (PL) [-3Gal?1-4GlcNAc?1-]n structures. We also examined binding of the H3N2 viruses using three different glycan microarrays: the synthetic Consortium for Functional Glycomics array; the defined N-glycan array designed to reveal contributions to binding based on sialic acid linkage type, branched structures, and core modifications; and the human lung shotgun glycan microarray. The results demonstrate that H3N2 viruses have progressively lost their capacity to bind nearly all canonical sialylated receptors other than a selection of biantennary structures and PL structures with or without sialic acid. Significantly, all viruses displayed robust binding to nonsialylated high-mannose phosphorylated glycans, even as the recognition of sialylated structures is decreased through antigenic drift.IMPORTANCE Influenza subtype H3N2 viruses have circulated in humans for over 50?years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of preexisting immunity to previously circulating H3N2 strains. The changes in hemagglutinin (HA) affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in the culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates.

Dataset Information

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

Publications

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

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