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

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

Journal of Clinical Microbiology, December 2016

27795346

Vasiliy P. Mishin, Tatiana Baranovich, Rebecca Garten, Anton Chesnokov, Anwar I. Abd Elal, Michelle Adamczyk, Jennifer LaPlante, Kirsten St. George, Alicia M. Fry, John Barnes, Stephanie C. Chester, Xiyan Xu, Jacqueline M. Katz, David E. Wentworth, Larisa V. Gubareva

Rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so 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 have developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of H3 clade 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a and 3C.3b viruses is based on the interrogation of five SNPs within three neighboring HA regions: 412-431; 465-481; and 559-571. 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 CT value < 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. Implementation of this approach enhanced virological surveillance and epidemiological studies from 2013-2016 when over 3000 A(H3N2) viruses were examined.