title: 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.
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: Vasiliy P
authors: VP
authors: Mishin
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.|Carter Consulting, Inc., Atlanta, Georgia, USA.
authors: Tatiana
authors: T
authors: Baranovich
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: Rebecca
authors: R
authors: Garten
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.|Battelle Memorial Institute, Atlanta, Georgia, USA.
authors: Anton
authors: A
authors: Chesnokov
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.|Battelle Memorial Institute, Atlanta, Georgia, USA.
authors: Anwar I
authors: AI
authors: Abd Elal
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.|Battelle Memorial Institute, Atlanta, Georgia, USA.
authors: Michelle
authors: M
authors: Adamczyk
authors: Wadsworth Center, New York State Department of Health (NYSDOH), Albany, New York, USA.
authors: Jennifer
authors: J
authors: LaPlante
authors: Wadsworth Center, New York State Department of Health (NYSDOH), Albany, New York, USA.
authors: Kirsten
authors: 0000-0001-5168-763X
authors: K
authors: St George
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: Alicia M
authors: AM
authors: Fry
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: John
authors: J
authors: Barnes
authors: Association of Public Health Laboratories, Silver Spring, Maryland, USA.
authors: Stephanie C
authors: SC
authors: Chester
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: Xiyan
authors: X
authors: Xu
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: Jacqueline M
authors: JM
authors: Katz
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA.
authors: David E
authors: DE
authors: Wentworth
authors: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention (CDC), Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Atlanta, Georgia, USA lgubareva@cdc.gov.
authors: Larisa V
authors: LV
authors: Gubareva
mesh_terms: D040961:Genetic Drift; D060005:Genotyping Techniques / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D007251:Influenza, Human / Q000821:virology*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus
keywords: A(H3N2); genotyping; influenza; pyrosequencing