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 midthroughput. 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 (C T ) values of Ͻ34 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.KEYWORDS A(H3N2), genotyping, influenza, pyrosequencing S ince their introduction into the human population in 1968, influenza A(H3N2) viruses have been responsible for seasonal epidemics and associated with both a prolonged duration of the epidemic season and a greater disease severity (1-4). The hemagglutinin (HA) glycoprotein is the major surface antigen of influenza viruses; it is also responsible for the receptor binding and membrane fusion required for entrance into susceptible host cells (5). Decades ago, this glycoprotein was named "hemagglutinin" for its ability to bind and bridge erythrocytes. The other surface antigen, neuraminidase (NA), is a receptor-destroying enzyme (6). As with other seasonal influenza viruses, A(H3N2) viruses undergo genetic and antigenic changes, called antigenic drift,

Influenza Vaccine Effectiveness in the United States during the 2015–2016 Season

“…In addition, A(H3N2) viruses were classified into hemagglutinin genetic groups by means of a pyrosequencing assay. 17,18 Selected viruses from each clade or genetic group were grown to high titer and antigenically characterized by means of hemagglutination-inhibition or focus-reduction (for A[H3N2]) assays against the 2015–2016 vaccine reference strains. 19 …”

Insights into the antigenic advancement of influenza A(H3N2) viruses, 2011–2018

“…In instances when HA sequence was not available for respiratory specimens, the H3 clade determination was performed using the previously described pyrosequencing method 40 . Pyrosequencing analysis was additionally used to assess nucleotide polymorphism at triplets encoding amino acid residues 158 and 160 in the HA sequences of influenza A(H3N2) virus isolates from clades 3C.2a and 3C.2a1.…”

“…Pyrosequencing analysis was additionally used to assess nucleotide polymorphism at triplets encoding amino acid residues 158 and 160 in the HA sequences of influenza A(H3N2) virus isolates from clades 3C.2a and 3C.2a1. The primers utilized for determining the proportion of virus population that lost the glycosylation sequon N 158 -X-T 160 were: H3-F370, H3-R645-biotin and H3clade-R2-F445 described previously 13,40 .…”

Design and validation of a universal influenza virus enrichment probe set and its utility in deep sequence analysis of primary cloacal swab surveillance samples of wild birds

“…First introduced in 2005, ‘high-throughput’ DNA sequencers, which can determine megabases of DNA sequence per run (Service, 2006), have evolved dramatically, increasing sequencing capacity by a factor of 100–1000 (Goodwin et al, 2016). They have been widely used in influenza research studies, including detecting IAV and norovirus infections in patients (Nakamura et al, 2009), uncovering mixed infection with 2009 pandemic influenza A viruses (Ghedin et al, 2011), high throughput sequencing of influenza B viruses (Zhou et al, 2014), evaluating genetic stability of influenza vaccine viruses (Laassri et al, 2015), revealing antigenic variants at low frequencies in IAV-infected patients (Dinis et al, 2016), and high-throughput identification of influenza A/H3N2 virus antigenic drift variants (Mishin et al, 2017). However, because of the limited amount of viral RNA in typical clinical samples, all of these studies employed virus-specific primers and virus-specific PCR amplification strategies to enrich for target influenza sequences.…”