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 (C_T) 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.

Keywords: A(H3N2); genotyping; influenza; pyrosequencing.

FIG 1

Hemagglutinin clades (genotypes) of influenza A(H3N2) viruses from the 2013 to 2014 and 2014 to 2015 influenza seasons. (A) Phylogenetic tree of HA gene sequences (1,653 nt). Influenza vaccine strain for the 2013 to 2014 influenza season is shown in bold italics. Selected amino acid differences among representative sequences of each clade are shown. Amino acids at position 159 (nucleotide triplet 475 to 477) are shown in bold. (B) HA sequence alignment of representative viruses from six clades. RT-PCR primers are indicated by solid arrows, RT-PCR amplicons by the black solid lines, sequencing primers by the broken arrows, and regions 1, 2, and 3 are denoted by black squares. The nucleotide triplet encoding the amino acid at position 159 is marked with bold black dots.

FIG 2

Algorithm for HA clade identification using the pyrosequencing assay. The assay resolves the short sequences within regions 1 to 3 of the HA. SNPs used for identification of clade 3C.2 are in red, for 3C.2a, 3C.2b, and 3C.3a are in green, and in purple for 3C.3 and 3C.3b. *, nucleotide variations, as listed in Table S3 in the supplemental material.

FIG 3

Determination of C_T value for original respiratory specimens. C_T values were determined using the CDC real-time RT-PCR diagnostic assay (detailed description of the assay is available at http://www.accessdata.fda.gov/cdrh_docs/pdf8/k080570.pdf).