Validation of two multiplex real-time PCR assays based on single nucleotide polymorphisms of the HA1 gene of equine influenza A virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates

Hanna Brister, Samantha M. Barnum, Stephanie Reedy, Thomas M. Chambers, Nicola Pusterla

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

We validated 2 multiplex real-time PCR (rtPCR) assays based on single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 (HA1) gene of H3N8 equine influenza A virus (EIV) to determine clade affiliation of prototype and field isolates. Initial validation of the 2 multiplex rtPCR assays (SNP1 and SNP2) was performed using nucleic acid from 14 EIV Florida sublineage clade 1 and 2 prototype strains. We included in our study previously banked EIV rtPCR-positive nasal secretions from 341 horses collected across the United States in 2012–2017 to determine their clade affiliation. All 14 EIV prototype strains were identified correctly as either Florida sublineage clade 1 or clade 2 using the 2 SNP target positions. Of 341 EIV rtPCR-positive samples, 337 (98.8%) and 4 (1.2%) isolates were classified as belonging to clade 1 and 2 Florida sublineage EIV, respectively. All clade 1 Florida sublineage EIV strains were detected in domestic horses, three clade 2 Florida sublineage EIV strains originated from horses recently imported into the United States, and one clade 2 Florida sublineage EIV strain originated from a healthy horse recently vaccinated with a modified-live intranasal EIV vaccine containing the American lineage strain A/eq/Kentucky/1991. EIV Florida sublineage clade differentiation using a fast and reliable multiplex rtPCR platform will help monitor the introduction of clade 2 Florida sublineage EIV strains into North America via international transportation.

Original languageEnglish
Pages (from-to)137-141
Number of pages5
JournalJournal of Veterinary Diagnostic Investigation
Volume31
Issue number1
DOIs
StatePublished - Jan 1 2019

Bibliographical note

Publisher Copyright:
© 2018 The Author(s).

Funding

We thank the Animal Health Trust, Newmarket, United Kingdom, and the University of Kentucky, Lexington, KY, for sharing nucleic acid from selected EIV Florida sublineage clade 1 and clade 2 prototype strains. TM Chambers and S Reedy were supported by a project of the Kentucky Agricultural Experiment Station (KY014053). This study was supported by Boehringer Ingelheim Vetmedica through an Advancement in Equine Research Award.

FundersFunder number
Boehringer Ingelheim Vetmedica
Kentucky Agricultural Experiment StationKY014053

    Keywords

    • Clade affiliation
    • equine influenza virus
    • horses
    • influenza A virus
    • multiplex rtPCR

    ASJC Scopus subject areas

    • General Veterinary

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