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. 2019 Jan;31(1):137-141.
doi: 10.1177/1040638718822693.

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  1   2 , Samantha M Barnum  1   2 , Stephanie Reedy  1   2 , Thomas M Chambers  1   2 , Nicola Pusterla  1   2
Affiliations

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 et al. J Vet Diagn Invest. 2019 Jan.

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.

Keywords: Clade affiliation; equine influenza virus; horses; influenza A virus; multiplex rtPCR.

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Conflict of interest statement

Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Nucleotide alignment of primers and probes for single nucleotide polymorphism (SNP)1 and SNP2 multiplex real-time PCR assays and partial nucleotide sequence of A/eq/Aboyne/1/2005 and A/eq/Kentucky/1991. Forward primer is highlighted in yellow, reverse primer in green, and probes are in blue. Nucleotide differences at positions 281/282 and 524/525 are in boldface.
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