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.
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.
Publication Date: 2019-02-26 PubMed ID: 30803412PubMed Central: PMC6505758DOI: 10.1177/1040638718822693Google Scholar: Lookup
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- Journal Article
- Validation Study
- Diagnosis
- Diagnostic Technique
- Disease Diagnosis
- Disease Management
- Disease Outbreaks
- Disease Surveillance
- Epidemiology
- Equine Diseases
- Equine Health
- Equine Science
- Genotyping
- H3N8
- Infectious Disease
- Influenza
- Molecular biology
- Polymerase Chain Reaction
- Real-Time PCR
- Veterinary Medicine
- Veterinary Research
- Viral Diseases
- Virology
Summary
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This study is about the validation of two specific PCR tests designed to detect and distinguish between two specific subtypes of the equine influenza A virus, based on the virus’s HA1 gene.
Overview of Research
The research focused on validating two multiplex real-time PCR (rtPCR) assays that are capable of determining the subtype (clade 1 or clade 2) of the H3N8 equine influenza A virus (EIV), especially within the Florida sublineage of the virus. These assays, referred to as SNP1 and SNP2, target single nucleotide polymorphisms (SNPs) of the hemagglutinin-1 (HA1) gene of the virus.
- The validation was initially conducted using 14 prototype strains of the virus belonging to the Florida sublineage.
- The study also incorporated nasal secretions from 341 horses collected across the United States between 2012 and 2017, as a way to determine the clade distribution of the virus within a real-world sample.
Findings of the Research
The study successfully validated the SNP1 and SNP2 assays, showing that they could accurately detect and categorize the H3N8 virus into either the clade 1 or clade 2 subtypes of the Florida sublineage.
- Among the 14 prototype strains, the assays correctly identified all as belonging to either clade 1 or clade 2.
- Among the 341 real-world samples, 337 (98.8%) were identified as clade 1, while the remaining 4 (1.2%) were clade 2.
- All clade 1 strains were detected in domestic horses, while three of the clade 2 strains were from horses recently imported to the US, and one was from a horse recently vaccinated with a strain of the virus included in a common vaccine.
Significance of the Research
The study’s findings are significant, as the development and validation of these assays could improve the efficiency and accuracy of detecting and differentiating between the clade 1 and clade 2 Florida sublineage EIV strains. This is particularly useful in monitoring the introduction and spread of clade 2 strains in North America through international horse transportation. Such a detection tool aids in the prevention, early detection, and response to potential outbreaks of the virus.
Cite This Article
APA
Brister H, Barnum SM, Reedy S, Chambers TM, Pusterla N.
(2019).
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.
J Vet Diagn Invest, 31(1), 137-141.
https://doi.org/10.1177/1040638718822693 Publication
Researcher Affiliations
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Brister, Barnum, Pusterla).
- Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Reedy, Chambers).
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Brister, Barnum, Pusterla).
- Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Reedy, Chambers).
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Brister, Barnum, Pusterla).
- Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Reedy, Chambers).
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Brister, Barnum, Pusterla).
- Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Reedy, Chambers).
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Brister, Barnum, Pusterla).
- Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (Reedy, Chambers).
MeSH Terms
- Animals
- Hemagglutinin Glycoproteins, Influenza Virus / analysis
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Influenza A Virus, H3N8 Subtype / isolation & purification
- Multiplex Polymerase Chain Reaction / methods
- Multiplex Polymerase Chain Reaction / veterinary
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / veterinary
- Orthomyxoviridae Infections / virology
- Polymorphism, Single Nucleotide
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
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.
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