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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 The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- 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
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
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.
References
This article includes 17 references
- Bernardino PN. Pyrosequencing as a fast and reliable tool to determine clade affiliation for equine influenza A virus.. J Vet Diagn Invest 2016;28:323–326.
- Brault AC. Multiplex qRT-PCR for the detection of Western equine encephalomyelitis, St. Louis encephalitis, and West Nile viral RNA in mosquito pools (Diptera: Culicidae).. J Med Entomol 2015;52:491–499.
- Bryant NA. Isolation and characterisation of equine influenza viruses (H3N8) from Europe and North America from 2008 to 2009.. Vet Microbiol 2011;147:19–27.
- Cook RF. Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV).. J Virol Methods 2002;105:171–179.
- Diallo IS. Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).. Vet Microbiol 2007;123:93–103.
- Fougerolle S. Genetic evolution of equine influenza virus strains (H3N8) isolated in France from 1967 to 2015 and the implications of several potential pathogenic factors.. Virology 2017;505:210–217.
- Gildea S. The molecular epidemiology of equine influenza in Ireland from 2007–2010 and its international significance.. Equine Vet J 2012;44:387–389.
- Guthrie AJ. The circumstances surrounding the outbreak and spread of equine influenza in South Africa.. Rev Sci Tech 1999;18:179–185.
- Harms C. Detection of modified-live equine intranasal vaccine pathogens in adult horses using quantitative PCR.. Vet Rec 2014;175:510.
- Legrand LJ. Genetic evolution of equine influenza strains isolated in France from 2005 to 2010.. Equine Vet J 2015;47:207–211.
- Olguin Perglione C. Molecular epidemiology and spatio-temporal dynamics of the H3N8 equine influenza virus in South America.. Pathogens 2016;5:4.
- Pusterla N. Detection of clade 2 equine influenza virus in an adult horse recently imported to the USA.. Equine Vet Educ 2014;26:453–455.
- Rash A. Evolution and divergence of H3N8 equine influenza viruses circulating in the United Kingdom from 2013 to 2017.. Pathogens 2017;6:6.
- Sreenivasan CC. Phylogenetic analysis and characterization of a sporadic isolate of equine influenza A H3N8 from an unvaccinated horse in 2018.. Viruses 2018;10:31.
- Watson J. The 2007 outbreak of equine influenza in Australia: lessons learned for international trade in horses.. Rev Sci Tech 2011;30:87–93.
- Weyer CT. Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.. J Virol Methods 2015;223:69–74.
- Wilson WC. Field evaluation of a multiplex real-time reverse transcription polymerase chain reaction assay for detection of vesicular stomatitis virus.. J Vet Diagn Invest 2009;21:179–186.
Citations
This article has been cited 6 times.- Zhang Y, Guo X, Yu M, Sun L, Qu Y, Guo K, Hu Z, Liu D, Zhang H, Wang X. Equine ANP32 proteins support influenza A virus RNA polymerase activity. Virol Sin 2023 Oct 27;38(6):951-60.
- Gonzalez-Obando J, Forero JE, Zuluaga-Cabrera AM, Ruiz-Saenz J. Equine Influenza Virus: An Old Known Enemy in the Americas. Vaccines (Basel) 2022 Oct 14;10(10).
- Oladunni FS, Oseni SO, Martinez-Sobrido L, Chambers TM. Equine Influenza Virus and Vaccines. Viruses 2021 Aug 20;13(8).
- Karam B, Wilson WD, Chambers TM, Reedy S, Pusterla N. Hemagglutinin inhibition antibody responses to commercial equine influenza vaccines in vaccinated horses. Can Vet J 2021 Mar;62(3):266-272.
- Islam MT, Alam ARU, Sakib N, Hasan MS, Chakrovarty T, Tawyabur M, Islam OK, Al-Emran HM, Jahid MIK, Anwar Hossain M. A rapid and cost-effective multiplex ARMS-PCR method for the simultaneous genotyping of the circulating SARS-CoV-2 phylogenetic clades. J Med Virol 2021 May;93(5):2962-2970.
- Chambers TM. Equine Influenza. Cold Spring Harb Perspect Med 2022 Jan 4;12(1).
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