A PCR based method for the identification of equine influenza virus from clinical samples.
Abstract: In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza virus (A2). We show that the assays are specific for the target genes chosen, and display sensitivity similar to virus isolation. The NP assay detects a variety of different influenza subtypes, whereas A1 and A2 assays are specific for influenza subtypes H7N7 and H3N8, respectively. Sequencing of amplicons obtained in the A2 assay yields information on antigenic regions of the haemagglutinin molecule, and use of this procedure in the routine surveillance of equine influenza will enable tentative characterisation of circulating viruses despite difficulties in isolating field strains of the H3N8 subtype. The A1 assay will be useful in ascertaining whether viruses of the H7N7 subtype still circulate amongst horses, or whether these are extinct.
Publication Date: 1999-07-27 PubMed ID: 10418871DOI: 10.1016/s0378-1135(99)00041-3Google 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
- Research Support
- Non-U.S. Gov't
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 research describes the creation of a procedure using a nested RT-PCR assay for quick diagnosis and characterization of equine influenza virus directly from clinical samples. The assay can detect different influenza subtypes and helps with the tentative characterization of circulating viruses.
Research Methodology and Strategy
- In the study, the researchers developed a nested RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) assay as a diagnostic tool for the rapid identification of equine influenza virus from clinical specimens.
- They extracted viral RNA from nasal swabs using the guanidine thiocyanate extraction method. This RNA was then reversed transcribed to form a complementary DNA (cDNA) that served as a template in a nested PCR reaction.
- The assay uses primer sequences specifically designed for three templates: the nucleoprotein gene, the haemagglutinin gene of the H7N7 equine influenza virus, and the haemagglutinin gene of the H3N8 equine influenza virus.
- The researchers validated the specificity and sensitivity of the assays against their selected target genes.
Findings of the Study
- The study found that the nucleoprotein (NP) assay could detect various influenza subtypes, while the H7N7 (A1) and H3N8 (A2) assays were designed to specifically identify their corresponding equine influenza subtypes.
- The A2 assay’s sequencing of the amplified products (amplicons) provides information regarding the antigenic regions of the haemagglutinin molecule. This is valuable for the characterization of circulating viruses, even in situations where it is difficult to isolate field strains of the H3N8 subtype.
- The A1 assay, designed for the H7N7 subtype, can potentially discern whether this subtype of the virus still exists among horses or if it has subsequently become extinct.
Implication of Research
- The diagnostic tool designed in this research offers a promising application for the rapid detection and characterization of equine influenza viruses.
- Its use in regular monitoring of equine influenza could provide key insights into the characteristics and prevalence of circulating viruses. This information can assist in controlling the disease’s spread and developing measures towards prevention and treatment.
- The discovery of whether the H7N7 subtype still circulates among horses, or whether these are extinct, will provide essential data for understanding the evolution and spread of influenza viruses among equine populations.
Cite This Article
APA
Oxburgh L, Hagström A.
(1999).
A PCR based method for the identification of equine influenza virus from clinical samples.
Vet Microbiol, 67(3), 161-174.
https://doi.org/10.1016/s0378-1135(99)00041-3 Publication
Researcher Affiliations
- Department of Veterinary Microbiology, Swedish University of Agricultural Sciences, Biomedical Centre, Uppsala. leif.oxburgh@vmm.slu.se
MeSH Terms
- Animals
- Base Sequence
- Chick Embryo
- DNA Primers / chemistry
- DNA, Viral / chemistry
- Electrophoresis, Agar Gel
- Fluorescent Antibody Technique / veterinary
- Hemagglutination Inhibition Tests / veterinary
- Hemagglutination Tests / veterinary
- Horse Diseases / diagnosis
- Horse Diseases / virology
- Horses
- Influenza A virus / classification
- Influenza A virus / isolation & purification
- Nose Diseases / diagnosis
- Nose Diseases / veterinary
- Nose Diseases / virology
- Orthomyxoviridae Infections / diagnosis
- Orthomyxoviridae Infections / veterinary
- Polymerase Chain Reaction / methods
- Polymerase Chain Reaction / veterinary
- RNA, Viral / isolation & purification
- Reverse Transcriptase Polymerase Chain Reaction / methods
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Sensitivity and Specificity
- Sequence Alignment
- Sequence Analysis, DNA
- Sequence Homology, Nucleic Acid
Citations
This article has been cited 6 times.- Singh RK, Dhama K, Karthik K, Khandia R, Munjal A, Khurana SK, Chakraborty S, Malik YS, Virmani N, Singh R, Tripathi BN, Munir M, van der Kolk JH. A Comprehensive Review on Equine Influenza Virus: Etiology, Epidemiology, Pathobiology, Advances in Developing Diagnostics, Vaccines, and Control Strategies.. Front Microbiol 2018;9:1941.
- Gora IM, Kwasnik M, Zmudzinski JF, Rozek W. Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus.. Virol J 2017 Jun 21;14(1):120.
- Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB. Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.. J Clin Microbiol 2009 Dec;47(12):3907-13.
- DiMenna LJ, Ertl HC. Pandemic influenza vaccines.. Curr Top Microbiol Immunol 2009;333:291-321.
- Quinlivan M, Cullinane A, Nelly M, Van Maanen K, Heldens J, Arkins S. Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus.. J Clin Microbiol 2004 Feb;42(2):759-63.
- Oxburgh L, Klingeborn B. Cocirculation of two distinct lineages of equine influenza virus subtype H3N8.. J Clin Microbiol 1999 Sep;37(9):3005-9.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
