A novel PCR protocol for detection and differentiation of neuropathogenic and non-neuropathogenic equid alphaherpesvirus 1.
Abstract: Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A→G) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A and G in neurologic and non-neurologic cases, respectively.
Publication Date: 2019-09-04 PubMed ID: 31477001PubMed Central: PMC6727124DOI: 10.1177/1040638719871975Google Scholar: Lookup
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- Journal Article
Summary
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This paper presents a newly developed method for detecting a specific virus in horses, Equid alphaherpesvirus 1 (EHV-1), and differentiating between its neuropathogenic and non-neuropathogenic types. The researchers tested this method against a previously established technique and found it to be more accurate.
Background
- Equid alphaherpesvirus 1 (EHV-1) is a virus that is known to cause serious conditions in horses like abortion, respiratory illness, or neurologic disease. This virus has both neuropathogenic and non-neuropathogenic versions, note the difference being a single nucleotide polymorphism (SNP).
- SNPs represent a variation of a single nucleotide that occurs at specific positions in the genome. In this case, there is a SNP (referred to as A→G) in the virus’s open reading frame (ORF) 30 – a sequence of DNA responsible for encoding the catalytic subunit of the DNA polymerase. This particular SNP can predict whether a virus is neuropathogenic.
- A method to determine the EHV-1 genotype (neuropathogenic or non-neuropathogenic) had been previously established using a technique called real-time PCR (rtPCR) but it had shown unsatisfactory results.
Research Development
- In order to address the issues with the initial rtPCR protocol, the researchers developed a new protocol that involved conventional PCR, sequencing, and restriction enzyme analysis (REA) – an method used to study DNA sequence.
- To evaluate their new PCR protocol, the researchers tested samples from 30 horses showing typical signs of EHV-1 infection.
- The results indicated that the novel PCR protocol, when combined with sequencing and REA, was more accurate in determining the EHV-1 genotype compared to the rtPCR technique.
Study Findings
- The researchers confirmed the earlier findings that the non-neuropathogenic genotype A is largely found in animals experiencing fever, respiratory signs, and abortions or perinatal mortality. On the other hand, the neuropathogenic genotype G was primarily found in animals exhibiting signs of neurologic disease.
- Interestingly, the study found indications of coinfection with both genotypes in some samples.
Further Research
- Although the study has presented a more reliable method to differentiate the EHV-1 genotypes, further investigation is required. The researchers suggest examining the significance of infections with genotype A and G in both neurologic and non-neurologic cases to gain deeper understanding of the behavior of the virus.
Cite This Article
APA
Lechmann J, Schoster A, Ernstberger M, Fouché N, Fraefel C, Bachofen C.
(2019).
A novel PCR protocol for detection and differentiation of neuropathogenic and non-neuropathogenic equid alphaherpesvirus 1.
J Vet Diagn Invest, 31(5), 696-703.
https://doi.org/10.1177/1040638719871975 Publication
Researcher Affiliations
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
- Institute of Virology (Lechmann, Fraefel, Bachofen), Equine Department, Clinic for Equine Internal Medicine (Schoster), Department of Farm Animals, Division of Herd Medicine and Outpatient Clinic (Ernstberger), Vetsuisse Faculty, University of Zurich, Zurich Switzerland.
- Swiss Institute of Equine Medicine ISME, Department of Clinical Veterinary Medicine, Vetsuisse Faculty, University of Bern, and Agroscope, Bern, Switzerland (Fouché).
MeSH Terms
- Animals
- DNA-Directed DNA Polymerase / genetics
- Female
- Genotype
- Herpesviridae Infections / veterinary
- Herpesviridae Infections / virology
- Herpesvirus 1, Equid / classification
- Herpesvirus 1, Equid / genetics
- Horse Diseases / virology
- Horses
- Nervous System Diseases / veterinary
- Nervous System Diseases / virology
- Open Reading Frames
- Polymorphism, Single Nucleotide
- Pregnancy
- Real-Time Polymerase Chain Reaction / methods
- Real-Time Polymerase Chain Reaction / veterinary
- Restriction Mapping / veterinary
Conflict of Interest Statement
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
References
This article includes 25 references
- Allen GP. Antemortem detection of latent infection with neuropathogenic strains of equine herpesvirus-1 in horses.. Am J Vet Res 2006 Aug;67(8):1401-5.
- Allen GP. Development of a real-time polymerase chain reaction assay for rapid diagnosis of neuropathogenic strains of equine herpesvirus-1.. J Vet Diagn Invest 2007 Jan;19(1):69-72.
- Allen GP. Risk factors for development of neurologic disease after experimental exposure to equine herpesvirus-1 in horses.. Am J Vet Res 2008 Dec;69(12):1595-600.
- Allen GP, Bolin DC, Bryant U, Carter CN, Giles RC, Harrison LR, Hong CB, Jackson CB, Poonacha K, Wharton R, Williams NM. Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky.. Equine Vet J 2008 Mar;40(2):105-10.
- Barbić L, Lojkić I, Stevanović V, Bedeković T, Starešina V, Lemo N, Lojkić M, Madić J. Two outbreaks of neuropathogenic equine herpesvirus type 1 with breed-dependent clinical signs.. Vet Rec 2012 Mar 3;170(9):227.
- Davison AJ. Evolution of the herpesviruses.. Vet Microbiol 2002 Apr 22;86(1-2):69-88.
- Diallo IS, Hewitson G, Wright LL, Kelly MA, Rodwell BJ, Corney BG. Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).. Vet Microbiol 2007 Jul 20;123(1-3):93-103.
- Fritsche AK, Borchers K. Detection of neuropathogenic strains of Equid Herpesvirus 1 (EHV-1) associated with abortions in Germany.. Vet Microbiol 2011 Jan 10;147(1-2):176-80.
- Goodman LB, Loregian A, Perkins GA, Nugent J, Buckles EL, Mercorelli B, Kydd JH, Palù G, Smith KC, Osterrieder N, Davis-Poynter N. A point mutation in a herpesvirus polymerase determines neuropathogenicity.. PLoS Pathog 2007 Nov;3(11):e160.
- Ma G, Azab W, Osterrieder N. Equine herpesviruses type 1 (EHV-1) and 4 (EHV-4)--masters of co-evolution and a constant threat to equids and beyond.. Vet Microbiol 2013 Nov 29;167(1-2):123-34.
- Maxwell LK, Bentz BG, Gilliam LL, Ritchey JW, Pusterla N, Eberle R, Holbrook TC, McFarlane D, Rezabek GB, Meinkoth J, Whitfield C, Goad CL, Allen GP. Efficacy of the early administration of valacyclovir hydrochloride for the treatment of neuropathogenic equine herpesvirus type-1 infection in horses.. Am J Vet Res 2017 Oct;78(10):1126-1139.
- Nugent J, Birch-Machin I, Smith KC, Mumford JA, Swann Z, Newton JR, Bowden RJ, Allen GP, Davis-Poynter N. Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks.. J Virol 2006 Apr;80(8):4047-60.
- Perkins GA, Goodman LB, Tsujimura K, Van de Walle GR, Kim SG, Dubovi EJ, Osterrieder N. Investigation of the prevalence of neurologic equine herpes virus type 1 (EHV-1) in a 23-year retrospective analysis (1984-2007).. Vet Microbiol 2009 Nov 18;139(3-4):375-8.
- Pronost S, Léon A, Legrand L, Fortier C, Miszczak F, Freymuth F, Fortier G. Neuropathogenic and non-neuropathogenic variants of equine herpesvirus 1 in France.. Vet Microbiol 2010 Oct 26;145(3-4):329-33.
- Pronost S, Legrand L, Pitel PH, Wegge B, Lissens J, Freymuth F, Richard E, Fortier G. Outbreak of equine herpesvirus myeloencephalopathy in France: a clinical and molecular investigation.. Transbound Emerg Dis 2012 Jun;59(3):256-63.
- Pronost S, Cook RF, Fortier G, Timoney PJ, Balasuriya UB. Relationship between equine herpesvirus-1 myeloencephalopathy and viral genotype.. Equine Vet J 2010 Nov;42(8):672-4.
- Pusterla N, Wilson WD, Mapes S, Finno C, Isbell D, Arthur RM, Ferraro GL. Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California.. Vet J 2009 Feb;179(2):230-9.
- Pusterla N, David Wilson W, Madigan JE, Ferraro GL. Equine herpesvirus-1 myeloencephalopathy: a review of recent developments.. Vet J 2009 Jun;180(3):279-89.
- Pusterla N, Mapes S, David Wilson W. Prevalence of latent alpha-herpesviruses in Thoroughbred racing horses.. Vet J 2012 Aug;193(2):579-82.
- Smith KL, Allen GP, Branscum AJ, Frank Cook R, Vickers ML, Timoney PJ, Balasuriya UB. The increased prevalence of neuropathogenic strains of EHV-1 in equine abortions.. Vet Microbiol 2010 Feb 24;141(1-2):5-11.
- Smith KL, Li Y, Breheny P, Cook RF, Henney PJ, Sells S, Pronost S, Lu Z, Crossley BM, Timoney PJ, Balasuriya UB. New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G2254 polymorphisms.. J Clin Microbiol 2012 Jun;50(6):1981-8.
- Tewari D, Del Piero F, Cieply S, Feria W, Acland H. Equine herpesvirus 1 (EHV-1) nucleotide polymorphism determination using formalin fixed tissues in EHV-1 induced abortions and myelopathies with real-time PCR and pyrosequencing.. J Virol Methods 2013 Nov;193(2):371-3.
- Van de Walle GR, Goupil R, Wishon C, Damiani A, Perkins GA, Osterrieder N. A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease.. J Infect Dis 2009 Jul 1;200(1):20-5.
- Vaz PK, Horsington J, Hartley CA, Browning GF, Ficorilli NP, Studdert MJ, Gilkerson JR, Devlin JM. Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1.. J Gen Virol 2016 Mar;97(3):747-755.
- Walter J, Seeh C, Fey K, Bleul U, Osterrieder N. Clinical observations and management of a severe equine herpesvirus type 1 outbreak with abortion and encephalomyelitis.. Acta Vet Scand 2013 Mar 5;55(1):19.
Citations
This article has been cited 1 times.- Sutton G, Garvey M, Cullinane A, Jourdan M, Fortier C, Moreau P, Foursin M, Gryspeerdt A, Maisonnier V, Marcillaud-Pitel C, Legrand L, Paillot R, Pronost S. Molecular Surveillance of EHV-1 Strains Circulating in France during and after the Major 2009 Outbreak in Normandy Involving Respiratory Infection, Neurological Disorder, and Abortion.. Viruses 2019 Oct 4;11(10).
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