Characterisation of gamma herpesviruses in the horse by PCR.
Abstract: A polymerase chain reaction (PCR) based on a combination of oligonucleotide primers selected using the octamer frequency disparity method with primers specific for EHV-5 (described by other authors) recognized all of a series of gamma herpesvirus field isolates. This PCR produced only three fragments: (1) one EHV-2-specific; (2) one EHV-5-specific; and (3) a fragment that occurred alone or in combination with the other two. Cloning and sequencing of four different isolates yielding only the last PCR product showed that this corresponds to a deletion/insertion mutant of EHV-2. The fact that this mutant was also plaque-purified from a culture producing all three PCR fragments demonstrated that the virus producing this fragment was distinct from the other two and that this specific DNA fragment was not an artefact due to PCR amplification. These data show that equine gamma herpesviruses are genetically more heterogeneous than previously assumed. The PCR failed to directly detect gamma herpesviruses from the DNA extracted from the same starting material used for the isolation of gamma herpesvirus by cocultivation with indicator cells. This demonstrates that the most reliable method for detection of equine gamma herpesviruses is the cocultivation with indicator cells.
Publication Date: 1997-12-31 PubMed ID: 9375003DOI: 10.1006/viro.1997.8825Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study explores the genetic diversities of gamma herpesviruses in horses using polymerase chain reaction (PCR). The researchers discovered more genetic variety than previously known and concluded that culturing virus samples in conjunction with indicator cells is the most accurate method of detection.
Research Methodology and Findings
- The research centered on using polymerase chain reaction (PCR), a lab technique used to amplify small or few segments of DNA. This tool was based on a combination of primers (short strands of DNA that serve as a starting point for DNA synthesis); some chosen through the octamer frequency disparity method and others specific for EHV-5 (a type of horse herpes virus).
- The research showed that the PCR recognized all gamma herpesvirus field isolates under study and produced three fragments: one EHV-2-specific (another type of horse herpes virus), one EHV-5-specific, and a third fragment that was found independently or in combination with the other two.
- After cloning and sequencing four different isolates yielding only the third fragment, researchers concluded this represented a mutation of EHV-2. This was affirmed by the fact that this mutant virus was also plaque-purified from a culture producing all three PCR fragments, proving that the third virus fragment was not a result of artificial amplification by the PCR but represented a distinct variant of the virus.
- These results illustrated that equine gamma herpesviruses are genetically more diverse than previously assumed, indicating a need for further research and understanding in this area.
PCR’s Limitation and the Most Reliable Detection Method
- The PCR technique failed to directly detect gamma herpesviruses from the DNA extracted from the same initial material used for isolating the virus in a traditional cultivation procedure.
- This led the researchers to conclude that the most reliable method for the detection of equine gamma herpesviruses is not PCR, rather cultivation of potential virus samples in harmony with indicator cells. Cocultivation facilitates the virus’s growth, making it easier to detect and isolate.
Cite This Article
APA
Franchini M, Akens M, Bracher V, von Fellenberg R.
(1997).
Characterisation of gamma herpesviruses in the horse by PCR.
Virology, 238(1), 8-13.
https://doi.org/10.1006/viro.1997.8825 Publication
Researcher Affiliations
- Institute of Veterinary Physiology, University of Zurich, Switzerland. mfra@vetphys.unizh.ch
MeSH Terms
- Animals
- Base Sequence
- Bronchoalveolar Lavage Fluid / virology
- Capsid / genetics
- DNA Primers
- Female
- Gammaherpesvirinae / genetics
- Gammaherpesvirinae / isolation & purification
- Horses / virology
- Lymphocytes / virology
- Male
- Molecular Sequence Data
- Polymerase Chain Reaction / methods
- Sequence Alignment
- Sequence Homology, Nucleic Acid
Citations
This article has been cited 6 times.- Temesgen T, Getachew Y, Negussie H. Molecular Identification of Equine Herpesvirus 1, 2, and 5 in Equids with Signs of Respiratory Disease in Central Ethiopia. Vet Med (Auckl) 2021;12:337-345.
- Thorsteinsdóttir L, Jónsdóttir S, Stefánsdóttir SB, Andrésdóttir V, Wagner B, Marti E, Torsteinsdóttir S, Svansson V. The effect of maternal immunity on the equine gammaherpesvirus type 2 and 5 viral load and antibody response. PLoS One 2019;14(6):e0218576.
- Dall Agnol AM, Beuttemmuller EA, Pilz D, Leme RA, Saporiti V, Headley SA, Alfieri AF, Alfieri AA. Detection of Equid gammaherpesvirus 2 and 5 DNA in the upper respiratory tract of asymptomatic horses from Southern Brazil. Braz J Microbiol 2019 Jul;50(3):875-878.
- Van Cleemput J, Poelaert KCK, Laval K, Nauwynck HJ. Unravelling the first key steps in equine herpesvirus type 5 (EHV5) pathogenesis using ex vivo and in vitro equine models. Vet Res 2019 Feb 18;50(1):13.
- Marenzoni ML, Stefanetti V, Danzetta ML, Timoney PJ. Gammaherpesvirus infections in equids: a review. Vet Med (Auckl) 2015;6:91-101.
- Marenzoni ML, Coppola G, Maranesi M, Passamonti F, Cappelli K, Capomaccio S, Verini Supplizi A, Thiry E, Coletti M. Age-dependent prevalence of equid herpesvirus 5 infection. Vet Res Commun 2010 Dec;34(8):703-8.
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