Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates
Background: Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently...
| Autores principales: | , , , , , , , , , |
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| Formato: | info:ar-repo/semantics/artículo |
| Lenguaje: | Inglés |
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BMC
2019
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| Acceso en línea: | https://bmcvetres.biomedcentral.com/articles/10.1186/1746-6148-9-111 http://hdl.handle.net/20.500.12123/4276 https://doi.org/10.1186/1746-6148-9-111 |
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| author | Maidana, Silvina Soledad Morano, Cintia Débora Cianfrini, Daniela Campos, Fabrício Souza Roehe, Paulo Michel Siedler, Bianca De Stefano, Gabriel Alejandro Mauroy, Axel Thiry, Etienne Romera, Sonia Alejandra |
| author_browse | Campos, Fabrício Souza Cianfrini, Daniela De Stefano, Gabriel Alejandro Maidana, Silvina Soledad Mauroy, Axel Morano, Cintia Débora Roehe, Paulo Michel Romera, Sonia Alejandra Siedler, Bianca Thiry, Etienne |
| author_facet | Maidana, Silvina Soledad Morano, Cintia Débora Cianfrini, Daniela Campos, Fabrício Souza Roehe, Paulo Michel Siedler, Bianca De Stefano, Gabriel Alejandro Mauroy, Axel Thiry, Etienne Romera, Sonia Alejandra |
| author_sort | Maidana, Silvina Soledad |
| collection | INTA Digital |
| description | Background: Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. The present work describes a multiplex PCR followed by REA for BoHV-5 subtyping.
Results: The method consists in the simultaneous amplification of glycoprotein B and UL54 gene fragments of 534 and 669 base pairs (bp), respectively, BstEII digestion of amplicons, separation of products in 1% agarose gels, and analysis of fragment length polymorphims. The multiplex PCR detected up to 227 BoHV-5 genome copies and 9.2 × 105 BoHV-5 genome copies when DNA was extracted from purified virus or infected tissue homogenates, respectively. The applicability of multiplex PCR-REA was demonstrated on 3 BoHV-5 reference strains. In addition, subtyping of two new isolates and seventeen previously reported ones (17 BHV-5a and 2 BHV-5b) by this method gave coincident results with those obtained with the classic BstEII REA assay.
Conclusions: Multiplex PCR-REA provides a new tool for the fast and simple diagnosis and subtyping of BoHV-5. |
| format | info:ar-repo/semantics/artículo |
| id | INTA4276 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | BMC |
| publisherStr | BMC |
| record_format | dspace |
| spelling | INTA42762019-01-16T15:25:40Z Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates Maidana, Silvina Soledad Morano, Cintia Débora Cianfrini, Daniela Campos, Fabrício Souza Roehe, Paulo Michel Siedler, Bianca De Stefano, Gabriel Alejandro Mauroy, Axel Thiry, Etienne Romera, Sonia Alejandra Enfermedades de los Animales Ganado Bovino Virus de los Animales Herpesviridae PCR Polimorfismo Herpes Virus Bovino Animal Diseases Cattle Animal Viruses Polymorphism Bovine Herpesvirus Herpesvirus Reacción en Cadena de la Polimerasa Background: Several types and subtypes of bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, making type/subtype differentiation essential to understand the pathogenesis and epidemiology of BoHV infections. BoHV-5 subtyping is currently carried out by BstEII restriction enzyme analysis (REA) of the complete virus genome. This method allowed the description of three subtypes, one of which is the most widespread while the remaining two have so far only been found in South America. The present work describes a multiplex PCR followed by REA for BoHV-5 subtyping. Results: The method consists in the simultaneous amplification of glycoprotein B and UL54 gene fragments of 534 and 669 base pairs (bp), respectively, BstEII digestion of amplicons, separation of products in 1% agarose gels, and analysis of fragment length polymorphims. The multiplex PCR detected up to 227 BoHV-5 genome copies and 9.2 × 105 BoHV-5 genome copies when DNA was extracted from purified virus or infected tissue homogenates, respectively. The applicability of multiplex PCR-REA was demonstrated on 3 BoHV-5 reference strains. In addition, subtyping of two new isolates and seventeen previously reported ones (17 BHV-5a and 2 BHV-5b) by this method gave coincident results with those obtained with the classic BstEII REA assay. Conclusions: Multiplex PCR-REA provides a new tool for the fast and simple diagnosis and subtyping of BoHV-5. Instituto de Virología Fil: Maidana, Silvina Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Morano, Cintia Débora Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Fil: Cianfrini, Daniela. Tecnovax SA; Argentina Fil: Campos, Fabrício Souza. Universidade Federal do Rio Grande do Sul. Institute of Basic Health Sciences. Department of Microbiology, Immunology and Parasitology. Virology Laboratory; Brasil Fil: Roehe, Paulo Michel. Universidade Federal do Rio Grande do Sul. Institute of Basic Health Sciences. Department of Microbiology, Immunology and Parasitology. Virology Laboratory; Brasil Fil: Siedler, Bianca. Universidade Federal de Pelotas. Laboratório de Bioprocessos; Brasil Fil: De Stefano, Gabriel Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Mauroy, Axel. University of Liège. Fundamental and Applied Research on Animal Health Center and Faculty of Veterinary Medicine. Veterinary Virology and Animal Viral Diseases; Bélgica Fil: Thiry, Etienne. University of Liège. Fundamental and Applied Research on Animal Health Center and Faculty of Veterinary Medicine. Veterinary Virology and Animal Viral Diseases; Bélgica Fil: Romera, Sonia Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad del Salvador. Cátedra de Inmunología; Argentina 2019-01-16T15:24:20Z 2019-01-16T15:24:20Z 2013-06 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion https://bmcvetres.biomedcentral.com/articles/10.1186/1746-6148-9-111 http://hdl.handle.net/20.500.12123/4276 1746-6148 https://doi.org/10.1186/1746-6148-9-111 eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-sa/4.0/ Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) application/pdf BMC BMC Veterinary Research 39 : 111 (2013) |
| spellingShingle | Enfermedades de los Animales Ganado Bovino Virus de los Animales Herpesviridae PCR Polimorfismo Herpes Virus Bovino Animal Diseases Cattle Animal Viruses Polymorphism Bovine Herpesvirus Herpesvirus Reacción en Cadena de la Polimerasa Maidana, Silvina Soledad Morano, Cintia Débora Cianfrini, Daniela Campos, Fabrício Souza Roehe, Paulo Michel Siedler, Bianca De Stefano, Gabriel Alejandro Mauroy, Axel Thiry, Etienne Romera, Sonia Alejandra Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title | Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title_full | Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title_fullStr | Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title_full_unstemmed | Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title_short | Multiplex PCR followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| title_sort | multiplex pcr followed by restriction length polymorphism analysis for the subtyping of bovine herpesvirus 5 isolates |
| topic | Enfermedades de los Animales Ganado Bovino Virus de los Animales Herpesviridae PCR Polimorfismo Herpes Virus Bovino Animal Diseases Cattle Animal Viruses Polymorphism Bovine Herpesvirus Herpesvirus Reacción en Cadena de la Polimerasa |
| url | https://bmcvetres.biomedcentral.com/articles/10.1186/1746-6148-9-111 http://hdl.handle.net/20.500.12123/4276 https://doi.org/10.1186/1746-6148-9-111 |
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