Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3)
- Autores
- Akhmedzhanov, Maksat; Scrochi, Mariela; Barrandeguy, Maria Edith; Vissani, Maria Aldana; Osterrieder, Nikolaus; Damiani, Armando Mario
- Año de publicación
- 2017
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión aceptada
- Descripción
- Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterizedby pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomicsequence of EHV-3 has been recently made available, its genomic content remains poorly characterizedand the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitategenetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenicregion between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologousrecombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporatedinto E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of theEHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from thoseof the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinantviruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli andin vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74)coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene,and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensablefor EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells;(iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic andtransmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning ofEHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis andhost immune responses.
Inst.de Virología
Fil: Scrochi, Mariela Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Virología; Argentina
Fil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina
Fil: Vissani, Aldana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina
Fil: Akhmedzhanov, Maksat. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania
Fil: Osterrieder, Nikolaus. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania
Fil: Damiani, Armando Mario. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania - Fuente
- Virus research 228 : 30-38. (January 2017)
- Materia
-
Enfermedades de los Animales
Genética
Herpes Virus Equino
Cromosomas
Animal Diseases
Genetics
Equine Herpesvirus
Chromosomes - Nivel de accesibilidad
- acceso restringido
- Condiciones de uso
- Repositorio
- Institución
- Instituto Nacional de Tecnología Agropecuaria
- OAI Identificador
- oai:localhost:20.500.12123/1361
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Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3)Akhmedzhanov, MaksatScrochi, MarielaBarrandeguy, Maria EdithVissani, Maria AldanaOsterrieder, NikolausDamiani, Armando MarioEnfermedades de los AnimalesGenéticaHerpes Virus EquinoCromosomasAnimal DiseasesGeneticsEquine HerpesvirusChromosomesEquine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterizedby pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomicsequence of EHV-3 has been recently made available, its genomic content remains poorly characterizedand the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitategenetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenicregion between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologousrecombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporatedinto E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of theEHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from thoseof the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinantviruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli andin vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74)coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene,and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensablefor EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells;(iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic andtransmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning ofEHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis andhost immune responses.Inst.de VirologíaFil: Scrochi, Mariela Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Virología; ArgentinaFil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Vissani, Aldana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Akhmedzhanov, Maksat. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; AlemaniaFil: Osterrieder, Nikolaus. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; AlemaniaFil: Damiani, Armando Mario. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania2017-09-29T15:00:36Z2017-09-29T15:00:36Z2017-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/1361http://www.sciencedirect.com/science/article/pii/S0168170216305536?via%3Dihub0168-1702 (Print)1872-7492 (Online)https://doi.org/10.1016/j.virusres.2016.11.012Virus research 228 : 30-38. (January 2017)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2025-09-29T13:44:11Zoai:localhost:20.500.12123/1361instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-29 13:44:11.762INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse |
dc.title.none.fl_str_mv |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
title |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
spellingShingle |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) Akhmedzhanov, Maksat Enfermedades de los Animales Genética Herpes Virus Equino Cromosomas Animal Diseases Genetics Equine Herpesvirus Chromosomes |
title_short |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
title_full |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
title_fullStr |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
title_full_unstemmed |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
title_sort |
Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3) |
dc.creator.none.fl_str_mv |
Akhmedzhanov, Maksat Scrochi, Mariela Barrandeguy, Maria Edith Vissani, Maria Aldana Osterrieder, Nikolaus Damiani, Armando Mario |
author |
Akhmedzhanov, Maksat |
author_facet |
Akhmedzhanov, Maksat Scrochi, Mariela Barrandeguy, Maria Edith Vissani, Maria Aldana Osterrieder, Nikolaus Damiani, Armando Mario |
author_role |
author |
author2 |
Scrochi, Mariela Barrandeguy, Maria Edith Vissani, Maria Aldana Osterrieder, Nikolaus Damiani, Armando Mario |
author2_role |
author author author author author |
dc.subject.none.fl_str_mv |
Enfermedades de los Animales Genética Herpes Virus Equino Cromosomas Animal Diseases Genetics Equine Herpesvirus Chromosomes |
topic |
Enfermedades de los Animales Genética Herpes Virus Equino Cromosomas Animal Diseases Genetics Equine Herpesvirus Chromosomes |
dc.description.none.fl_txt_mv |
Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterizedby pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomicsequence of EHV-3 has been recently made available, its genomic content remains poorly characterizedand the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitategenetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenicregion between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologousrecombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporatedinto E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of theEHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from thoseof the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinantviruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli andin vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74)coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene,and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensablefor EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells;(iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic andtransmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning ofEHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis andhost immune responses. Inst.de Virología Fil: Scrochi, Mariela Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Cátedra de Virología; Argentina Fil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Vissani, Aldana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Akhmedzhanov, Maksat. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania Fil: Osterrieder, Nikolaus. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania Fil: Damiani, Armando Mario. Freie Universität Berlin. Institut für Virologie. Zentrum für Infektionsmedizin Robert von Ostertag-Haus; Alemania |
description |
Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterizedby pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomicsequence of EHV-3 has been recently made available, its genomic content remains poorly characterizedand the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitategenetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterialartificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenicregion between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologousrecombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporatedinto E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of theEHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from thoseof the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinantviruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli andin vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74)coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene,and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensablefor EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells;(iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic andtransmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning ofEHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis andhost immune responses. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-09-29T15:00:36Z 2017-09-29T15:00:36Z 2017-01 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
acceptedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/20.500.12123/1361 http://www.sciencedirect.com/science/article/pii/S0168170216305536?via%3Dihub 0168-1702 (Print) 1872-7492 (Online) https://doi.org/10.1016/j.virusres.2016.11.012 |
url |
http://hdl.handle.net/20.500.12123/1361 http://www.sciencedirect.com/science/article/pii/S0168170216305536?via%3Dihub https://doi.org/10.1016/j.virusres.2016.11.012 |
identifier_str_mv |
0168-1702 (Print) 1872-7492 (Online) |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/restrictedAccess |
eu_rights_str_mv |
restrictedAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
Virus research 228 : 30-38. (January 2017) reponame:INTA Digital (INTA) instname:Instituto Nacional de Tecnología Agropecuaria |
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INTA Digital (INTA) |
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INTA Digital (INTA) |
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Instituto Nacional de Tecnología Agropecuaria |
repository.name.fl_str_mv |
INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria |
repository.mail.fl_str_mv |
tripaldi.nicolas@inta.gob.ar |
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