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
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/1361
Acceder
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oai_identifier_str oai:localhost:20.500.12123/1361
network_acronym_str INTADig
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network_name_str INTA Digital (INTA)
spelling 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/restrictedAccess2025-10-23T11:16:23Zoai: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:l2025-10-23 11:16:24.046INTA 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
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str 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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