Microbiological image
Recombinant Bovine herpervirus 5 expressing enhanced green fluorescent protein
Recombinante de herpesvirus bovino 5 expresando proteina verde fluorescente
Carolina A. Silvestro
, Ana C. Bratanich
Corresponding author
Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Virología, Argentina
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The <span class="elsevierStyleItalic">clusters</span> CI, CII, CIII, CIV, CV, CVI, CVII y CVIII are indicate in the dendogram. The banding patterns obtained by PCR-based methodology are in the right of the dendogram. The attached table contains demographic and drug resistant date of the cases.</p>" ] ] ] "autores" => array:1 [ 0 => array:2 [ "autoresLista" => "Carlos A. Tello, Nelly Borja, Ronnie G. Gavilan, Ruth Garcia-de-la-Guarda" "autores" => array:4 [ 0 => array:2 [ "nombre" => "Carlos A." "apellidos" => "Tello" ] 1 => array:2 [ "nombre" => "Nelly" "apellidos" => "Borja" ] 2 => array:2 [ "nombre" => "Ronnie G." "apellidos" => "Gavilan" ] 3 => array:2 [ "nombre" => "Ruth" "apellidos" => "Garcia-de-la-Guarda" ] ] ] ] ] "idiomaDefecto" => "en" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S032575411730041X?idApp=UINPBA00004N" "url" => "/03257541/0000004900000003/v1_201708180123/S032575411730041X/v1_201708180123/en/main.assets" ] "en" => array:17 [ "idiomaDefecto" => true "cabecera" => "<span class="elsevierStyleTextfn">Microbiological image</span>" "titulo" => "Recombinant <span class="elsevierStyleItalic">Bovine herpervirus 5</span> expressing enhanced green fluorescent protein" "tieneTextoCompleto" => true "paginas" => array:1 [ 0 => array:2 [ "paginaInicial" => "301" "paginaFinal" => "302" ] ] "autores" => array:1 [ 0 => array:4 [ "autoresLista" => "Carolina A. Silvestro, Ana C. Bratanich" "autores" => array:2 [ 0 => array:4 [ "nombre" => "Carolina A." "apellidos" => "Silvestro" "email" => array:1 [ 0 => "carolina@silvestro.com.ar" ] "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor0005" ] ] ] 1 => array:2 [ "nombre" => "Ana C." "apellidos" => "Bratanich" ] ] "afiliaciones" => array:1 [ 0 => array:2 [ "entidad" => "Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Virología, Argentina" "identificador" => "aff0005" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor0005" "etiqueta" => "⁎" "correspondencia" => "Corresponding author." ] ] ] ] "titulosAlternativos" => array:1 [ "es" => array:1 [ "titulo" => "Recombinante de herpesvirus bovino 5 expresando proteina verde fluorescente" ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1390 "Ancho" => 1858 "Tamanyo" => 264345 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">CRFK cells were infected with the BoHV-5 recombinant (ΔLR +GFP) at a MOI of 0.1. Twenty-four hours later they were fixed and nuclear DNA was stained with 4′,6,-diaminodino-2-phenylindole (DAPI) (Invitrogen™). DAPI and GFP expression were monitored under a fluorescent microscope; the obtained images were merged into the same picture using the ImageJ program. (A) Observed expression of GFP in all the cytoplasms of infected cells with the presence of multinucleated cells (arrow), and cytoplasmic inclusion bodies (triangle). (B) Advanced infections showed evidence of apoptosis with cell shrinkage, condensed and pyknotic nuclei and apoptotic bodies.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><p id="par0005" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Bovine alpha herpesvirus 5</span> (BoHV-5) induces neurological disease in cattle, including tremors, nystagmus, teeth grinding, circling, ataxia, recumbence, paddling, and death<a class="elsevierStyleCrossRefs" href="#bib0055"><span class="elsevierStyleSup">2,6,7,9</span></a>. Nonsuppurative meningoencephalitis is a hallmark of this infection. Like the majority of the alpha herpes viruses, it generates latency in trigeminal ganglia<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">5</span></a>. The latency-related (LR) gene is the main responsible for the maintenance of latency<a class="elsevierStyleCrossRefs" href="#bib0050"><span class="elsevierStyleSup">1,3,4,8</span></a>. To evaluate the LR function within the viral infection, a recombinant virus was constructed deleting the LR promoter to avoid its expression without affecting overlapping genes in the antisense strand (the immediate early <span class="elsevierStyleItalic">bicp0</span> gene).</p><p id="par0010" class="elsevierStylePara elsevierViewall">The enhanced green fluorescent protein (EGFP) was used as a reporter to facilitate recombinant virus detection in the cell culture plates. For that purpose, EGFP was PCR-amplified from pEGFP-C1 (Clontech™) and subsequently cloned into a plasmid surrounded by selected 5′ and 3′ recombination arms of BoHV-5. The recombinant plasmid was linearized with <span class="elsevierStyleItalic">ScaI</span> and cotransfected with full-length BoHV-5 DNA previously extracted with DNazol<span class="elsevierStyleSup">®</span> (Invitrogen™) into CRFK (Crandell Feline Kidney) cells plated in a 24-well plate dish at 90% of cell density. Twenty-four hours later cells were reseeded into a 60<span class="elsevierStyleHsp" style=""></span>mm-plate and covered with 1% methylcellulose medium overlaid after their attachment. EGFP positive plaques were identified under fluorescent microscope, isolated and reseeded in a fresh monolayer. Viral clones were purified six times before being aliquoted and frozen at −80<span class="elsevierStyleHsp" style=""></span>°C. The correct site of recombination and removal of the promoter target was confirmed by sequencing of the recombination ends toward the reporter gene. The recombinant virus was denominated BoHV-5 ΔLR +GFP. To our knowledge, this is the first bovine herpesvirus in which the LR gene has been replaced by EGFP, making this recombinant a powerful tool for <span class="elsevierStyleItalic">in vitro</span> and <span class="elsevierStyleItalic">in vivo</span> studies.</p><p id="par0015" class="elsevierStylePara elsevierViewall">In this section we showed the recombinant BoHV-5 ΔLR +GFP-infecting CRFK cells. <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a> shows typical viral plaque morphology. In <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a> we used DAPI to contrast the nuclei and to facilitate visualization of cytopathic effects.</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><elsevierMultimedia ident="fig0010"></elsevierMultimedia><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0005">Ethical responsibilities</span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Protection of human and animal subjects</span><p id="par0020" class="elsevierStylePara elsevierViewall">The authors state that for this investigation no experiments have been performed on humans or animals.</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Confidentiality of data</span><p id="par0025" class="elsevierStylePara elsevierViewall">The authors state that in this article there are no patient data.</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Right to privacy and informed consent</span><p id="par0030" class="elsevierStylePara elsevierViewall">The authors state that in this article there are no patient data.</p></span></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Funding</span><p id="par0035" class="elsevierStylePara elsevierViewall">This research was supported by the <span class="elsevierStyleGrantSponsor" id="gs1">Agencia Nacional de Promocion Cientifica y Tecnologica</span> (<span class="elsevierStyleGrantNumber" refid="gs1">PICT 2010-0925</span>). CS is recipient of a Fellowship for the conclusion of the PhD degree from the Conicet (Consejo Nacional de Investigaciones Cientificas y Tecnicas).</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Conflict of interest</span><p id="par0040" class="elsevierStylePara elsevierViewall">The authors declare that they have no conflicts of interest.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:5 [ 0 => array:3 [ "identificador" => "sec0005" "titulo" => "Ethical responsibilities" "secciones" => array:3 [ 0 => array:2 [ "identificador" => "sec0010" "titulo" => "Protection of human and animal subjects" ] 1 => array:2 [ "identificador" => "sec0015" "titulo" => "Confidentiality of data" ] 2 => array:2 [ "identificador" => "sec0020" "titulo" => "Right to privacy and informed consent" ] ] ] 1 => array:2 [ "identificador" => "sec0025" "titulo" => "Funding" ] 2 => array:2 [ "identificador" => "sec0030" "titulo" => "Conflict of interest" ] 3 => array:2 [ "identificador" => "xack295021" "titulo" => "Acknowledgements" ] 4 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-12-30" "fechaAceptado" => "2017-03-30" "multimedia" => array:2 [ 0 => array:7 [ "identificador" => "fig0005" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 1417 "Ancho" => 1886 "Tamanyo" => 417075 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">CRFK cells were infected with the BoHV-5 ΔLR +GFP at a MOI of 0.1 and later overlaid with 1% methylcellulose medium. Forty-eight hours later, when the cytopathic effect was evident, the plates were visualized under a fluorescent microscope to detect GFP expression.</p>" ] ] 1 => array:7 [ "identificador" => "fig0010" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 1390 "Ancho" => 1858 "Tamanyo" => 264345 ] ] "descripcion" => array:1 [ "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">CRFK cells were infected with the BoHV-5 recombinant (ΔLR +GFP) at a MOI of 0.1. Twenty-four hours later they were fixed and nuclear DNA was stained with 4′,6,-diaminodino-2-phenylindole (DAPI) (Invitrogen™). DAPI and GFP expression were monitored under a fluorescent microscope; the obtained images were merged into the same picture using the ImageJ program. (A) Observed expression of GFP in all the cytoplasms of infected cells with the presence of multinucleated cells (arrow), and cytoplasmic inclusion bodies (triangle). (B) Advanced infections showed evidence of apoptosis with cell shrinkage, condensed and pyknotic nuclei and apoptotic bodies.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bibs0005" "bibliografiaReferencia" => array:9 [ 0 => array:3 [ "identificador" => "bib0050" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Meningoencephalitis caused by IBR virus in calves in Argentina" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:6 [ 0 => "B.J. Carrillo" 1 => "A. Ambrogi" 2 => "A.A. Schudel" 3 => "M. Vazquez" 4 => "E. Dahme" 5 => "A. 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Thiry" 6 => "E. Thiry" 7 => "S.A. Romera" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1186/1751-0147-53-37" "Revista" => array:5 [ "tituloSerie" => "Acta Vet Scand" "fecha" => "2011" "volumen" => "53" "paginaInicial" => "37" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/21651813" "web" => "Medline" ] ] ] ] ] ] ] ] 6 => array:3 [ "identificador" => "bib0080" "etiqueta" => "7" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Meningoencefalite em bovinos causada por herpesvirus bovino-5 no Mato Grosso do Sul e São Paulo" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:5 [ 0 => "S.C. Salvador" 1 => "R.A.A. Lemos" 2 => "F. Riet-Correa" 3 => "P.M. Roeche" 4 => "A.L.R. Osório" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:5 [ "tituloSerie" => "Pesq Vet Brasil" "fecha" => "1998" "volumen" => "18" "paginaInicial" => "76" "paginaFinal" => "83" ] ] ] ] ] ] 7 => array:3 [ "identificador" => "bib0085" "etiqueta" => "8" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "The latency-related gene of <span class="elsevierStyleItalic">Bovine herpesvirus</span> types 1 and 5 and its modulation of cellular processes" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:2 [ 0 => "C. Silvestro" 1 => "A. Bratanich" ] ] ] ] ] "host" => array:1 [ 0 => array:2 [ "doi" => "10.1007/s00705-016-3067-5" "Revista" => array:6 [ "tituloSerie" => "Arch Virol" "fecha" => "2016" "volumen" => "161" "paginaInicial" => "3299" "paginaFinal" => "3308" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/27659677" "web" => "Medline" ] ] ] ] ] ] ] ] 8 => array:3 [ "identificador" => "bib0090" "etiqueta" => "9" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Bovine encephalitis herpesvirus" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:1 [ 0 => "M.J. Studdert" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:5 [ "tituloSerie" => "Vet Rec" "fecha" => "1989" "volumen" => "125" "paginaInicial" => "584" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/2603348" "web" => "Medline" ] ] ] ] ] ] ] ] ] ] ] ] "agradecimientos" => array:1 [ 0 => array:4 [ "identificador" => "xack295021" "titulo" => "Acknowledgements" "texto" => "<p id="par0045" class="elsevierStylePara elsevierViewall">We thank Centro de Virología Animal, Instituto de Ciencia y Tecnología Dr. Cesar Milstein, CONICET, Ciudad de Buenos Aires, Argentina for the use of the fluorescence microscope and Carlos Palacios for his assistance.</p>" "vista" => "all" ] ] ] "idiomaDefecto" => "en" "url" => "/03257541/0000004900000003/v1_201708180123/S032575411730055X/v1_201708180123/en/main.assets" "Apartado" => array:4 [ "identificador" => "41742" "tipo" => "SECCION" "en" => array:2 [ "titulo" => "Imagen microbiológica" "idiomaDefecto" => true ] "idiomaDefecto" => "en" ] "PDF" => "https://static.elsevier.es/multimedia/03257541/0000004900000003/v1_201708180123/S032575411730055X/v1_201708180123/en/main.pdf?idApp=UINPBA00004N&text.app=https://www.elsevier.es/" "EPUB" => "https://multimedia.elsevier.es/PublicationsMultimediaV1/item/epub/S032575411730055X?idApp=UINPBA00004N" ]
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| 2021 June | 15 | 4 | 19 |
| 2021 May | 24 | 5 | 29 |
| 2021 April | 92 | 29 | 121 |
| 2021 March | 33 | 7 | 40 |
| 2021 February | 18 | 11 | 29 |
| 2021 January | 23 | 18 | 41 |
| 2020 December | 31 | 16 | 47 |
| 2020 November | 29 | 18 | 47 |
| 2020 October | 20 | 16 | 36 |
| 2020 September | 16 | 21 | 37 |
| 2020 August | 27 | 37 | 64 |
| 2020 July | 11 | 17 | 28 |
| 2020 June | 13 | 12 | 25 |
| 2020 May | 14 | 20 | 34 |
| 2020 April | 15 | 9 | 24 |
| 2020 March | 15 | 12 | 27 |
| 2020 February | 18 | 9 | 27 |
| 2020 January | 13 | 9 | 22 |
| 2019 December | 14 | 9 | 23 |
| 2019 November | 11 | 9 | 20 |
| 2019 October | 25 | 9 | 34 |
| 2019 September | 24 | 11 | 35 |
| 2019 August | 5 | 6 | 11 |
| 2019 July | 10 | 12 | 22 |
| 2019 June | 38 | 20 | 58 |
| 2019 May | 99 | 41 | 140 |
| 2019 April | 50 | 12 | 62 |
| 2019 March | 11 | 4 | 15 |
| 2019 February | 13 | 4 | 17 |
| 2019 January | 5 | 3 | 8 |
| 2018 December | 5 | 8 | 13 |
| 2018 November | 14 | 1 | 15 |
| 2018 October | 15 | 22 | 37 |
| 2018 September | 16 | 5 | 21 |
| 2018 August | 11 | 1 | 12 |
| 2018 July | 6 | 6 | 12 |
| 2018 June | 12 | 2 | 14 |
| 2018 May | 16 | 6 | 22 |
| 2018 April | 8 | 3 | 11 |
| 2018 March | 9 | 1 | 10 |
| 2018 February | 9 | 3 | 12 |
| 2018 January | 10 | 3 | 13 |
| 2017 December | 16 | 2 | 18 |
| 2017 November | 23 | 3 | 26 |
| 2017 October | 32 | 2 | 34 |
| 2017 September | 19 | 1 | 20 |
| 2017 August | 19 | 6 | 25 |
| 2017 July | 0 | 1 | 1 |
Show all
