A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Anais da Academia Brasileira de Ciências (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652014000401749 |
Resumo: | Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis. |
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A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolatereverse geneticsdengue virusmolecular cloninginfectious cloneDengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.Academia Brasileira de Ciências2014-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652014000401749Anais da Academia Brasileira de Ciências v.86 n.4 2014reponame:Anais da Academia Brasileira de Ciências (Online)instname:Academia Brasileira de Ciências (ABC)instacron:ABC10.1590/0001-3765201420130332info:eu-repo/semantics/openAccessSANTOS,JEFFERSON J.S.CORDEIRO,MARLI T.BERTANI,GIOVANI R.MARQUES,ERNESTO T.A.GIL,LAURA H.V.G.eng2015-10-27T00:00:00Zoai:scielo:S0001-37652014000401749Revistahttp://www.scielo.br/aabchttps://old.scielo.br/oai/scielo-oai.php||aabc@abc.org.br1678-26900001-3765opendoar:2015-10-27T00:00Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC)false |
dc.title.none.fl_str_mv |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
title |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
spellingShingle |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate SANTOS,JEFFERSON J.S. reverse genetics dengue virus molecular cloning infectious clone |
title_short |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
title_full |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
title_fullStr |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
title_full_unstemmed |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
title_sort |
A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate |
author |
SANTOS,JEFFERSON J.S. |
author_facet |
SANTOS,JEFFERSON J.S. CORDEIRO,MARLI T. BERTANI,GIOVANI R. MARQUES,ERNESTO T.A. GIL,LAURA H.V.G. |
author_role |
author |
author2 |
CORDEIRO,MARLI T. BERTANI,GIOVANI R. MARQUES,ERNESTO T.A. GIL,LAURA H.V.G. |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
SANTOS,JEFFERSON J.S. CORDEIRO,MARLI T. BERTANI,GIOVANI R. MARQUES,ERNESTO T.A. GIL,LAURA H.V.G. |
dc.subject.por.fl_str_mv |
reverse genetics dengue virus molecular cloning infectious clone |
topic |
reverse genetics dengue virus molecular cloning infectious clone |
description |
Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-12-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652014000401749 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652014000401749 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/0001-3765201420130332 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Academia Brasileira de Ciências |
publisher.none.fl_str_mv |
Academia Brasileira de Ciências |
dc.source.none.fl_str_mv |
Anais da Academia Brasileira de Ciências v.86 n.4 2014 reponame:Anais da Academia Brasileira de Ciências (Online) instname:Academia Brasileira de Ciências (ABC) instacron:ABC |
instname_str |
Academia Brasileira de Ciências (ABC) |
instacron_str |
ABC |
institution |
ABC |
reponame_str |
Anais da Academia Brasileira de Ciências (Online) |
collection |
Anais da Academia Brasileira de Ciências (Online) |
repository.name.fl_str_mv |
Anais da Academia Brasileira de Ciências (Online) - Academia Brasileira de Ciências (ABC) |
repository.mail.fl_str_mv |
||aabc@abc.org.br |
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1754302860348096512 |