Construction and characterisation of a complete reverse genetics system of dengue virus type 3
Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades....
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2013-12-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
| Subjects: | |
| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000800983&lng=en&tlng=en |
| _version_ | 1827844690484920320 |
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| author | Jefferson Jose da Silva Santos Marli Tenorio Cordeiro Giovani Rota Bertani Ernesto Torres de Azevedo Marques Laura Helena Vega Gonzales Gil |
| author_facet | Jefferson Jose da Silva Santos Marli Tenorio Cordeiro Giovani Rota Bertani Ernesto Torres de Azevedo Marques Laura Helena Vega Gonzales Gil |
| author_sort | Jefferson Jose da Silva Santos |
| collection | DOAJ |
| description | Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis. |
| first_indexed | 2024-03-12T08:46:29Z |
| format | Article |
| id | doaj.art-11b198a05c6e4955a2a89aac5c21d1ae |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T08:46:29Z |
| publishDate | 2013-12-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-11b198a05c6e4955a2a89aac5c21d1ae2023-09-02T16:26:12ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602013-12-01108898399110.1590/0074-0276130298S0074-02762013000800983Construction and characterisation of a complete reverse genetics system of dengue virus type 3Jefferson Jose da Silva SantosMarli Tenorio CordeiroGiovani Rota BertaniErnesto Torres de Azevedo MarquesLaura Helena Vega Gonzales GilDengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000800983&lng=en&tlng=enreverse geneticsdengue virusmolecular cloning |
| spellingShingle | Jefferson Jose da Silva Santos Marli Tenorio Cordeiro Giovani Rota Bertani Ernesto Torres de Azevedo Marques Laura Helena Vega Gonzales Gil Construction and characterisation of a complete reverse genetics system of dengue virus type 3 Memorias do Instituto Oswaldo Cruz reverse genetics dengue virus molecular cloning |
| title | Construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| title_full | Construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| title_fullStr | Construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| title_full_unstemmed | Construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| title_short | Construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| title_sort | construction and characterisation of a complete reverse genetics system of dengue virus type 3 |
| topic | reverse genetics dengue virus molecular cloning |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762013000800983&lng=en&tlng=en |
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