Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.
Identifieur interne : 001301 ( PubMed/Checkpoint ); précédent : 001300; suivant : 001302Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.
Auteurs : Sjoerd H E. Van Den Worm [Pays-Bas] ; Klara Kristin Eriksson ; Jessika C. Zevenhoven ; Friedemann Weber ; Roland Züst ; Thomas Kuri ; Ronald Dijkman ; Guohui Chang ; Stuart G. Siddell ; Eric J. Snijder ; Volker Thiel ; Andrew D. DavidsonSource :
- PloS one [ 1932-6203 ] ; 2012.
Descripteurs français
- KwdFr :
- ADN complémentaire, Analyse de séquence d'ADN, Animaux, Cellules dendritiques (virologie), Clonage moléculaire, Données de séquences moléculaires, Génome viral, Humains, Lignée cellulaire, Méthode des plages virales, Ordre des gènes, Recombinaison génétique, Régulation de l'expression des gènes viraux, Réplication virale, Virus de la vaccine (génétique), Virus du SRAS (croissance et développement), Virus du SRAS (génétique), Virus recombinants (génétique).
- MESH :
- croissance et développement : Virus du SRAS.
- génétique : Virus de la vaccine, Virus du SRAS, Virus recombinants.
- virologie : Cellules dendritiques.
- ADN complémentaire, Analyse de séquence d'ADN, Animaux, Clonage moléculaire, Données de séquences moléculaires, Génome viral, Humains, Lignée cellulaire, Méthode des plages virales, Ordre des gènes, Recombinaison génétique, Régulation de l'expression des gènes viraux, Réplication virale.
English descriptors
- KwdEn :
- Animals, Cell Line, Chlorocebus aethiops, Cloning, Molecular, DNA, Complementary, Dendritic Cells (virology), Gene Expression Regulation, Viral, Gene Order, Genome, Viral, Humans, Molecular Sequence Data, Reassortant Viruses (genetics), Recombination, Genetic, SARS Virus (genetics), SARS Virus (growth & development), Sequence Analysis, DNA, Vaccinia virus (genetics), Viral Plaque Assay, Virus Replication.
- MESH :
- chemical : DNA, Complementary.
- genetics : Reassortant Viruses, SARS Virus, Vaccinia virus.
- growth & development : SARS Virus.
- virology : Dendritic Cells.
- Animals, Cell Line, Chlorocebus aethiops, Cloning, Molecular, Gene Expression Regulation, Viral, Gene Order, Genome, Viral, Humans, Molecular Sequence Data, Recombination, Genetic, Sequence Analysis, DNA, Viral Plaque Assay, Virus Replication.
Abstract
Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.
DOI: 10.1371/journal.pone.0032857
PubMed: 22412934
Affiliations:
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pubmed:22412934Le document en format XML
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<front><div type="abstract" xml:lang="en">Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.</div>
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<Abstract><AbstractText>Severe acute respiratory syndrome (SARS) is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV) that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime) as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV). Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs). In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E). Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.</AbstractText>
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