Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.
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-...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22412934/?tool=EBI |
| _version_ | 1818388384554418176 |
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| author | Sjoerd H E van den Worm 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 Davidson |
| author_facet | Sjoerd H E van den Worm 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 Davidson |
| author_sort | Sjoerd H E van den Worm |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-14T04:24:59Z |
| format | Article |
| id | doaj.art-1aa9c7d4d5c342dd91842e1140dd5d4d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T04:24:59Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1aa9c7d4d5c342dd91842e1140dd5d4d2022-12-21T23:17:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0173e3285710.1371/journal.pone.0032857Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.Sjoerd H E van den WormKlara Kristin ErikssonJessika C ZevenhovenFriedemann WeberRoland ZüstThomas KuriRonald DijkmanGuohui ChangStuart G SiddellEric J SnijderVolker ThielAndrew D DavidsonSevere 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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22412934/?tool=EBI |
| spellingShingle | Sjoerd H E van den Worm 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 Davidson Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. PLoS ONE |
| title | Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. |
| title_full | Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. |
| title_fullStr | Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. |
| title_full_unstemmed | Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. |
| title_short | Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination. |
| title_sort | reverse genetics of sars related coronavirus using vaccinia virus based recombination |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22412934/?tool=EBI |
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