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-...
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Public Library of Science (PLoS)
2012
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oai:doaj.org-article:1aa9c7d4d5c342dd91842e1140dd5d4d2021-11-18T07:25:50ZReverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.1932-620310.1371/journal.pone.0032857https://doaj.org/article/1aa9c7d4d5c342dd91842e1140dd5d4d2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22412934/?tool=EBIhttps://doaj.org/toc/1932-6203Severe 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.Sjoerd H E van den WormKlara Kristin ErikssonJessika C ZevenhovenFriedemann WeberRoland ZüstThomas KuriRonald DijkmanGuohui ChangStuart G SiddellEric J SnijderVolker ThielAndrew D DavidsonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 3, p e32857 (2012) |
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DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q 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. |
| 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. |
| format |
article |
| 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 |
| title |
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_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_sort |
reverse genetics of sars-related coronavirus using vaccinia virus-based recombination. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2012 |
| url |
https://doaj.org/article/1aa9c7d4d5c342dd91842e1140dd5d4d |
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