Reverse Genetics of SARS-Related Coronavirus Using Vaccinia Virus-Based Recombination
Autor: | Stuart G. Siddell, Guohui Chang, Roland Züst, Andrew D. Davidson, Friedemann Weber, Sjoerd H. E. van den Worm, Eric J. Snijder, Ronald Dijkman, Jessika C. Zevenhoven, Volker Thiel, Thomas Kuri, Klara K. Eriksson |
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Jazyk: | angličtina |
Rok vydání: | 2012 |
Předmět: |
Viral Diseases
viruses Viral Plaque Assay medicine.disease_cause Global Health Virus Replication chemistry.chemical_compound Chlorocebus aethiops Gene Order Cloning Molecular skin and connective tissue diseases Coronavirus Recombination Genetic 0303 health sciences Multidisciplinary Vaccination virus diseases 3. Good health Infectious Diseases Severe acute respiratory syndrome-related coronavirus Medicine Reassortant Viruses Research Article Gene Expression Regulation Viral Human coronavirus 229E DNA Complementary Science Molecular Sequence Data Vaccinia virus Genome Viral Biology Microbiology Virus Cell Line 03 medical and health sciences Complementary DNA Virology medicine Animals Humans Gene 030304 developmental biology 030306 microbiology fungi Immunity Dendritic Cells Sequence Analysis DNA biology.organism_classification COVID-19 Nucleotide sequencing Recombinant vaccines Homologous recombination SARS coronavirus Viral replication DNA recombination Viral genomics Reverse genetics respiratory tract diseases chemistry Clinical Immunology Vaccinia |
Zdroj: | PLoS ONE PloS one PLoS ONE, Vol 7, Iss 3, p e32857 (2012) PLOS ONE, 7(3):e32857 PLoS ONE, 7(3) |
ISSN: | 1932-6203 |
Popis: | 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. |
Databáze: | OpenAIRE |
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