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
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