title: Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.
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.
authors.affiliation: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Sjoerd H E
authors.initials: SH
authors.lastname: van den Worm
authors.forename: Klara Kristin
authors.initials: KK
authors.lastname: Eriksson
authors.forename: Jessika C
authors.initials: JC
authors.lastname: Zevenhoven
authors.forename: Friedemann
authors.initials: F
authors.lastname: Weber
authors.forename: Roland
authors.initials: R
authors.lastname: Züst
authors.forename: Thomas
authors.initials: T
authors.lastname: Kuri
authors.forename: Ronald
authors.initials: R
authors.lastname: Dijkman
authors.forename: Guohui
authors.initials: G
authors.lastname: Chang
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Siddell
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Snijder
authors.forename: Volker
authors.initials: V
authors.lastname: Thiel
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Davidson
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary; D003713:Dendritic Cells / Q000821:virology; D015967:Gene Expression Regulation, Viral; D023061:Gene Order; D016679:Genome, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016865:Reassortant Viruses / Q000235:genetics*; D011995:Recombination, Genetic; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA; D014616:Vaccinia virus / Q000235:genetics*; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary