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.

LY6E impairs coronavirus fusion and confers immune control of viral disease

“…Recombinant HCoV-229E 31 , HCoV-229E-Rluc (expressing Renilla luciferase [Rluc] by replacing the majority of HCoV-229E ORF4) 32 , HCoV-OC43 33 , MHV strain A59 34 , MHV-Gluc, strain A59…”

Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure

“…One year later a third reverse genetic system for coronaviruses was described that is based on the use of vaccinia virus as eukaryotic cloning vector. This system has been first described for the generation of recombinant HCoV-229E and was subsequently applied to the generation of recombinant Mouse hepatitis virus (MHV; Coley et al, 2005), IBV (Casais et al, 2001), feline infectious peritonitis virus (FIPV; Tekes et al, 2008), and SARS coronavirus (van den Worm et al, 2012). As a large DNA virus, vaccinia virus has the following advantages: 1) Vaccinia virus itself can accommodate large fragments of foreign genes (at least the size of coronavirus genomes), and the virus' own infectivity and replication ability are not affected.…”

“…The fragment corresponding to the IBV-YN 3′end contained a polyA sequence of 25 nts followed by a NotI restriction endonuclease recognition site. This strategy has been successful for other full-length coronavirus cDNA clones (Casais et al, 2001;van den Worm et al, 2012;Tekes, 2016). It allows to generate an in vitro transcribed RNA containing one additional G nucleotide at the 5′-end (required as transcription start site for the T7 RNA polymerase) and a polyA tail at the 3′-end (Fig.…”

Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase

“…YFV, GFP, and firefly and Renilla luciferases) have been reported for distinct viruses and used for various purposes, e.g. on the evaluation of virus-host interactions, viral tropism, and antiviral and neutralization assays (Roth et al 2009, Schoggins et al 2012, van den Worm et al 2012, Dag et al 2013, De Baets et al 2015, Zhang et al 2015. These studies include the development of recombinant flavivirus expressing reporter genes, such as Dengue virus expressing GFP and firefly and Renilla luciferases (Zou et al 2011, Schoggins et al 2012.…”

“…In addition, infectious clones or replicon systems expressing reporter genes (e.g. firefly and Renilla luciferases, green and yellow fluorescent proteins) have been developed and used mainly on molecular virology studies, to construct viral vectors and on sophisticated antiviral assays (De Baets et al 2015, Patkar et al 2009, Roth et al 2009, Schoggins et al 2012, van den Worm et al 2012, Dag et al 2013, Kato et al 2014, Ding et al 2015, Zhang et al 2015. Among the reporter genes, Gaussia luciferase (GLuc) started to be used in in vivo and in vitro assays due to some advantages such as high stability and sensitivity, and the fact that it is secreted, facilitating its detection in several experiments (Tannous et al 2005, Venisnik et al 2007.…”