Coronavirus reverse genetic systems: Infectious clones and replicons

Abstract

Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last thirteen years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV).

Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination

“…Combining homologous recombination in S. cerevisiae with other modern molecular methods (i.e: Gibson Assembly ® ) may further shorten the construction time of viral infectious clone systems (Gibson et al, 2010). Interestingly two other methods of coronavirus genome assembly (Targeted RNA recombination, and use of a Vaccinia virus backbone) both involve a variation of the homologous recombination mechanism (Almazán et al, 2014). Homologous recombination in S. cerevisiae builds upon these technologies because it allows for single-step construction of the viral cDNA genome in a BAC vector.…”

“…Performing polymerase chain reaction (PCR), especially reverse transcription PCR (RT-PCR) on coronavirus genomes proves troublesome, as the error rate of DNA polymerases increases in relation to copy number and amplicon size (Eckert and Kunkel, 1991). In combination, these factors make the assembly of coronavirus genome's by traditional molecular cloning tedious and time consuming (Almazán et al, 2014;Enjuanes, 2005). Virologists have developed several alternative methods to overcome these obstacles; however, none of them allow for rapid construction and expression of a coronavirus infectious clone system.…”

“…We hypothesize that construction of a coronavirus genome by homologous recombination within S. cerevisiae, will prove an additional efficient means to molecular cloning coronaviruses while preserving the strengths of the plasmid based method: long term stability and efficient expression ( Fig. 1c) (Almazán et al, 2014).…”

The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing

“…Second, using this information and promoted by rapidly advancing methods in structural biology, X-ray or NMR structures were obtained for numerous (recombinant) full-length CoV nsps or domains thereof, in particular for SARS-CoV (Neuman et al, 2014b). Third, multiple techniques for the targeted mutagenesis of CoV genomes were developed and refined, which was a specific technical challenge due to the exceptionally large size of the CoV RNA genome (Almazan et al, 2014). By launching engineered mutant genomes in susceptible cells, the RNA and protein players in the CoV replication cycle can now be interrogated directly, to reveal their importance, function(s) and/or interactions in vivo.…”

Virulence factors in porcine coronaviruses and vaccine design

“…The first CoV infectious cDNA clone was obtained for TGEV, as a bacterial artificial chromosome (BAC) (Almazan et al, 2000). Since then, additional cDNA clones were obtained for TGEV and PEDV, based either on BACs or in vitro ligation (Almazan et al, 2014; Beall et al, 2016; Jengarn et al, 2015). This review will focus on the study of enteric CoVs interaction with the host to determine virulence genes, since the modification or deletion of these genes may lead to virus attenuation, and to the generation of vaccine candidates to prevent infection by porcine CoVs.…”