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Researchers Rapidly Reconstruct SARS-CoV-2 Virus Using Synthetic Genomics

NEW YORK – Researchers in Switzerland and Germany have developed a yeast-based synthetic genomics platform capable of rapidly reconstructing diverse RNA viruses, including SARS-CoV-2.

The ability to reconstruct viruses from pieces of their genomes has allowed researchers to gain insights into viral pathogenesis and vaccine development, the researchers wrote in a study published on Monday in Nature. But large RNA virus genomes, such as those of coronaviruses, are cumbersome to clone and manipulate in Escherichia coli because they're too large and can be occasionally unstable. Therefore, as an alternative, they developed a yeast-based synthetic genomics platform to genetically reconstruct RNA viruses, including members of the Coronaviridae, Flaviviridae, and Paramyxoviridae families.

They generated viral subgenomic fragments using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, which they reassembled in one step in Saccharomyces cerevisiae using transformation associated recombination (TAR) cloning. This technique maintained each viral genome as a yeast artificial chromosome (YAC).

"Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-2 in only a week after receipt of the synthetic DNA fragments," the authors wrote. "The technical advance we describe here allows a rapid response to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants — in real-time — during an outbreak."

During the early phase of the SARS-CoV-2 outbreak, isolates of the virus were urgently needed to develop diagnostics, antivirals, and vaccines, and to establish appropriate in vivo disease models, but they weren't available to health authorities and the scientific community, the researchers said. Generating SARS-CoV-2 from chemically synthesized DNA could bypass the limited availability of virus isolates and would allow for genetic modifications and functional characterization.

The researchers first attempted a genome-wide reassembly of the approximately 1.1 megabase-long Mycoplasma genome using E. coli as an intermediate host, but the maintenance of 100-kbp DNA fragments was difficult in this host. They then moved on to S. cerevisiae to clone, assemble, and mutagenize the entire Mycoplasma genome because of yeast's ability to recombine overlapping DNA fragments internally, which led to the development of the TAR cloning technique.

The team first tested the mouse hepatitis virus (MHV) strain A59 containing the gene for the green fluorescent protein (MHV-GFP), which has an established vaccinia virus-based reverse genetics platform. Viral RNA was prepared from MHV-GFP-infected murine cells and used to amplify seven overlapping DNA fragments by RT-PCR. All DNA fragments were simultaneously transformed into S. cerevisiae and the resulting clones were screened for correct assembly of the YAC containing the MHV genome by multiplex PCR covering the junctions between recombined fragments. This screen revealed that more than 90 percent of the clones tested were positive, indicating a highly efficient assembly in yeast, the researchers said.

They then rescued MHV-GFP to generate capped viral genomic RNA, transfected it into BHK-MHV-N cells, and found that GFP-expressing cells were readily detectable within 48 hours, indicating successful recovery of infectious virus. Finally, the researchers assessed the replication kinetics of the recovered viruses, which were indistinguishable from the parental MHV-GFP.

To address if the synthetic genomics platform could be applied to coronaviruses, and if it could be used for rapid mutagenesis, the investigators performed a similar experiment using MERS-CoV, along with several other coronaviruses and viruses of other families, such as Zika virus and human respiratory-syncytial-virus. They were able to successfully clone these viral genomes in yeast irrespective of the virus source, the nucleic acid template, or the number of DNA fragments.

"Of note, we cloned hRSV-B without any prior information on the virus genotype directly from a clinical sample (nasopharyngeal aspirate) by designing RSV consensus primers to amplify four overlapping DNA fragments," the authors wrote. "Collectively, these results demonstrate that the synthetic genomics platform provides the technical advance to rapidly generate molecular clones of diverse RNA viruses by using virus isolates, cloned DNA, synthetic DNA, or clinical samples as starting material."

One main advantage of the TAR cloning system is that the viral genomes can be fragmented into at least 19 overlapping pieces and reassembled with remarkable efficacy, they added.