On May 4th, local time, Nature, the world’s leading academic journal, published an online study by “Rapid Rei of SARS-CoV-2 using a synthetic sedition sedition” in the form of “Accelerated Article Preview”, a study by a number of scientific institutions in Switzerland, Germany and Russia.
Based on the known genetic sequence of the new coronavirus, the team quickly constructed a live new coronavirus in the experiment using reverse genetics in the yeast.
The paper is written by Volker Thiel of the Institute of Virology and Immunology at the University of Bern in Switzerland and Joerg Jores of the Department of Infectious Diseases and Pathology at the University of Veterinary Medicine. The study was published online on February 21st, local time, on bioRxiv, a pre-printed website, without peer review.
It is worth noting that this is a basic study of viral reconstruction based on known viral genomes, and the results have nothing to do with one of the previous rumors that the new coronavirus is synthetic, and that the new coronavirus built in the laboratory in this study is a viral reconstruction study based on the published viral genome sequence after the outbreak.
The team noted that the chemical synthesis of new coronaviruses, especially before the new outbreak virus has been successfully isolated, can help scientists provide infectious virus strains to health departments and laboratories as soon as possible, as well as genetic modification and functional characterization of individual genes, in order to buy time to respond quickly to outbreaks.
Reverse genetics is considered an indispensable tool that has revolutionized our understanding of the pathogenesis of viruses and vaccine development, the paper said. Large RNA virus genomes, such as coronavirus genomes, are difficult to clone and operate in E. coli hosts due to their large and unstable genomes. The team reported on a yeast-based synthetic genomics platform for gene reconstruction of a variety of RNA viruses, including members of the coronavirus, yellow and paravisor virus escopherology departments.
The team first tested the accuracy of a yeast-based synthetic genomics platform in other RNA viruses, such as rat hepatitis virus MHV, and the team tested the ability of the mouse hepatitis virus A59 strain to clone genes containing green fluorescent protein (MHV GFP), which showed that 90 percent of the MHV genome was properly assembled in the cloning of the test, indicating that the virus was highly efficient in assembling in yeast.
It was also used by the platform that the researchers engineered and resurrected the new coronavirus within a week of receiving the synthetic DNA fragment.
Specifically, the team divided the virus genome into 12 fragments, at 0.5kbp-3.4Kbp. At the same time, to facilitate serological diagnosis and tracking during cell culture, the team designed the synthesis of new coronaviruses to express GFP (green fluorescent protein). As a result, the team divided fragment 11 into three subclips containing the GFP sequence, which was inserted into ORF7a (Open Reading Box, ORF), resulting in a total of 14 fragments.
The team chemically synthesized the 14 DNA fragments, which were placed on January 14 and received 12 of them on February 4. Fragments 5 and 7 have some problems with cloning in E. coli that cannot be completed. However, the team, which also obtained a new coronavirus sample from a patient in Munich (SARS-CoV-2/M?nchen 1.1/2020/929), decided to use RT-PCR amplification to obtain fragments 5 and 7.
Using TAR clones, the researchers obtained properly assembled molecular clones for all six new coronavirus constructs. These DNA sequences are then stitched together using the homogenous recombination system of yeast, using the homogenous recombination system of yeast, according to the repeated sequence at the end. After obtaining the complete virus sequence, this DNA sequence is transcribed into viral RNA using T7 RNA polymerase, which is introduced to VeroE6 (monkey kidney cells) by using the use of T7 RNA polymerase, so that the cells are infected and the superfluids (containing the released virus particles) of these cells are injected into other media, which can infect other cells.
The team went on to say, “We were able to engineer and resuscitate the chemical synthesis clones of the recently popular new coronavirus only a week after we acquired the synthetic DNA fragments of the virus.” “The recombination of viruses is highly efficient and accurate, and usually more than 90% of clones are correct.
Notably, in addition to the new coronavirus, the team also reported using this technology to synthesize the construction of MHV (rat hepatitis virus, a coronavirus) and MERS-Cov, hCov-229E and Zika virus construction is still in the experimental.