Modern Approaches to Molecular Genetics of Viruses in the Study of the Members of the Family Coronaviridae

Abstract

The existence of certain suspicions about the artificial origin of the COVID-19 pandemic and about the possible use of reverse genetics technology to create the SARS-CoV-2 virus require an understanding of its capabilities in the design of new viruses. The aim of this work is to show how the use of reverse genetics allows the design of previously non-existent coronaviruses, technologies and the main achievements in their creation. Only the information in the public domain was used for the preparation of this article. The technology is called «reverse genetics» because when obtaining RNA viruses capable of replication, the process is going not from DNA to RNA, as usual, but on the contrary, from the RNA of the virus to its complementary DNA (cDNA), and from it with the help of T7 RNA polymerase – «back» to the infectious RNA. Since the resulting plus-RNA of the coronavirus genome mimics cellular messenger RNA (mRNA), it is immediately recognized by the cell's translation machine and triggers the formation of its own infectious viral particles. Two systems of reverse genetics have been developed, involving the production of an infectious plusRNA, in vitro and in vivo. The problem of obtaining a full-length cDNA of the giant genome of coronaviruses is solved by fragmentation and subsequent stitching of fragments using standard molecular biology approaches. The article provides the examples of how this technology makes it possible to obtain synthetic coronaviruses that are indistinguishable from those isolated from nature, to change the range of their hosts, to enhance virulence and resistance to specific antibodies, and to influence the pathogenesis of the disease. The article also shows the prospects for the use of recombinant viruses in cellular screening analyses and infection models in vivo for the identification of preventive and therapeutic approaches to the virus disease treatment.