Genetic manipulations of Coronavirus
Advancements in Biotechnology will be impossible without Genetics which is dealt with in Journal of Biochemistry and Biotechnology. Genetic manipulation of CoV sequences is challenging because of the large size (27–32 kbp) of the RNA genomes. However, two approaches have been developed to allow researchers to introduce mutations, deletions, and reporter genes into CoV genomes. These approaches are (1) targeted RNA recombination and (2) reverse genetics using infectious cDNA constructs of CoV.
The first approach exploits high-frequency copy-choice recombination to introduce mutations of interest into the 3′ end of the CoV gRNA. It includes targeted RNA recombination; a cDNA clone encoding the region from the spike glycoprotein to the 3′ end of the RNA is generated. Next, RNA is transcribed from the plasmid DNA and the RNA is transfected into cells co-infected with the CoV of interest. RNA recombination occurs between the replicating CoV and the transfected substrate RNA, and viruses with the 3′ end sequences derived from the transfected substrate RNA will be generated. The recombinant viruses are generated by high-frequency copy-choice recombination, but the challenge is to sort or select for the recombinant virus of interest from the background of wild-type virus.
The second approach for manipulating CoV sequences, generating infectious cDNA constructs of CoV, has been developed in several laboratories. Full-length CoV sequences have been cloned and expressed using bacterial artificial chromosomes (BACs), vaccinia virus vectors, and from an assembled set of cDNA clones representing the entire CoV genome. The generation of a full-length cDNA and subsequent generation of a full-length CoV gRNA allows for reverse genetic analysis of CoV sequences. Successful reverse genetics systems are now in place to study the replication and pathogenesis of SARS-CoV.
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