Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biolog ical interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of th ose cDNAs in bacteria. Herein, we show that the application of two strategi es, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is us eful for the engineering of large RNA molecules. A cDNA encoding an infecti ous coronavirus RNA genome has been cloned as a bacterial artificial chromo some. The rescued coronavirus conserved ail of the genetic markers introduc ed throughout the sequence and showed a standard mRNA pattern and the antig enic characteristics expected for the synthetic virus. The cDNA was transcr ibed within the nucleus, and the RNA translocated to the cytoplasm. Interes tingly, the recovered virus had essentially the same sequence as the origin al one, and no splicing was observed. The cDNA was derived from an attenuat ed isolate that replicates exclusively in the respiratory tract of swine. D uring the engineering of the infectious cDNA, the spike gene of the virus w as replaced by the spike gene of an enteric isolate. The synthetic virus re plicated abundantly in the enteric tract and was fully virulent, demonstrat ing that the tropism and virulence of the recovered coronavirus can be modi fied. This demonstration opens up the possibility of employing this infecti ous cDNA as a Vector for Vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.