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

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Ediacaran biota from Sonora, Mexico.

The Ediacaran biota is the earliest diverse community of macroscopic animals and protoctists. Body and trace fossils in the Clemente Formation of northwestern Sonora extend downward the geologic range of Ediacaran forms. Taxa present in the Clemente Formation include cf. Cyclomedusa plana, Sekwia sp., an erniettid (bearing an air mattress-like "pneu" body construction), and the trace fossils Lockeia ichnosp. and Palaeophycus tubularis. The trace fossils confirm the presence of sediment-dwelling animals in this shallow marine community. The body fossils are headless, tailless, and appendageless. Some may be body fossils of animals but others may be fossils of large protoctists. These body and trace fossils, recovered from thinly bedded sandstones and siltstones, occur 75 meters lower in the Sonoran stratigraphic section than a distinctive Clemente Formation oolite. The stratigraphic position of the fossils below this oolite permits long-distance correlation between fossiliferous Proterozoic strata of Mexico and the United States. Correlations utilizing both the Clemente Formation oolite and a trace fossil (Vermiforma antiqua) confirm the antiquity (600 million years or more) of this body fossil-rich community of macroscopic eukaryotes. The recently discovered body fossils are the oldest known remains of the Ediacaran biota.