The long term objectives are to (1) understand the molecular details of coronavirus structure and replication, (2) develop cloned genes (DNA copies of the viral RNA genes) to be used for (i) probes to examine virus replication, (ii) improved methods of virus diagnosis, and (iii) improved vaccines, and (3) understand the mechanism(s) of persistent coronavirus infection in chronic human disease, especially chronic intestinal disease.
The specific aims of this proposal are the following: (1) To complete the cDNA cloning and to establish the primary sequence for as much as possible of the remaining portion (18 Kb) of the genome (20 Kb) for each the porcine transmissible gastroenteritis coronavirus (TGEV) and the bovine enteric coronavirus (BCV). For this, synthetic oligonucleotides of now established sequences will be used to prime cDNA synthesis of the genome for further cloning. Sequencing will employ both the Maxam and Gilbert, and Sanger methods. Protein sequences will be deduced and concensus sequences, particularly at intergenic regions, will be sought. (2) To isolate the intracellular mRNA sequences for each TGEV and BVC and (a) translate them to identify their protein products, and (b) sequence their 5' terminal regions to (i) establish a gene map, and (ii) identify potential transcriptional control sequences. (3) To continue a concentrated characterization of the genetics, structure, synthesis, and glycosylation of the BCV hemagglutinin glycoprotein (gp 140). Continued work on TGEV and BVC as model enteric coronaviruses is needed because, (1) both are important pathogens in animals and some evidence suggests that they both may be zoonotic, (2) each is closely related to a different human respiratory coronavirus, (3) human enteric coronaviruses cannot yet be readily cultured in vitro, and (4) each possesses unique structural features so far not described in the highly studied murine or avian coronaviruses, namely, (a) 10 polyadenylated intracellular RNA species for TGEV, and (b) an unusual dimeric glycoprotein of 14O Kd (gp140, the hemagglutinin) for BCV.
