IDENTIFICATION AND CHARACTERIZATION OF NE W AND UNKNOWN CORONAVIRUSESBY USING RT-PCR AND DEGENERATED PRIMERS

A modified method of the reverse transcription followed by polymerase chain reaction (RT-PCR) was developed in order to examine the genome o f a recently discovered virus, the porcine epidermic diarrhoea virus ( PEDV), which resembled morphologically the coronaviruses. The publishe d sequences of the genomes of various coronaviruses were compared. On the level of the acid sequence, conserved regions, common to all coron aviruses, were found in the gene encoding the nonstructural protein 1b as well as genes coding for the major structural proteins (S, M, and N). Due to the degeneration of the genetic code, some amino acids may be encoded by different nucleotide triplets. In order to compensate fo r this degeneration, mixtures of primers were synthesized, containing a variety of nucleotide sequences which together represented all possi ble codons for the conserved amino acid sequences. This method allowed to amplify and clone approximately 4000 base pairs of the genome of P EDV, An analysis of the genomic sequences revealed that PEDV holds an interesting intermediate position between human coronavirus 223E and T ransmissible Gastroenteritis virus. We postulate that the method prese nted in this contribution may be useful to study and characterize othe r unknown viruses, especially viruses for which no cell cultures for p ropagation are available.