[Identification and characterization of new and unknown coronaviruses using RT-PCR and degenerate primers].
A modified method of the reverse transcription followed by polymerase chain reaction (RT-PCR) was developed in order to examine the genome of a recently discovered virus, the porcine epidemic diarrhoea virus (PEDV), which resembled morphologically the coronaviruses. The published sequences of the genomes of various coronaviruses were compared. On the level of the amino acid sequence, conserved regions, common to all coronaviruses, were found in the gene encoding the nonstructural protein 1b as well as in the 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 for this degeneration, mixtures of primers were synthesized, containing a variety of nucleotide sequences which together represented all possible codons for the conserved amino acid sequences. This method allowed to amplify and clone approximately 4000 base pairs of the genome of PEDV. An analysis of the genomic sequences revealed that PEDV holds an interesting intermediate position between human coronavirus 229E and Transmissible Gastroenteritis virus. We postulate that the method presented in this contribution may be useful to study and characterize other unknown viruses, especially viruses for which no cell cultures for propagation are available.
Tobler
K
K
Institut für Virologie, Veterinãr-medizinische Fakultät, Universität Zürich.
Ackermann
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017934:Coronavirus / Q000235:genetics / Q000302:isolation & purification*; D028941:Coronavirus 229E, Human; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D017931:DNA Primers; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012194:RNA-Directed DNA Polymerase; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013553:Swine Diseases; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry
D017931:DNA Primers; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase
Identifikation und Charakterisierung von neuen und unbekannten Coronaviren mit Hilfe von RT-PCR und degenerierten Primern.