Detection of related positive-strand RNA virus genomes by reverse transcription/polymerase chain reaction using degenerate primers for common replicase sequences.
A set of degenerate sense and antisense primers were designed on the basis of short segments with identical amino acids in the predicted ORF 1b replicase proteins of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus, strain Lelystad virus (PRRSV-LV), which are members of a new group of positive-strand RNA viruses. Reverse transcription/polymerase chain reaction amplification using this set of degenerate primers yielded products of the expected size from the genomes of all three viruses. It also yielded a product of appropriate size from the genome of another strain of PRRSV (VR2332), the ORF 1b sequence of which is unknown, but the 3' end of the genome of which differs from that of the PRRSV-LV genome by about 50%. No products were generated from the genome of simian hemorrhagic fever virus (SHFV), another member of this virus group. However, an appropriate product was generated with a second set of degenerate primers which was designed from the same ORF 1b segments of LDV, EAV and PRRSV-LV as the first set but on the basis of human codon preferences. Sequence analysis showed that the amplified SHFV ORF 1b segment exhibited about 50% nucleotide identity with the corresponding segments of ORF 1b of LDV, EAV and PRRSV. The results show that these and other degenerate primer sets might be useful for the search of related viruses in other mammalian species.
Chen
Z
Z
Department of Microbiology, University of Minnesota, Minneapolis 55455, USA.
Plagemann
P G
PG
D000595:Amino Acid Sequence; D000818:Animals; D018018:Arterivirus / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D007240:Equartevirus / Q000235:genetics*; D018397:Equidae; D006801:Humans; D007772:Lactate dehydrogenase-elevating virus / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014158:Transcription, Genetic
D017931:DNA Primers; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase