Detection of novel RNA viruses: morbilliviruses as a model system

Abstract

Recent advances in molecular detection of viral-specific nucleic acid sequences have facilitated the search for pathogenic viruses in clinical samples. Primers for the polymerase chain reaction can be targeted to unique or conserved regions of the viral genome. While the design of primers must be based on known sequences, it has been demonstrated in bacteria that amplification with primers which hybridize by highly-conserved regions of the genome can be exploited to detect new agents. By taking advantage of sequence conservation among diverse members of a viral genus, we designed PCR primers capable of hybridizing to three different members of the genus Morbillivirus for which sequence information is available. Simultaneous amplification with primers for the porphobilinogen deaminase gene, a constitutively expressed gene in all cells, confirmed the presence of amplifiable RNA. Sequence alignments for the matrix and nucleoprotein genes suggested a highly conserved region of the nucleoprotein gene as a likely target for gene amplification. Morbilivirus primer pairs were chosen from nucleoprotein gene, which successfully amplified measles virus, canine distemper virus, and phocine distemper virus. In a single reaction which combines cDNA synthesis by reverse transcription of the RNA genome with the polymerase chain reaction (RT PCR reaction), amplification with these primers detected as little as 0·1 pfu (plaque forming unit) of measles virus and 1 TCID 50 of canine distemper virus. These Morbillivirus primers can be used to search for novel members of the genus which are likely to share sequence similarity in these highly conserved regions. Simultaneous amplification with the porphobilinogen deaminase primers verifies the presence of amplifiable RNA. thus permitting confident interpretation of negative results for the Morbillivirus amplification.