Selective Amplification of the Nucleocapsid Gene of Porpoise and Dolphin Morbillivirus by Conventional Reverse Transcription Polymerase Chain Reaction
Over the past two decades, various epizootics of porpoise (PMV) and dolphin (DMV) morbilliviruses have caused high mortality in cetaceans. These viruses have also been isolated from stranded Mediterranean monk seals (Monachus monachus). There is considerable debate as to whether PMV and DMV are separate viral species or different strains of the same virus. Sequence analyses of their complete nucleocapsid (N) genes showed that they share 88.9% and 93.5% identity in their nucleotide and amino acid sequences, respectively. The carboxyl (C) terminal region of the N gene is highly divergent among all known morbilliviruses and in the case of PMV and DMV, has the potential for use in their differential diagnosis. Pair-wise alignments (GCG Gap) from nucleotide positions 1251 to 1650 within the C-terminal region, showed only an 82.8 percent identity. Viral RNA from tissue culture grown PMV and DMV was reverse transcribed using a specific N gene oligonucleotide primer, and the complete N genes were amplified by PCR, cloned, and sequenced. The sequences were used to develop primers for PCR to differentiate between the two cetacean morbilliviruses. Our results have confirmed that these primers are specific for the detection of these viruses, and a similar strategy is being employed to differentially amplify N gene sequences from canine (CDV) and phocine (PDV) distemper viruses. The use of conventional RT-PCR for the amplification of PMV and DMV genes has been described, requiring further sequencing of amplified DNA fragments to determine identity. Selective amplification of N gene fragments may allow for faster detection and identification of all known marine morbilliviruses.