Differential Detection of Marine Mammal Morbilliviruses by Real-Time Reverse Transcription Polymerase Chain Reaction
Morbillivirus epizootics have caused high mortality in cetaceans and pinnipeds. The first recognized marine morbillivirus outbreaks occurred in 1987 in Lake Baikal, Russia and along the Atlantic coast of the United States in which thousands of Siberian seals (Phoca sibirica) and bottlenose dolphins (Tursiops truncatus) died, respectively. The four known marine morbilliviruses are: Porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), phocine distemper virus (PDV), and canine distemper virus (CDV). The carboxyl-terminal region of the nucleocapsid (N) protein gene is highly divergent among all known morbilliviruses, and may allow for the differentiation and specific detection of their genomic sequences. However, the use of molecular probes in real-time RT-PCR, that unambiguously differentiates between genomic sequences of the four morbilliviruses in infected marine mammal tissues, based on the carboxyl-terminal region of the N gene, has not been previously described.
Viral RNA from PMV, DMV, PDV, and CDV was reverse transcribed with random primers and the complete ORF of the N genes was amplified by PCR, cloned, and sequenced to verify gene integrity. Multiple alignments of the four N genes revealed non-conserved sequences at the carboxyl-terminal end of the genes. These sequences were used to design oligonucleotide primers to specifically amplify 200 base pair DNA fragments from each viral genome in conventional RT-PCR.
A real-time RT-PCR was developed using these primer sets and molecular dual-labeled locked nucleic acid (LNA) probes that specifically hybridized to one of the four morbilliviruses. Complementary DNA of DMV, PMV, PDV and CDV were rapidly and accurately identified at Ct values of 17.99, 26.45, 17.38 and 16.97, respectively. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as a control of RNA quality and the designed fluorogenic probe was the consensus of GAPDH sequences generated from 11 marine mammal species. The newly developed real-time RT-PCR assays allow for a faster, specific and sensitive detection of all known morbilliviruses that affect marine mammals compared to the conventional RT-PCR.