| Over the past two decades, various epizootics of marine morbilliviruses have caused high mortality in cetaceans and pinnipeds. These viruses are: dolphin morbillivirus (DMV), porpoise morbillivirus (PMV), phocid distemper virus (PDV), and canine distemper virus (CDV). We have developed real-time RT-PCR assays to specifically and sensitively differentiate infections caused by DMV, PMV, PDV, and CDV targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total RNA, extracted from DMV, PMV, PDV, and CDV infected Vero cell cultures, exhibited high specificity and produced positive cycle threshold (CT) values after the 17th, 25th, 17th, and 16th cycles, respectively. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as a control of RNA quality using known primers and a consensus GAPDH probe that reacted with tissues of 11 different marine mammal species. Further, a generic real-time morbillivirus assay targeted a conserved region within the N gene and detected DMV, PMV, CDV, PDV, rinderpest virus (RPV) and measles virus (MV).Finally, a study comparing the expressed N protein of DMV in a baculovirus (Autographa californica) expression system and also a yeast (Kluyveromyces lactis) expression system, with one recombinant yeast strain containing a secretion domain and one without, found the proteins to be &sim57-kDa by SDS-PAGE and western blot analysis. Further, a baculovirus and a yeast recombinant, lacking the yeast secretory domain, expressed the first fully assembled DMV-N recombinant proteins. Both recombinant proteins were visualized by transmission electron microscopy having similar diameters of &sim22 nm and helical morphology. Purified baculovirus DMV-N protein was used as antigen in an indirect ELISA (iELISA) to detect antibodies against morbilliviruses in 31 of 96 sera samples collected from several wild marine mammal species, humans, and domestic dogs. It is anticipated that the purified yeast expressed DMV-N protein can also be used as antigen to detect morbillivirus antibodies in sera in an iELISA.In conclusion, these molecular and serological assays will advance the diagnostic capabilities of laboratories by improving the response time from stranding to diagnosis to treatment. Simultaneously, these assays may provide valuable information about previous or current infections of these deadly viruses in wild marine mammal populations. |
