Detection of morbillivirus infection by RT-PCR RFLP analysis in cetaceans and carnivores

• Published in: Journal of virological methods 2017-09, Vol.247, p.22-27
• Main Authors: Verna, Federica, Giorda, Federica, Miceli, Ilaria, Rizzo, Giovanna, Pautasso, Alessandra, Romano, Angelo, Iulini, Barbara, Pintore, Maria Domenica, Mignone, Walter, Grattarola, Carla, Bozzetta, Elena, Varello, Katia, Dondo, Alessandro, Casalone, Cristina, Goria, Maria
• Format: Article
• Language: English
• Subjects: Animals; Canine distemper virus; Carnivora; Carnivore; Cetacea; Cetacean; Dolphin morbillivirus; Morbillivirus - genetics; Morbillivirus - isolation & purification; Morbillivirus Infections - veterinary; Polymorphism, Restriction Fragment Length; Reverse Transcriptase Polymerase Chain Reaction - methods; RFLP; RT-PCR
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2017.05.009

•Morbillivirus isolation and identification are challenging in wild animals.•This study describes an improved RT-PCR RFLP based on degenerate primers.•The use of degenerate primers increases the sensitivity of a PCR.•RT-PCR RFLP allows to detect, differentiate and characterize CDV and DMV.•RT-PCR RFLP could be useful in routine labs, in absence of a sequencer. Morbillivirus genus comprises several members related to specific hosts, such as canine distemper virus (CDV) and cetacean morbillivirus (CeMV) in which the dolphin morbillivirus (DMV) is included. Both CDV and DMV are able to cause serious outbreak associated with high morbidity and mortality representing an important conservation threat for terrestrial and aquatic mammalian species. This paper describes a new RT-PCR RFLP technique based on a RT-PCR with degenerate primers targeting a 287 bp fragment located on the conserved N terminus of the morbillivirus NP gene, followed by MseI RFLP, in order both to confirm the detection of the virus and to distinguish DMV from CDV. Both carnivores and cetaceans tissues (brain, lung and lymph node) presenting evidence of morbillivirus infection (MI) were analyzed. RT-PCR positive samples were typed by RFLP analysis and then sequenced to confirm the RFLP results. This method was applied during the last morbillivirus cetacean die-off occurred in the Mediterranean basin in 2013, when there was the urgent need of a rapid and economic method to investigate among causes of death on stranded cetaceans. This new technique has proved to be a valuable, reliable, simple and relatively inexpensive diagnostic tool easily applicable also in limited-resource laboratories.