title: Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.
abstract: Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply.
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Centelleghe
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Beffagna
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Zanetti
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Zappulli
authors.affiliation: University of Teramo, Faculty of Veterinary Medicine, Località Piano d'Accio, Teramo, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Di Guardo
authors.affiliation: Department of Comparative Biomedicine and Food Science, University of Padua, Legnaro, Padua, Italy. Electronic address: cinzia.centelleghe@gmail.com.
authors.forename: Sandro
authors.initials: S
authors.lastname: Mazzariol
mesh_terms: D000818:Animals; D000835:Animals, Wild / Q000821:virology; D001921:Brain / Q000821:virology; D002591:Cetacea / Q000821:virology; D004289:Dolphins / Q000821:virology*; D005814:Genes, Viral; D007558:Italy; D008168:Lung / Q000821:virology; D008221:Lymphoid Tissue / Q000821:virology; D018103:Morbillivirus / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D018185:Morbillivirus Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000821:virology
chemical_list: D012367:RNA, Viral
keywords: Cetaceans; Dolphin morbillivirus; Haemagglutinin gene; Nested RT-PCR