Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology

Abstract

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family

“…According to our findings and previous literature data, CDV is present in 4 out of the 8 species of the Mustelidae family in our region [ 9 , 10 , 13 , 40 ]. Considering the relevance of these animals in conservation biology, vaccination in wildlife rescue centers may be an important tool in the conservation of rare and protected mustelids [ 62 ].…”

“…The complete genome sequencing was performed with MinION Nanopore sequencing technology (Oxford Nanopore Technologies, UK). We used a previously published universal amplicon-based sequencing method designed for CDV [ 12 , 13 ]. The detailed protocol and the primers are available at our laboratory protocols.io page [ 81 ].…”

“…They found that CDV seroprevalence is an indicator for the population trend of these animals, supporting our hypothesis that CDV may be an important wildlife disease [ 28 ]. In Europe, CDV has been reported among multiple species to date, including the stone marten ( Martes foina ), pine marten ( Martes martes ), Eurasian badger ( Meles meles ), Eurasian otter ( Lutra lutra ), European mink ( Mustela lutreola ), European polecat ( Mustela putorius ) and the American mink ( Mustela vison ) [ 13 , 29 – 34 ].…”

“…In Hungary, three different CDV genotypes (Europe, Arctic-like and European wildlife lineages) was described so far based on the H gene nucleotide sequences. In addition to dogs, CDV infection was detected in other carnivores, including the red fox and raccoon ( Procyon lotor ), Eurasian otter and ferret ( Mustela furo ) [ 9 – 13 ]. These genotypes have significantly different history, geographic distribution and known host range.…”

“…Detection and investigation of viral diseases are important factors for conserving protected and rare species; however, the elusive nature of several mustelids hampers our understanding of their viruses. Monitoring road-killed animals is a general practice for population genetic studies on rare species [ 53 , 54 ], but it also gives a good opportunity to get data about pathogens of these animals [ 13 , 55 ]. Herein we present the results of a post-mortem retrospective surveillance study to detect CDV RNA among road-killed mustelids and perform complete genomic sequencing, phylogenetic and recombination analyses on these virus sequences.…”