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Canine distemper virus (CDV) is a single-stranded negative-sense RNA virus, which belongs to the Paramyxoviridae family in the Morbillivirus genus. Based on nucleotide sequence analysis of the hemagglutinin (H) gene several distinct genotypes are known in different hosts and geographical areas worldwide. A wide range of domestic animals and wild carnivores are at risk. The Eurasian otter (Lutra lutra) is a piscivorous top predator of aquatic habitats. Although, the otter is a widely distributed and well-studied, flagship species in Europe, there is a considerable lack of knowledge in virological context. The understanding of CDV epidemiology in Eurasian otter is of great importance for veterinary health and nature conservation point of view. Therefore, our aim was to perform a retrospective study, spanning a ∼20 years time period to reveal the set of CDV genotypes in connection to Eurasian otter infections. Altogether 329 lung tissue samples of otters collected in Hungary between 2002 and 2021were examined. All samples are derived from road-killed animals, therefore we do not have the data of existing symptoms at the time of death. The samples were RT-PCR screened for Canine Distemper Virus. The positive samples were sequenced with a pan-genotype CDV-specific amplicon-based sequencing method developed by our research group for Oxford Nanopore Technologies platform.. The detailed protocol is available at our laboratory protocol.io page. Two animals out of the 329 were positive for CDV RNA. One positive sample is from 2006 and the other is from 2010. In one case, complete genome sequencing was performed with a MinION nanopore sequencing, Oxford Nanopore technology. In the other case, the virus titer was too low to subject for complete genome sequencing. We applied additional PCR, targeting the hemagglutinin (H) gene of CDV and sequenced on regular Sanger Sequencer platform. In addition to the description and characterization of CDV relatively little information is available on the ecological background of the virus, or even their effects on otter populations. We present the importance of CDV infections in managing stable Eurasian otter populations and highlight the reliability of road-killed animals for virological studies. [ABSTRACT FROM AUTHOR]

Our main goal was to develop novel sampling protocol and study design for viral discovery and monitoring in bats to support multi purposal research activities (ie. detection, sequencing, serology, isolation) with minimal conservational effect and disturbance. We tested this method within the framework of Lloviu cuevavirus (LLOV) surveillance program, wich is a barely known member of Filoviridae. Live animal sampling events were performed between 2018 and 2021 at the site of LLOV re-emergence in Hungary (Northeast Hungary, Zemplén Mountains), when samples (blood, urine, faeces, ectoparasites) were collected from Schreiber's bats (Miniopterus schreibersii). Whole blood (maximum of 50 µL) was taken from the uropatagium vein from each animal by Minivette® POCT (Sarstedt, Germany) disposable microtubes. The blood samples were collected in 1.5-ml Eppendorf tubes where serum was separated for serological assays by low-speed centrifugation (5000 rpm) for five minutes. Cell pellets were complemented with viral transport medium to 200 μl and were aliquoted. 100 μl aliquot were used for nucleic acid extraction (Direct-Zol RNA MiniPrep, Zymo Research) and RT-PCR detection of LLOV RNA on site with MygoPro PCR platform. During the live sampling events, whole blood samples were taken from captured bats after which they were left hanging separately in disposable paper bags (air permeable) for approximately 2 hours, while on-site qRT-PCR (qRT-PCR Brilliant III Probe Master Mix,Agilent) analysis for the presence of LLOV RNA in from blood samples was performed. This methodology permitted the observation and re-sampling of bats with active LLOV infection. Detailed protocol is available at our protocols.io page: https://www.protocols.io/edit/protocol-for-the-field-detection-of-the-european-f-bqwxmxfn/description We were able to determine the prevalence of active infection of LLOV among the sampled individuals with a minimally invasive blood sampling method. Our workflow was adequate to detect and sequence pathogens (with an amplicon based sequencing method: https://www.protocols.io/view/lloviu-cuevavirus-sequencing-protocol-bmz3k78n) in the viremic phase of an infection as well as maintain the possibility of isolation. In the past years more than 11000 bats were culled for filovirus research purposes. Novel protocols are urgently needed realize bat sampling in the context of nature conservation and zoonoses research. [ABSTRACT FROM AUTHOR]