We present the genomic sequencing and phylogenetic analysis of 19 complete genomes of CDV strains from red foxes during an epizootic event in Hungary, 2021. We provided novel, complete genomic sequence data and showed the reliability of NGS sequencing in genomic epidemiological studies which may support rapid response actions during future epizootic situations. A total of 21 of the 24 foxes were positive for CDV with one-step RT-PCR. As reposted by the animal rescue centre, the 3 negative animals died with similar symptoms as the other 21, so it is conceivable that it was not possible to detect viral RNA in the collected swab samples39–41. In addition to the observed symptomatic animals, there may have been more undetected cases in the region. Notably a limitation of our study is the lack of source information about the investigated animals. However the phylogenetic relatedness and the elevated case number as experienced by the rescue center supports the idea of a more widespread epizootic event. Based on the phylogenetic analysis the sequences from the foxes belonged to the Europe lineage and showed the greatest similarity with an H gene of CDV which was detected in Hungarian dog sample from 2004 in the same area10.
Across Europe, episodes of canine distemper outbreaks in non-dog host species with Europe lineage have been reported. In Germany, numerous wild red foxes exhibiting neurological signs suggestive of canine distemper and several badgers and were found dead. After H gene of CDV sequences were analyzed from five foxes and one badger were confirmed with Europe lineage from 200814. In Italy, similar to the current epidemic in Hungary, at least 30 foxes with altered behavior were seen near human habitations and facilities in 2009. Most foxes were juveniles during the epizootic. Then the presence of the European lineage in three infected foxes was confirmed by H gene sequencing13. In Switzerland, numerous wild carnivores, including red foxes, Eurasian badgers, stone and pine martens, and one Eurasian lynx were found with CDV lesions between 2009 and 2010. The first 50 animals confirmed CDV positive. This epidemic was detected in a large spectrum of affected species, and high morbidity and mortality, especially in red foxes and badgers15. In Denmark, a major outbreak of canine distemper virus was detected in farmed minks (Neovison vison) from mink farms and a high number of species such as foxes, raccoon dogs (Nyctereutes procyonoides), and wild ferret (Mustela putorius), between 2012 and 201316. The Europe lineage of CDV in wildlife has continued to be reported from nearby countries, first in Italy from wild species, mainly foxes and badgers, between 2006 and 200917, thereafter in Germany from raccoons (Procyon lotor) from 2015 and fox from 201618, and recently in Northern Italy from foxes, badgers, and stone martens between 2018 and 201919. Based on these epizootic events, it can be assumed that this lineage will be present among European wild animals, and detected from wadings for a long time to come and may reappear frequently in Europe.
Understanding the evolution of enzootic strains and the transmission risk from wildlife to domestic animals are highly important to mitigate the effect of spillover events on household animals. During the last years, several studies recognized the importance of providing genetic data42–45. Host jump events from wildlife to domestic animals were supposedly connected to substitutions at the amino acid positions 530 and 549 in the signaling lymphocytic activation molecule SLAM binding region. It was hypothesized in multiple studies that the substitutions at the residues G/E 530 to R/D/N and Y549H may have a crucial role in the inter-species transmission from domestic dogs to non-dog hosts2. In contrast, the current study reports that all epizootic sequences from red foxes presented the 530G and 549Y, at the amino acid level. In this term, other studies support our current observations, since the CDV hemagglutinin gene sequences of red foxes in Germany, Denmark and Italy contain a 549H and a 549Y amino acid, indicating that both versions were found in red foxes2,13,14,16. Based on the data available so far, it needs to be reconsidered whether these amino acid substitutions and constellations correspond to the host or not.
The importance of sequencing data to better understand CDV evolution is increasingly recognized in other studies as well. Apart from the limitation of our study, namely the lack of different CDV lineages to extensively verify the method, we present a novel NGS-based sequencing performance to aid future studies. We designed the method to be applicable for sequencing multiple genetic lineages. Notably this is the first NGS-based method for targeted CDV genomic sequencing where virus propagation is not necessary. Next-generation sequencing methods were previously used in relation to CDV research. In a study, CDV infection was identified in a dog that was imported to Italy from Cuba. CDV was detected and isolated from the infected brain tissue. Subsequently, this isolate was subjected for Next-Generation Sequencing using the MinION Nanopore technology34. Another recent study presented complete genomic data which was acquired by Sanger sequencing method. These papers well represent the increasing need for rapid and specific genomic data generation46. The main advantage of our method is the overcome of in vitro isolation, which greatly facilitates the possibility of wide scale use. Using the amplicon-based NGS sequencing technology is not unique in epidemics, but it was fairly used in veterinary health-related events to date. We highlight the importance of similar methods to aid future investigations of epizootic events or even supporting surveillance efforts. In addition, as presented on the phylogenetic analysis, there is a significant lack of genetic sequence information about enzootic and non-enzootic CDV strains. However we designed the application to be specific for several genetic lineages, the NGS workflow of the current study needs to be tested on other lineages as well in the future.
From a nature conservation point of view, it is of paramount importance to learn more about diseases in animal species susceptible to CDV infection and prepare or ait mitigation efforts during epizootic events. Foxes’ social behavior during the reproductive season and the dispersion of juvenile animals can played a major role in epizootic CDV amplification and diffusion in a wide geographic range, as discussed before13,19. CDV is known to easily cross species barriers and is able to infect different animal species. Notably, to better understand recurring epizootics of enzootic CDV strains needs the perspective of OneHealth concept. We need to better understand environmental and animal behavioral factors, among many others.