Discovery and detection of novel parvoviruses in Danish wild red foxes (Vulpes vulpes)

Introduction: Parvoviridae includes multi-host viruses of health relevance for canids. Wild species inhabiting human-dominated landscapes (e.g., foxes) can facilitate parvovirus transmission between wild and domestic animals. However, data on parvoviruses in wildlife remains limited. This study aimed to identify which parvoviruses circulate among Danish red foxes (Vulpes vulpes) and investigate their molecular epidemiology.

Methods: One stool and 81 spleen samples from foxes were included. Initially, metagenomic sequencing with a method designed to enrich for parvoviruses was performed on the stool sample. DNA was then extracted from spleen samples and screened using a pan-amdoparvovirus PCR and PCRs targeting viruses identified by metagenomics. Sanger sequencing was used to confirm positives and study virus diversity in Denmark. Global phylogenetic analyses were performed by maximum likelihood.

Results: Two complete parvoviral genomes were recovered from the metagenomic analysis: a novel hamaparvoviral species (48.7% non-structural (NS) protein 1 identity to its closest relative) that was, however, not detected in spleen samples, and the recently described fox parvovirus (Protoparvovirus carnivoran4) detected also in two spleens (virus prevalence: 3.7%). Additionally, we identified fragments of the recently discovered newlavirus (Protoparvovirus carnivoran5), later detected with a prevalence of 37.8% (31/82). Finally, a novel amdoparvoviral species (74.3% NS1 identity to its closest relative) was discovered via pan-genus PCR and detected with a prevalence of 6.1% (5/82). Danish amdoparvoviruses and fox parvoviruses were highly conserved (94.4-99.9% and 96.7-100% pairwise identities, respectively), while newlaviruses were highly variable (identities: 73.2-96.0%). Interestingly, newlaviruses clustered by country of origin within NS1 phylogenies, but no geographical clustering was observed with capsid sequences.

Conclusions: Our multi-method parvovirus discovery approach allowed us to discover two novel species and identify two other recently discovered parvoviruses. This suggests that additional, uncharacterized parvoviruses could be circulating. Further research is needed to thoroughly study parvovirus epidemiology in canids and examine cross-species transmission.