Cyclovirus-Related DNA in Bovine Feces from Northern Portugal: Molecular Detection and Phylogenetic Characterization

Cycloviruses are circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses detected in a wide range of biological and environmental samples, although the natural hosts and biological significance of many members of the genus remain poorly understood. Information regarding their occurrence in cattle, particularly in fecal material, remains limited. In this study, 100 bovine fecal samples collected from 21 farms in northern Portugal were screened for circovirus- and cyclovirus-related DNA using a nested PCR assay targeting a conserved region of the replication-associated protein (rep) gene. Amplicons were characterized by bidirectional Sanger sequencing and maximum-likelihood phylogenetic analysis. Cyclovirus-related DNA was detected in 14/100 samples, corresponding to a molecular occurrence of 14.0% (cluster-bootstrap 95% CI: 5.6–26.5%), with positive animals originating from 7/21 farms. The obtained partial rep sequences showed 94.8–100% nucleotide identity with Crocidura russula feces-associated Cyclovirus MAVG5 (PQ576920), and five sequences were identical to the corresponding homologous region. Phylogenetic analysis grouped all bovine-derived sequences with MAVG5 within a broader lineage containing Human-associated Cyclovirus 6. In an exploratory GEE analysis accounting for within-farm clustering, no statistically significant association was observed between cyclovirus detection and diarrhea (GEE: OR = 0.76, 95% CI: 0.31–1.86; p = 0.549). These findings expand current knowledge of Cyclovirus-related DNA in livestock-associated fecal material and indicate the occurrence of closely related Cyclovirus sequences in distinct mammalian-associated matrices within the Iberian Peninsula. However, detection in feces alone does not establish cattle as the biological host or demonstrate active infection. Complete-genome characterization and broader host and environmental surveillance are required to clarify the taxonomy, host range, and ecological significance of these viruses.