Higher viral microbiome diversity in household than stray pets: insights into urban community assembly

Urbanization may alter companion animal viromes in ways relevant to zoonotic surveillance, but the viral microbiome dynamics and assembly drivers of urban household/stray pets remain elusive. Challenging the conventional view that stray pets necessarily harbor greater viral richness, we characterized vertebrate-associated viromes of cats and dogs across Shanghai to compare vOTU richness and community composition, identify putative novel viral lineages, and investigate key assembly factors. Using an integrated meta-omics approach, we profiled vertebrate-associated viromes of household and stray cats/dogs in Shanghai. A total of 245 fecal or rectal swabs were processed through VLP enrichment, co-extraction of viral DNA and RNA, amplification, and Illumina paired-end sequencing. Viral contigs were identified with four complementary tools (VirSorter2, DeepVirFinder, VirFinder, geNomad), validated by CheckV, and analyzed via taxonomy, phylogenetics, and statistics (alpha/beta diversity, RDA, network co-occurrence). Our analysis identified 123 vertebrate viral operational taxonomic units (vOTUs) from families including Coronaviridae , Poxviridae , and Parvoviridae . Contrary to conventional expectations, household dogs exhibited the highest Chao1 richness among the four host groups and had significantly higher richness than household cats, stray cats, and stray dogs (BH-FDR-adjusted Wilcoxon rank-sum tests, p < 0.05). However, household and stray dogs did not differ significantly in overall viral community composition (PERMANOVA, R 2 = 0.07, p = 0.115), indicating that the household-stray contrast in dogs was richness-specific rather than a broad compositional shift. In addition, household dogs harbored 73.7% of all host-specific vOTUs (14/19), all of which belong to Coronaviridae , and showed the highest vOTU richness of vertebrate-associated viruses from families with recognized zoonotic potential. We further identified 59 putative novel viral lineages exhibiting <80% amino acid similarity to known lineages. Viral communities demonstrated remarkable host specificity (15% vOTU overlap between cats and dogs) and clear modular separation in network analysis. Multivariate modeling revealed that viral community composition was significantly influenced by socio-environmental factors (population density, green space coverage), highlighting the roles of host density and urban greening in viral community assembly. These findings distinguish elevated viral richness from compositional differentiation in urban companion-animal viromes and highlight the need to incorporate both host identity and urban environmental context into One Health surveillance frameworks. Video Abstract