Disentangling production and persistence of extracellular virions in grassland soils with SIP-viromics

Viruses are abundant and ecologically important in soils, yet the persistence and production dynamics of extracellular virions remain poorly understood. We applied genome-resolved stable isotope probing viromics (SIP-viromics), combining H ₂ ¹⁸ O labeling with viral metagenomics, to track virion turnover in seasonally dry grassland soils following rewetting. We identified 354 viral populations (vOTUs) using individual-sample and combined virome assemblies. Only 22% of vOTUs exhibited significant ¹⁸ O enrichment, indicating active replication and new virion production during the 1-week incubation; the majority (78%) persisted without detectable replication, consistent with a viral seed bank. Active vOTUs accounted for 4.76–5.15% of total virions per gram of soil, with viral loads ranging from 3.15 × 10 ¹⁰ to 6.59 × 10 ¹⁰ virions per gram. Probabilistic and deterministic sensitivity analyses spanning viral DNA fraction and genome length reinforced that persistent virions represented the majority of the extracellular viral pool post-wet-up, regardless of parameter assumptions. Host predictions linked both active and persistent vOTUs primarily to Actinomycetota and Pseudomonadota—bacterial groups known to rapidly resuscitate following rewetting—suggesting that some viruses exhibit rapid turnover, while others persist over longer timescales, forming a stable viral pool capable of reinitiating infections during favorable conditions. These results demonstrate that SIP-viromics can distinguish newly produced from persistent virions and reveal predicted host-associated, lineage-level patterns consistent with lytic infection and virion production. Our findings advance understanding of soil virus-host interactions and highlight the ecological role of persistent virions as a genetic reservoir contributing to microbial turnover and biogeochemical cycling following environmental disturbance.