Metatranscriptomic discovery of a novel viral class from the Yangshan deep-water port virosphere

RNA viruses are highly abundant and diverse in marine ecosystems, yet the currently discovered viral species represent only a minute fraction of the total diversity, with viral communities in port ecosystems particularly understudied. To bridge this gap, in summer, approximately 100 L of surface water was collected from Yangshan Port. Viral particles were concentrated using tandem filtration and 50-kDa tangential-flow ultrafiltration, followed by metatranscriptomic sequencing of extracted RNA. This approach yielded 74 full-length RNA-dependent RNA polymerase (RdRp) sequences, which clustered into 72 distinct genus-level (or higher) taxonomic units-significantly expanding the known diversity of seaport RNA viruses. Phylogenetic and EFI-EST network analyses showed that 93% of these sequences belong to the two dominant phyla, Pisuviricota and Kitrinoviricota, forming a seasonally stable backbone consistent with a previously obtained winter dataset. Notably, five sequences formed a maximally supported (99% bootstrap) basal clade within Kitrinoviricota. They share only 8-24% amino-acid identity with representatives of the four ICTV-recognized classes and exhibit DALI Z-scores ≤ 20, precluding assignment to any currently established class. Three of these candidate novel-class viruses were already detectable in the winter metatranscriptome (bit-score ≥ 40, ≥ 98% amino-acid identity), indicating year-round persistence of this lineage in Yangshan Port. Their taxonomic novelty thus reflects a gap in the current ICTV framework rather than seasonal sporadicity. Two sequences represent true seasonal debutants absent from public databases. These sequences not only fill a notable gap in the summer virome but also lack a class-level taxonomic anchor within the ICTV hierarchy, jointly occupying a "season-new" and "taxon-new" niche that prioritizes them for downstream isolation and functional characterization.