Molecular identification and analysis of a novel picorna-like virus

Scylla paramamosain, commonly known as the mud crab, is an economically important crustacean species. However, the diversity and molecular characteristics of RNA viruses infecting S. paramamosain remain largely unexplored. This study aimed to identify RNA viruses in S. paramamosain and to characterize the genomic features, phylogenetic position, and virus-derived small interfering RNA (vsiRNA) profiles of a newly discovered virus. In this study, high-throughput sequencing and de novo transcriptome assembly were performed on S. paramamosain collected from a market in Zhejiang, China. Among the assembled contigs, three RNA viruses were identified, including two previously reported viruses (a reovirus and a toti-like virus), as well as a novel picorna-like virus designated Scylla paramamosain picorna-like virus 1 (SpPLV1). The complete genome was obtained by RT-PCR, rapid amplification of cDNA ends (RACE), and Sanger sequencing. Bioinformatic analyses were conducted for open reading frame prediction, conserved domain annotation, and phylogenetic reconstruction. Small RNA sequencing was further performed to analyze the length distribution and strand bias of vsiRNAs. The complete genome of SpPLV1 is 9,596 nucleotides in length and contains two open reading frames (ORFs), encoding conserved structural and non-structural domains characteristic of the order Picornavirales. Mapping coverage analysis showed a higher accumulation of viral reads in the 3′ region, particularly within ORF2, suggesting active transcription or replication. Phylogenetic analysis based on the RNA-dependent RNA polymerase domain revealed that SpPLV1 clusters with Wenzhou picorna-like viruses 6 and 7, forming an unclassified group closely related to the family Marnaviridae. Small RNA analysis revealed that 125,720 and 226,399 vsiRNAs were derived from the toti-like virus and the reovirus, respectively, with a predominant length of 22 nt and balanced sense and antisense strand distribution. In contrast, only 44 small RNAs were mapped to SpPLV1, without a clear length peak or strand bias. SpPLV1 represents a novel picorna-like virus identified in S. paramamosain, exhibiting typical genomic features of the order Picornavirales and forming a distinct lineage closely related to members of the family Marnaviridae. The identification of SpPLV1 provides new insights into the RNA virome of S. paramamosain and highlights the need for further studies on its potential role in host-virus interactions.