Molecular and biological characterization of a novel virus representing a novel genus of Alphaflexiviridae infecting different accessions of seashore paspalum, a turfgrass, widely grown in the United States

Background Seashore paspalum ( Paspalum vaginatum ), a widely cultivated turfgrass in the United States, is valued for its salinity tolerance and environmental adaptability. Despite its economic importance, viral infections in paspalum remain poorly characterized. This study reports the discovery and characterization of a novel virus infecting paspalum, tentatively named Paspalum latent virus (PaLV), representing a new taxon within the family Alphaflexiviridae . Methods High-throughput sequencing (HTS), 5′ RACE, and Sanger sequencing were used to obtain complete genome sequences of PaLV from multiple paspalum accessions. Phylogenetic analysis was performed using replicase protein sequences. Structural modeling of the coat protein (CP) was conducted using AlphaFold2 and TM-align. RT-PCR assays were developed for virus detection and host range studies. Genetic diversity and population structure were assessed using CP sequences from 11 PaLV isolates. Results PaLV possesses a 6,995 nt genome with five ORFs and lacks the AlkB domain found in Lolium latent virus (LoLV). The genome exhibits unique features, including overlapping start-stop codons between ORF4 and ORF5 and a second in-frame AUG codon in the CP region. Phylogenetic analysis placed PaLV in a distinct clade clearly separated from Potexvirus and Lolavirus . Structural modeling revealed conserved CP domains despite low sequence identity. PaLV was detected in 90% of tested paspalum accessions and confirmed to infect Zea mays , Sorghum spp. , Setaria italica , and Lolium multiflorum , without visible symptoms. Genetic analysis indicated low diversity and strong purifying selection, with limited spatial structure (FST = 0.124). Conclusions PaLV represents a novel viral species and likely a new sub-genus ( Paspalovirus ) within Alphaflexiviridae . Its latent infection profile and broad host range underscore the need for routine molecular diagnostics in turfgrass management. The RT-PCR assay developed here provides a reliable tool for PaLV detection, and further studies are warranted to assess its impact on plant physiology and breeding.