Characterization and Construction of Full-Length cDNA Infectious Clone of a Novel BCMV Isolate in Pathogroup III

<i>Bean common mosaic virus</i> (BCMV; <i>Potyvirus phaseovulgaris</i>) is one of the primary viruses that severely impacts the yield and quality of common beans (<i>Phaseolus vulgaris</i> L.) and has a worldwide distribution. Utilizing small RNA sequencing and RT-PCR validation, this study identified widespread co-infection by multiple viruses in field-collected common bean samples, with BCMV being the dominant viral species. A novel isolate, designated DY9, was obtained from these field samples. Pathotype characterization confirmed DY9 as pathotype PG-III, while previous studies reported all other PG-III members as <i>Bean common mosaic necrosis virus</i> (BCMNV). Whole-genome sequencing and phylogenetic analysis revealed that DY9 was genetically closer to BCMV and diverged significantly from known PG-III isolates. Based on these findings, we constructed an infectious clone of DY9. To address the genetic instability of <i>Potyvirus</i> in the <i>Escherichia coli</i> (<i>E. coli</i>) expression system, we discovered that inserting Intron 2 (derived from the <i>NiR</i> gene of <i>P. vulgaris</i>, GenBank: U10419.1) at position 2431 of the HC-Pro gene and targeting Intron 1 (derived from the <i>ST LS1</i> gene of <i>Solanum tuberosum</i>, GenBank: X04753.1) at position 4240 of the CI gene significantly improved the stability of the cloning vector. The clone was verified to systemically infect common bean plants and induce typical mosaic symptoms. Infectivity was validated through RT-PCR, RT-qPCR, Western blotting, and transmission electron microscopy. This study represents the first successful construction of an infectious clone for pathotype PG-III BCMV, providing a critical reverse genetics tool for dissecting viral pathogenesis and identifying resistance genes. These findings not only expand the genetic diversity of BCMV but also offer a methodological reference for constructing infectious clones of <i>Potyvirus</i> species.