The increasing global movement of plant material and the complexity of viral communities associated with cultivated crops complicate routine plant virus diagnostics. High-throughput sequencing (HTS) has therefore become an important tool for the comprehensive characterization of plant viromes. In this study, symptomatic pepper (Capsicum annuum) samples submitted to our laboratory between 2020 and 2025 were investigated using HTS following unsuccessful routine diagnostic assays, despite the presence of virus-like symptoms. Virome analysis revealed the presence of multiple viruses with distinct biological characteristics. Eggplant mottled dwarf virus (EMDV) sequences were identified, representing, to our knowledge, the first sequence data from Hungary. In addition, sequences related to tobacco vein clearing virus (TVCV) showed highest similarity to endogenous viral element present in Capsicum annuum genome assemblies. Persistent viruses, including bell pepper alphaendornavirus (BPEV) and pepper cryptic virus 2 (PCV2), were also detected. These findings demonstrate the complex viral communities associated with cultivated pepper and highlight the limitations of strictly targeted diagnostic approaches. The results emphasize the value of HTS for comprehensive virome characterization in horticultural crops.

The 2b protein of cucumoviruses is an RNA silencing suppressor (RSS), and its activity is associated with its nucleocytoplasmic localization. However, the contribution of nuclear localization signals (NLSs), nuclear export signals (NESs), and their sequence context remains poorly understood. Here, we compare cucumber mosaic virus (CMV) and peanut stunt virus (PSV) 2b proteins and their NLS- and NES-containing regions using recombinant proteins. The subcellular localization of PSV 2b has not been characterized, and no protein structural model has been reported previously. CMV 2b displayed a balanced nucleocytoplasmic distribution and strong RSS activity, whereas PSV 2b showed predominantly nuclear localization and weaker RSS activity. Exchange of the N-terminal region and NLSs resulted in pronounced changes in localization and RSS activity. PPC 2b, containing the CMV C-terminal region preceded by PSV sequence, showed the highest RSS activity, while the corresponding recombinant virus caused the most severe symptoms. AlphaFold analysis demonstrated lower structural confidence and higher predicted flexibility in the NES-containing C-terminal regions of CMV 2b and PPC 2b, compared with PSV 2b and CCP 2b, containing the PSV C-terminal region preceded by CMV sequence. These differences may influence NES accessibility and nuclear export.