Citrus Vein Enation Virus Acts as the Dominant Contributor to Host Transcriptional Reprogramming During Synergistic Mixed Infection with Citrus Yellow-Vein Associated Virus in 'Mexican' Lime

Plant viruses in mixed infections can interact in a synergistic or antagonistic fashion, and have the capacity to directly impact their host. In Citrus spp., citrus vein enation virus (CVEV) and citrus yellow-vein associated virus (CYVaV) induce a mixed infection phenotype displaying an attenuation of CVEV symptom expression, enhancement of CYVaV symptom expression, and increased viral titers of both pathogens. RNA-seq was used to investigate the potential virus-host interactions between CVEV, CYVaV, and their native citrus host in single and mixed infections. Single infections of CVEV induced a broad, strong suppressive effect on host systems, whereas single infections of CYVaV depended on a dual strategy of defense system suppression and immune evasion. The CVEV – CYVaV mixed infection provoked a robust host response, generating approximately 1,500 unique differentially expressed genes (DEGs) with stronger gene set enrichment scores linked to immune-pathway manipulation and secondary metabolite pathways not seen in the single infection RNA-seq data. In non-unique up- and downregulated DEGs, the mixed infection retained features of both viruses, however CVEV contributes more from its broad suppression strategy, including the strong downregulation of host defenses and metabolic pathways. Collectively, this indicates that CVEV acts as the dominant contributor to host transcriptional remodeling during mixed infection with CYVaV, revealing a functional decoupling between transcriptional dominance, viral titer, and the disease phenotype. These findings emphasize the complexity of viral pathogenesis mechanisms and underscore the importance of studying virus-virus and virus-host interactions to understand both pathogen and host dynamics in mixed infections.