The rules in co-infection of multiple viruses across diverse lineages in a fungal host

ABSTRACT Viruses, ubiquitous non-cellular organisms, pose significant threats to human health and to the agricultural productivity of both livestock and crops. Emerging evidence indicates that multiple viruses can infect a single host, and viral co-infection can exert a profound influence on host physiology. However, our understanding of the prevalence of co-infection and the compatibility of phylogenetically distant viruses is still limited. In this study, we surveyed 406 field strains of the plant fungal pathogen Botrytis cinerea and identified 76 mycoviruses. Strikingly, 404 strains were co-infected with two or more viruses, with some harboring up to 25 viruses simultaneously. We discerned significant preference patterns among viruses in their host. Specifically, we identified “one-to-one” and “two-to-one” rules, wherein one or two viruses could be used to reliably predict the presence or absence of other viruses in the same host, and validated these predicted rules by using five B. cinerea strains. Furthermore, through the RNA-sequencing approach, we uncovered B. cinerea genes associated with the differences caused by different sets of co-infecting viruses. These are implicated in integral components of membrane, transmembrane transporter activity, autophagy pathways, mitophagy pathway, fatty acid biosynthetic process, sphingolipid metabolism, and glycosphingolipid biosynthesis. Our findings underscore the high prevalence of co-infection by multiple viruses in a fungal host within a population and highlight compatibility dynamics among phylogenetically diverse viruses. These insights contribute to our understanding of viral ecology and hold promise for informing strategies to manage viral diseases effectively.IMPORTANCEViruses, pervasive threats to both humans and agriculture, often infect hosts concurrently, profoundly impacting physiology. Despite this, the prevalence and compatibility of co-infecting viruses remain poorly understood. In the study of 406 Botrytis cinerea strains, we discovered a striking phenomenon: 404 out of the 406 strains hosted multiple viruses, some with up to 25 at once. Through rigorous analysis, we unveiled distinct preference patterns among these viruses within hosts, identifying predictive co-infection rules validated by experimentation. Furthermore, we identified genes linked to these dynamics, shedding light on critical cellular processes involved in the regulation of the co-infection rules. These findings highlight the widespread nature of viral co-infection and offer insights crucial for effectively managing viral diseases.