Evaluation of long-term immunity following inoculation with highly diverse orthomarburgvirus isolates in Egyptian rousette bats ( <i>Rousettus aegyptiacus</i> )

Viral coinfections and their impact on long-term immunity represent an understudied area in disease ecology and infectious disease research. Coinfections can influence the host's susceptibility to future infections, alter host and pathogen population dynamics, modify infection and shedding patterns, impose evolutionary pressures, and affect the risk of zoonotic spillover. Egyptian rousette bats (ERB; <i>Rousettus aegyptiacus</i>; common name: Egyptian rousettes) are a natural reservoir host for Marburg virus (MARV) and Ravn virus (RAVV), as well as a vertebrate reservoir for Kasokero virus (KASV) and a putative reservoir for Sosuga virus (SOSV). Viral coinfections have been documented in numerous free-ranging bat species as well as ERBs, raising questions about how these interactions influence immune responses, viral shedding, and pathogen maintenance within the natural host population. This is particularly critical given the genetic diversity among co-circulating viral species (e.g., MARV and RAVV) and the potential implications for the development of protective immunity and subsequent viral inoculation outcomes. In this study, ERBs previously infected with MARV alone or KASV + MARV were inoculated approximately 8 months later with homotypic (MARV) or heterotypic (RAVV) orthomarburgvirus isolates. The results demonstrated no viral replication or shedding post-inoculation, and all bats displayed strong secondary immune responses consistent with sterilizing immunity. These findings suggest that both MARV monoinfection and KASV + MARV coinfection confer robust protection against reinfection, regardless of the viral isolate. This research enhances our understanding of immune responses during viral coinfections in bats and their potential role in mitigating zoonotic pathogen spillover.IMPORTANCELong-term immunity elicited in bats during coinfection (i.e., simultaneous infection) with viruses they naturally host is not well understood. These interactions could affect susceptibility to subsequent reinfection and pathogen spread. Egyptian rousette bats, natural hosts for several pathogenic zoonotic viruses, including Marburg and Kasokero viruses, can be found multiply infected in the wild, but the immune consequences of being coinfected remain unclear. Here, bats previously infected with either Marburg virus alone or with both Kasokero and Marburg viruses were later challenged with Marburg virus or the related Ravn virus. No reinoculated bats showed signs of virus replication, and all mounted strong immune responses. These results suggest that this coinfection combination still provides robust protection against reinfection, even with diverse orthomarburgviruses. This study helps improve our understanding of how bats manage viral coinfections and may inform how these interactions influence zoonotic spillover risk.