Rotavirus is a non-enveloped RNA virus traditionally thought to exit host cells through lytic or conventional vesicular pathways, but recent evidence shows that it also exploits host microvesicles for non-lytic transmission. To characterize this transmission pathway, we combined high-resolution microscopy (TEM, confocal microscopy, immunogold labeling) with biochemical and infectivity assays. Our work revealed that rotavirus infection robustly increased microvesicle production, and individual vesicles frequently contained multiple viral particles. Quantitative analysis revealed striking differential partitioning of particle types. Mature triple-layered particles predominated within infected cells, whereas extracellular microvesicles were strongly enriched for immature double-layered particles. Although free double-layered particles lack the outer capsid required for classical receptor-mediated entry, microvesicle-associated particles initiated productive infections, whereas disruption of the vesicular membrane abolished infectivity. These findings suggest an evolutionarily advantageous dual transmission strategy. By using host-derived membranes to transmit otherwise non-infectious intermediates, rotavirus can bypass outer-capsid-dependent entry, potentially reducing the energetic cost of producing fully mature infectious particles while enabling collective transmission. Maintaining a free-virus pathway may nevertheless be important because transmission by individual mature virions imposes population bottlenecks that limit the propagation of defective or cheating genomes and preserve high-fitness genotypes. Thus, partitioning viral progeny between vesicle-associated double-layered particles and free triple-layered particles may balance the immediate benefits of collective spread with long-term genetic quality control. Whether double-layered particle enrichment arises from active sorting or spatial coupling between viral assembly and microvesicle biogenesis remains unresolved.
Significance We demonstrate that rotavirus employs a dual-transmission strategy to optimize viral transmission and replication. While mature triple-layered particles predominate within cells, we show that rotavirus exploits host-derived microvesicles to preferentially package and transmit double-layered particles. This envelopment allows otherwise non-infectious viral intermediates to spread efficiently between cells. Evolutionarily, this reduces the energetic burden of building outer capsids for every genome, freeing resources to maximize viral replication. Concurrently, the virus maintains an independent transmission route via free, mature particles, preventing the accumulation of defective “cheater” genomes inherent to collective vesicle infection. These findings highlight a sophisticated evolutionary balancing act, revealing how viruses deploy structurally distinct particles across varied transmission routes to maximize both local spread and genetic integrity.
