Mammalian Orthoreoviruses were first isolated in the 1950s from the stool samples of healthy children in Mexico and Cincinnati [1]. Serology reports have estimated an exposure of > 50 % in children five years or younger and up to > 80 % in adults, reflecting a high prevalence of natural infection among individuals of all age groups [2]. However, no known human diseases have been convincingly linked to reoviral infection, thus the name “REO” (Respiratory Enteric Orphan) was given to highlight its presence in the respiratory and enteric tracts and inability to cause any severe symptoms (orphan of a disease) [1]. Four serotypes of mammalian reoviruses have been described to date: Type 1 Lang (T1L), Type 2 Jones (T2J), Type 3 Dearing and Abney (T3D, T3A), and more recently Type 4 Ndelle (T4N) [3]. Since mammalian reoviruses are largely asymptomatic in humans [4], they quickly became invaluable tools in virology and molecular biology. Notably, research on reovirus has helped unravel key mechanisms of RNA synthesis and mRNA translation initiation [5]. The minimal toxicities of reovirus T3D have also been demonstrated in multiple clinical trials with patients receiving high viral doses [6], whereby no maximal tolerated doses have yet been reported. It is noteworthy that, despite their generally weak pathogenicity in humans, some reovirus strains have been associated with intestinal and central nervous system conditions, particularly in newborns and young infants. More recently, reovirus has been implicated in triggering immune responses associated with gluten intolerance and celiac disease in adults, potentially by disrupting intestinal immune homeostasis or promoting inflammatory pathways [7], [8]. Nonetheless, the amount of clinical data accumulated on reoviruses as an oncolytic immunotherapy provides strong rationale to continue the development of this unique viral treatment. (The initiated, completed, and currently ongoing clinical trials with reovirus are listed in Table 1 and Fig. 1 graphically summarizes the review.)

Reovirus is a non-enveloped virus featuring an icosahedral capsid that encases a segmented genome of 10 double-stranded RNA (dsRNA) segments [9]. Each segment encodes a specific protein critical for the life cycle of the virus. The three large segments (L1, L2, and L3) produce proteins λ3, λ2, and λ1, where λ3 functions as the RNA-dependent RNA polymerase (RdRp), and λ2 serves both as the capping enzyme and as a core structural protein. The three medium segments (M1, M2, and M3) encode for proteins like μ1, which is essential for membrane penetration, μ2, and μNS, a non-structural protein key to forming viral replication centers (viral factories). Meanwhile, the four small segments (S1, S2, S3, and S4) generate proteins including σ1, the cell attachment protein (which can also produce a smaller variant by alternative translation initiation, σ1 s), along with σ2, σ3, and σNS that aid in core assembly and modulate host interactions [10]. Together, these components drive the infection cycle through stages of attachment, entry, replication, and assembly [11].

Naturally reovirus enters the body via oral-faecal routes [12]. Despite facing harsh conditions along this route, such as stomach acid and digestive enzymes, the virus remains highly stable in these environments due to its rigid double layered protein-based capsids, which allow it to reach the small intestine where the infection takes place [13], [14]. The intestinal Microfold (M) cells in Peyer's patches, a key component of gut-associated lymphoid tissue (GALT), are the primary entry point of reovirus [15]. Once infection has been successfully established, the virus can enter deeper layers of the intestinal tissue through M cells, where it replicates and spreads. Thereafter, reovirus usually remains localized in the gut, causing mild or asymptomatic infections. In newborn mice reovirus can cross the intestinal barrier and spread systematically through the lymphatic system and bloodstream (viremia), potentially reaching distal organs like the liver, lungs, or brain particularly [12].

In the initial stage of infection, reovirus particles are processed by cysteine proteases cathepsin B/L, which removes portions of the outer capsid to form an infectious subviral particle (ISVP) with an exposed receptor-binding protein σ1 [16]. This modification enables the virus to attach to host cells expressing junctional adhesion molecule A (JAM-A) and presenting sialic acid on their surface [16]. Following attachment, interactions between the remaining capsid protein λ2 and cellular β1 integrins promote endocytosis. In the late endosomes, further proteolytic disassembly allows the virus core to breach the endosomal membrane and access the cytoplasm [16]. Once in the cytoplasm, transcription is initiated within the viral core by the RdRp, λ3. This enzyme synthesizes capped mRNAs, which exit the core through channels formed by the λ2 protein and stay bound to it during early replication [17]. These initial transcripts, along with their protein products, drive the synthesis of complementary negative strand RNAs and additional structural proteins, culminating in the assembly of new virions. Concurrently, viral components aggregate to form specialized viral factories in the cytoplasm, which serve as dedicated sites for genome replication and particle assembly. Ultimately, progeny virions are released from the host cell either via direct lysis or through apoptosis-induced cell lysis and other mechanisms [10], [18], [19], [20].

As a line of defense against reovirus infection, the mucosal immune system employs not only innate but also adaptive immune mechanisms [21]. The innate immune response is triggered when macrophages detect reovirus through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) [22]. These cells release interferons (IFN-α and IFN-β) and pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α, which create an antiviral state, limiting viral replication and alerting other immune cells [22]. Moreover, reoviral antigens are collected from the gut lumen by M cells, which are then transported to dendritic cells (DCs), antigen-presenting cells (APCs) [12]. This allows further recruitment and education of T cells which mobilizes a cell-mediated adaptive immune response. In addition, naïve B cells residing in the germinal centers are then activated by the helper T-cells and subsequently mature to produce secretory neutralizing IgA (sIgA) into the gut mucosa, preventing reovirus from further infecting epithelial cells. As the immune response develops, memory B and T cells are generated, which provide long-term protection against future infections by reoviruses [21].