Rabbit Rotavirus

Overview and Taxonomy of Rabbit Rotavirus

Taxonomic Classification and Virological Basis

Rabbit rotavirus (RRV), more precisely referred to as lapine rotavirus A (RVA), is a member of the family Reoviridae, subfamily Sedoreovirinae, genus Rotavirus. The genus Rotavirus is classified into nine species (Rotavirus A through I, and a putative species J), with Rotavirus A (RVA) being the predominant pathogen associated with acute gastroenteritis in both humans and a wide array of animal species, including rabbits (Oryctolagus cuniculus). The classification of rotaviruses is fundamentally rooted in the antigenic properties of the inner capsid protein VP6, which defines the group (A–I), and the serological or genotypic characterization of the two outer capsid proteins, VP7 (the glycoprotein, defining G-genotypes) and VP4 (the protease-sensitive protein, defining P-genotypes). This dual genotyping system, formally recognized by the Rotavirus Classification Working Group (RCWG), is the cornerstone of rotavirus taxonomy and epidemiology. For lapine strains, the most historically and epidemiologically significant G/P combinations have been G3P[1] and G3P[2], although recent discoveries have expanded this paradigm [3, 4, 5].

The virion itself is a non-enveloped, triple-layered icosahedral particle approximately 70–75 nm in diameter, a morphology consistent across all rotavirus species and first elucidated in early comparative studies using immune electron microscopy on porcine and calf isolates [6]. The genome consists of 11 segments of double-stranded RNA (dsRNA), each encoding a single viral protein, with the exception of segment 11, which encodes NSP5 and NSP6. These 11 segments code for six structural proteins (VP1–VP4, VP6, VP7) and six non-structural proteins (NSP1–NSP6). The segmented nature of the genome is of paramount evolutionary importance, as it facilitates genetic reassortment, a process whereby co-infection of a single cell with two distinct rotavirus strains can yield progeny viruses with novel combinations of genome segments. This mechanism is a primary driver of rotavirus diversity, host range expansion, and the emergence of novel strains with pandemic or zoonotic potential [3, 4, 7].

Historical Context and Early Characterization

The etiological role of rotaviruses in neonatal diarrhea of rabbits was recognized in parallel with studies in other livestock species. Early experimental infections demonstrated that conventionally reared calves were fully susceptible to rotavirus isolates originating from heterologous hosts, including rabbits, underscoring the potential for cross-species transmission and the shared pathogenic mechanisms across mammalian rotaviruses [2]. Cross-neutralization studies from this era revealed a reciprocal antigenic correlation between bovine and rabbit isolates, suggesting a close antigenic relationship that predates the molecular era of genotyping [2]. These foundational studies established rabbits not only as a natural host for rotavirus but also as a valuable experimental model for understanding rotavirus pathogenesis, immunity, and vaccine development. Indeed, the production of polyclonal antibodies against recombinant VP6 protein in rabbits has been a standard methodology for decades, leveraging the rabbit's robust humoral immune response to generate high-titer, specific reagents for diagnostic assay development, including ELISA and lateral flow immunoassays [8, 1].

The Classical Lapine Genotypes: G3P[1] and G3P[2]

For over two decades, the molecular epidemiology of lapine RVA has been dominated by two genotype constellations: G3P[1] and G3P[2]. The G3 genotype is remarkably promiscuous, having been identified in a wide range of hosts including humans, simians, canines, felines, bats, and rabbits [5, 9]. The P[1] genotype, in contrast, is considered a classic lapine or "rabbit-like" genotype, though it has also been sporadically detected in humans, often in the context of direct zoonotic transmission [3, 10]. The G3P[1] combination, therefore, represents a well-documented lapine lineage with demonstrated zoonotic potential.

Complete genome sequencing of G3P[1] strains from rabbits and from human infants with gastroenteritis has provided profound insights into their evolutionary history. For instance, the human strain BE5028, isolated from a two-year-old boy in Belgium, possessed a genotype constellation of G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3, which was nearly identical to lapine strains circulating in the same geographic region [10]. Phylogenetic analyses of all 11 gene segments confirmed that BE5028 was of almost complete lapine origin, representing a direct rabbit-to-human interspecies transmission event capable of causing severe disease in a human child [10]. This finding was not an isolated incident; a similar lapine-like strain, B4106, had been identified in a Belgian child in 2000, indicating sustained circulation and repeated spillover of this lineage into the human population [10]. Furthermore, the NSP5 gene segment of BE5028 contained a head-to-tail partial duplication combined with two short insertions and a deletion, a unique molecular signature indicative of continuous, long-term circulation within the rabbit population [10]. This molecular clock-like feature underscores the stability and endemicity of G3P[1] strains in rabbits.

The G3P[2] genotype is another major lapine lineage, with P[2] being considered a rabbit-specific genotype. The first complete genome sequence of a G3P[2] strain from Korea (Rab1404) revealed a constellation of G3-P[2]-I2-R3-C3-M3-A9-N2-T3-E3-H3 [7]. Intriguingly, Rab1404 exhibited a complex reassortant history. While its VP4, VP6, and NSP5 genes were closely related to other lapine strains, its VP1, VP2, VP3, and VP7 genes were closely related to a bat rotavirus strain (LZHP2), and its NSP1–NSP4 genes were closely related to the simian strain RRV [7]. This mosaic genome suggests that Rab1404 is a product of multiple interspecies transmission and reassortment events involving bat, simian, and potentially bovine or canine/feline rotaviruses [7]. Similarly, the Chinese G3P[2] strain Z3171 displayed a distinct constellation (G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3), differing from other lapine strains in its NSP3 and other segments, further supporting the notion of ongoing reassortment and the existence of undetected genotypes circulating in the global rabbit population [4].

Emerging and Atypical Genotypes: Evidence of Zoonotic Spillover and Reassortment

The classical view of lapine rotavirus diversity has been challenged by the recent identification of novel and atypical G/P combinations, most notably G3P[9] and G3P[1] with unusual internal gene constellations. The detection of a G3P[9] strain (C-3/15) from a diarrheic rabbit in Mexico represents a landmark finding [5]. The P[9] genotype is the most common human P-genotype worldwide and had never before been reported in rabbits. Phylogenetic analysis of both the VP7 and VP4 genes of C-3/15 revealed a close genetic relationship to human, not lapine, rotaviruses [5]. This finding strongly suggests a reverse-zoonotic event, the transmission of a human rotavirus into a rabbit, or a reassortment event where a lapine strain acquired a human P[9] gene. The clinical significance of this finding is twofold: it demonstrates that rabbits are susceptible to infection with human rotavirus strains, and it raises the possibility that rabbits could serve as a reservoir or intermediate host for the evolution of novel human-pathogenic strains.

Further evidence of the dynamic nature of lapine rotavirus evolution comes from the complete genome analysis of an Italian G3P[1] strain, which displayed a genotype constellation shared with other G3P[1] strains described in both rabbits and humans [3]. This study, published in 2025, reinforces the ongoing circulation and genetic stability of this lineage while also highlighting the need for continuous surveillance [3]. The detection of bat-like G3 rotavirus genes in children with gastroenteritis in the Dominican Republic, where the VP7 sequences were highly similar to those of bat rotaviruses from Bulgaria, further illustrates the complex web of interspecies transmission that characterizes rotavirus ecology [9]. While this study focused on humans, it underscores the potential for bat rotaviruses to contribute genetic material to strains circulating in other mammals, including rabbits, as suggested by the bat-like genes in the Korean lapine strain Rab1404 [7, 9].

Epidemiological Significance and Economic Impact

Rabbit rotavirus is a significant contributor to the complex etiology of enteric disease in commercial rabbitries worldwide. Digestive disorders are the primary cause of economic losses in rabbit farming, and rotavirus is frequently identified as a co-pathogen in multifactorial disease outbreaks [11]. Large-scale epidemiological surveys, such as those conducted on the Iberian Peninsula, have demonstrated that rotavirus is commonly detected in growing rabbits (approximately 15–35 days old), often in co-infection with enteropathogenic Escherichia coli (EPEC), Clostridium spiroforme, and Eimeria spp. [11]. In these settings, simple infections are the exception rather than the rule; mixed infections involving two or three pathogens are the most prevalent scenario, complicating diagnosis, treatment, and control [11]. The economic impact is exacerbated by the fact that antibiotic treatment, often the first line of defense, is ineffective against viral pathogens and may contribute to antimicrobial resistance.

The global distribution of lapine rotavirus is now well-documented, with molecular characterization studies emerging from Europe (Italy, Belgium, Spain), Asia (China, South Korea), and the Americas (Mexico) [1, 2, 4-6, 8]. The seroprevalence of rotavirus antibodies in rabbit populations is high, as demonstrated by serological surveillance using advanced multiplex assays like the Luminex xMAP and x-TAG platforms, which have been developed for the simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus, and rabbit rotavirus [12, 13]. These high-throughput tools are essential for routine monitoring in laboratory animal facilities and commercial farms, where the pathogen status of rabbits can directly affect the validity of experimental results and the profitability of production [12, 13].

Implications for Zoonotic Risk and Public Health

The World Health Organization (WHO) recognizes rotavirus as the leading cause of severe diarrheal disease in children under five years of age globally, responsible for hundreds of thousands of deaths annually, primarily in low-income countries. While the primary burden is from human-specific strains, the zoonotic potential of animal rotaviruses, including lapine strains, is a growing public health concern. The documented cases of G3P[1] lapine strains causing gastroenteritis in human infants in Belgium provide unequivocal evidence that rabbit rotaviruses can cross the species barrier and cause disease in humans [10]. The close genetic relationship between these human isolates and contemporaneous lapine strains suggests that such spillover events may be more common than previously recognized, particularly in regions with close human-animal contact.

Furthermore, the segmented genome of rotavirus allows for reassortment between human and animal strains, potentially generating novel viruses with pandemic potential. The detection of a human P[9] genotype in a rabbit in Mexico [5] and the presence of bat-like genes in a Korean lapine strain [7] illustrate the bidirectional flow of genetic material between species. The Centers for Disease Control and Prevention (CDC) and the World Organisation for Animal Health (WOAH) emphasize the importance of a One Health approach to surveillance, integrating human, animal, and environmental health data to monitor and mitigate the emergence of zoonotic rotaviruses. The continued characterization of lapine rotavirus strains, including their full genome sequences, is therefore not merely an academic exercise but a critical component of global rotavirus surveillance and pandemic preparedness. The development of reverse genetics systems for rotavirus, while still challenging, holds promise for future studies on the molecular determinants of host range and virulence, which could inform risk assessment and vaccine development [14].

Genomic Diversity and Reassortment Patterns

The genomic architecture of group A rotaviruses (RVAs) is inherently modular, comprising 11 discrete double-stranded RNA (dsRNA) segments, each of which can theoretically reassort independently during co-infection. This fundamental property drives a near-boundless capacity for genetic diversification and is the central mechanism by which lapine rotaviruses (LRVs) are shaped, disseminated, and, critically, propelled across species barriers. For rabbit populations, the consequences of this genomic plasticity are profound, yielding constellations that range from the seemingly stable, host-adapted lineages to complex chimeras that reflect the convergence of viral gene pools from humans, bovines, simians, and even bats. The totality of evidence from whole-genome sequencing efforts conducted over the past decade now paints a picture of the LRV genome not as a static entity, but as a dynamic assemblage whose patterns of reassortment reveal a hidden epidemiology of interspecies contact and viral trafficking [1, 2, 4-6].

The Canonical Constellations and the Emergence of Novel Lineages

For years, the characterization of LRVs was largely restricted to partial sequencing of the outer capsid genes, leading to the long-held assumption that the predominant circulating genotypes were the G3P[1] and G3P[2] combinations [5, 10]. However, the advent of complete genome sequencing has radically revised this perspective, revealing that the entire constellation, the full set of 11 Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx genotypes, is the relevant unit of evolutionary analysis. Early deep sequencing of the Italian G3P[1] strain highlighted a constellation shared with other lapine and human G3P[1] strains, suggesting a degree of genetic coherence within this lineage [3]. Yet, the discovery of the Chinese isolate Z3171 shattered the notion of a single, unified LRV genome. With its constellation of G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3, Z3171 was shown to differ substantially in both gene content and sequence from reference strains like N5 and Rab1404, providing unequivocal genomic evidence for a reassortment event between human and lapine strains or, equally compelling, the circulation of previously undetected genotypes within the rabbit reservoir [4].

The Korean isolate Rab1404 serves as perhaps the most illustrative example of the multi-species genomic chimerism that defines modern LRV evolution. Its constellation, G3-P[2]-I2-R3-C3-M3-A9-N2-T3-E3-H3, did not arise from a simple rabbit-to-rabbit lineage. Instead, phylogenetic dissection revealed a startling mosaic: the VP1, VP2, VP3, and VP7 genes were closely allied with a bat strain (LZHP2), while the NSP1, NSP2, and NSP4 genes traced their ancestry to a simian strain (RRV). The VP4, VP6, and NSP5 segments retained a lapine origin, and yet, the VP6 (I2), NSP1 (N2), and NSP5 (H3) segments bore signatures of bovine RVA [7]. This astonishing level of genomic compartmentalization implies that Rab1404 is the product of at least two, and likely more, sequential reassortment events. The presence of simian RRV-like genes is particularly telling; RRV itself is a known reassortant bearing bovine, canine, and feline genetic elements, suggesting that the rabbit virus acquired these segments indirectly through an intermediary host that had already been infected with a complex virus [7]. This highlights a critical epidemiological pathway: the rabbit is not merely a terminal host but can act as a sink or, potentially, a mixing vessel for genetic material from disparate mammalian reservoirs.

Direct Zoonotic Spillover and Zoonotic Reassortment

The genomic data now unequivocally support the role of LRVs in both zoonotic spillover (rabbit-to-human) and the acquisition of human genes within the lapine host. The most dramatic evidence for the former comes from the identification of the Belgian human strain BE5028. A two-year-old child presenting with acute gastroenteritis was found to harbor a G3P[1] strain whose entire genotype constellation, G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3, was unequivocally of lapine origin [10]. The close phylogenetic relationship of BE5028 to a previous lapine-like human isolate from 2000 (B4106) confirmed that this lineage of rabbit rotavirus has repeatedly jumped into the human population [10]. Critically, the NSP3 segment of BE5028 was identified as T6, a genotype typical of bovine RVAs, indicating that within the rabbit population, a further reassortment with a bovine strain had occurred before the zoonotic transmission event [10]. This demonstrates that reassortment can pre-adapt or at least precede a species jump, creating a virus that, while retaining its lapine backbone, carries a necessary bovine segment that may facilitate replication in a human host.

The converse phenomenon, a human rotavirus infecting a rabbit, was documented in Mexico with the strain C-3/15. This isolate presented a combination never before seen in rabbits: G3P[9]. The P[9] genotype is considered a human-adapted VP4 specificity, and both the VP7 and VP4 genes of C-3/15 were shown by phylogenetic analysis to cluster with human, not lapine, lineages [5]. The presence of a fully human-like outer capsid on an otherwise unknown internal genome context strongly suggests an interspecies transmission event from a human source into the rabbit population, likely followed by, or coincident with, a reassortment that allowed the virus to replicate in the rabbit enteric tract [5]. These reciprocal zoonotic events underscore a bidirectional genomic traffic, emphasizing that rabbits and humans share a closer ecologic and virologic relationship than previously appreciated, a point of significant concern for public health authorities and veterinary epidemiologists monitoring zoonotic emergence.

The Role of VP6 and Interspecies Homogeneity

While diversity is rampant in the outer capsid genes, a remarkable conservation is observed at the level of the inner capsid protein VP6, a key antigenic target encoded by segment 6. The VP6 protein, which comprises the middle layer of the triple-layered particle, is the defining antigen for group A classification and is highly immunogenic, as demonstrated by the generation of robust polyclonal antisera in rabbits immunized with recombinant VP6 [8, 1]. Phylogenetic studies of LRVs consistently assign the VP6 gene to the I2 genotype, a genotype widely disseminated across bovine, simian, and some human strains [4, 10, 7]. This suggests that the I2 VP6 lineage is a "public good" in the rotavirus metagenome, easily exchanged and readily functional in heterologous backgrounds. The functional basis for this exchangeability is rooted in the protein’s structure; VP6 contains a trimerization domain and a carboxyl-terminal assembly domain that are conserved across mammalian RVAs, allowing a VP6 from a bovine or human source to assemble seamlessly onto the core of a lapine virus [15]. This structural compatibility lowers the barrier to reassortment for this segment, facilitating the formation of viable chimeric viruses that can then go on to acquire new outer capsid genes.

Genomic Signatures of Long-Term Circulation and Diagnostic Implications

The genomic record also carries signatures of prolonged, uninterrupted circulation within rabbit populations. The most compelling of these is the unusual rearrangement observed in the NSP5 gene of the lapine-like human strain BE5028. Sequence analysis revealed a head-to-tail partial duplication combined with two short insertions and a deletion [10]. Such rearrangements in the NSP5 gene, which encodes a non-structural protein essential for viroplasm formation and genome replication [16], are rarely observed in transient human infections. Their presence in the lapine lineage BE5028 is a clear indicator of long-term, continuous viral replication within a closed or semi-closed rabbit population. This genetic decay or duplication, which would likely be selected against in a highly competitive, diverse human epidemic, can persist in the more stable, less diverse environment of a rabbitry, serving as a molecular clock of endemicity.

From a diagnostic and surveillance perspective, the vast genomic diversity of LRVs poses both a challenge and an opportunity. The high seroprevalence of RVA in commercial rabbitries, as detected by serological tools like the Luminex xMAP assay [12] and confirmed by molecular methods such as the x-TAG assay [13], indicates widespread exposure. However, the presence of reassortant strains containing non-lapine genes (e.g., human P[9] or bovine T6) can lead to false negatives in molecular assays that are designed against conserved regions of canonical rabbit strains. The WHO and WOAH have acknowledged the importance of comprehensive strain surveillance in animal reservoirs as a component of pandemic preparedness, and the data from rabbit populations make a strong case for incorporating LRV sequencing into routine One Health monitoring programs. The detection of a P[1] strain of full lapine origin in a human infant [10], and a human P[9] strain in a rabbit with enteritis [5], are textbook examples of the types of events that precede the emergence of novel human rotavirus genotypes. Therefore, the genomic diversity of the LRV is not an abstract curiosity; it is a measurable, actionable parameter of viral risk that demands continuous, high-resolution surveillance across the agronomic and wildlife interfaces.

Molecular Pathogenesis of Rabbit Rotavirus

The molecular pathogenesis of rabbit rotavirus (LRV) represents a complex interplay between viral genetic determinants, host cellular factors, and dynamic evolutionary forces that drive interspecies transmission and reassortment. Understanding these mechanisms at the molecular level is critical not only for comprehending lapine disease but also for assessing the zoonotic potential of these viruses, a concern recognized by global health authorities including the World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) given the increasing evidence of rotavirus spillover events.

Genomic Architecture and Genotype Diversity as Determinants of Pathogenesis

Rabbit rotaviruses, classified within species Rotavirus A (RVA), possess a segmented double-stranded RNA genome comprising 11 segments, each encoding either a structural protein (VP1-VP4, VP6, VP7) or a non-structural protein (NSP1-NSP5/6). The molecular pathogenesis of LRV is fundamentally rooted in its genotype constellation, which dictates host range, cellular tropism, and virulence. Historically, the most prevalent and epidemiologically significant lapine genotypes have been G3P[1] and G3P[2] [3, 4, 5]. However, recent comprehensive genomic analyses have revealed a far more complex landscape. The G3 genotype, defined by the VP7 outer capsid glycoprotein, is remarkably promiscuous across host species, having been identified in humans, rabbits, bats, pigs, and monkeys, suggesting that this VP7 lineage possesses structural features that facilitate cross-species adaptation [5, 9]. Conversely, the P[1] and P[2] genotypes, defined by the VP4 protease-sensitive hemagglutinin, have been considered more host-restricted, with P[2] initially believed to be exclusive to rabbits [4, 7].

The molecular basis for this differential host restriction lies in the VP8* subunit of VP4, which mediates initial attachment to sialic acid receptors on intestinal epithelial cells. The discovery of a G3P[9] strain (C-3/15) in a Mexican rabbit with enteritis fundamentally challenges previous assumptions about host range [5]. The P[9] genotype had been considered exclusively human; its detection in a rabbit, with both VP7 and VP4 genes showing close phylogenetic relatedness to human rotaviruses, provides compelling molecular evidence for a direct interspecies transmission event from humans to rabbits [5]. This finding has profound implications for understanding the molecular pathogenesis of LRV, as it demonstrates that the lapine intestinal epithelium expresses receptors compatible with human rotavirus VP4 variants, and that the rabbit host can support replication of a virus bearing a human-derived attachment protein. From a public health perspective, this bidirectional transmission potential underscores the need for surveillance frameworks aligned with WOAH guidelines for emerging zoonotic pathogens.

Reassortment as a Driver of Pathogenic Evolution

The segmented nature of the rotavirus genome renders LRV highly susceptible to reassortment, a process whereby co-infection of a single cell with two distinct rotavirus strains generates progeny viruses with novel gene segment combinations. This mechanism is arguably the most powerful driver of molecular pathogenesis in LRV, enabling rapid acquisition of genetic material from heterologous hosts. Complete genome sequencing of lapine strains has unveiled a mosaic of genetic origins that speaks to extensive reassortment history. The Korean strain Rab1404 (G3-P[2]-I2-R3-C3-M3-A9-N2-T3-E3-H3) exemplifies this phenomenon: while its VP4, VP6, and NSP5 genes are of lapine origin, its VP1, VP3, VP7, NSP3, and NSP4 genes share close phylogenetic relationships with canine/feline rotaviruses, and its VP6, NSP1, and NSP5 genes are closely related to bovine rotaviruses [7]. Furthermore, eight of its eleven gene segments showed close relatedness to the simian strain RRV, itself believed to be a reassortant between bovine-like and canine/feline strains [7]. This genetic chimerism suggests that Rab1404 arose through multiple sequential reassortment events involving viruses from diverse mammalian reservoirs before ultimately adapting to the rabbit host.

Similarly, the Chinese isolate Z3171 (G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3) exhibited a genotype constellation distinct from previously characterized LRV strains, with its NSP2 (N2) and NSP3 (T1) genotypes differing from those of other lapine isolates [4]. The authors of that study proposed that either a reassortment event occurred between human and rabbit rotaviruses, or that undetected genotypes are circulating in rabbit populations [4]. The human lapine-like strain BE5028, isolated from a Belgian child with gastroenteritis, provides direct evidence for the zoonotic consequences of reassortment. This strain possessed a G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3 constellation, and phylogenetic analyses indicated that its NSP3 segment was of bovine(-like) origin, suggesting that a bovine NSP3 had been introduced into the lapine rotavirus population through reassortment within the preceding 12 years [10]. Critically, BE5028 also harbored a head-to-tail partial duplication in its NSP5 gene, combined with two short insertions and a deletion, indicative of continuous circulation and evolution within the rabbit population before its transmission to a human infant [10]. These molecular signatures of replication fidelity errors and recombination within the lapine host underscore the dynamic nature of LRV genome evolution.

Molecular Mechanisms of Enterocyte Damage and Diarrheal Pathogenesis

The clinical hallmark of LRV infection is acute gastroenteritis, particularly in neonatal and weanling rabbits, where it contributes significantly to the enteric disease complex often involving co-infections with enteropathogenic Escherichia coli (EPEC), Clostridium spiroforme, and Eimeria spp. [11]. The molecular pathogenesis of rotavirus-induced diarrhea is multifactorial, involving both direct virus-mediated enterocyte destruction and the action of the viral enterotoxin NSP4.

At the cellular level, rotavirus infection targets mature enterocytes lining the villi of the small intestine. The initial step involves VP4-mediated attachment to cell surface receptors, followed by VP7-dependent entry. Once internalized, viral replication occurs within cytoplasmic inclusion bodies called viroplasms, whose formation is orchestrated by the non-structural proteins NSP2 and NSP5. NSP2, a nonspecific RNA-binding protein, assembles into 10S multimers that interact with the viral RNA-dependent RNA polymerase VP1, forming complexes that coordinate genome replication and packaging [16]. The VP6 protein, which forms the middle capsid layer, is essential for particle stability and transcriptional activation; its trimerization domain resides between amino acid residues 105 and 328, while a distinct carboxyl-terminal domain (residues 251-397) is required for assembly onto single-shelled particles [15]. The high immunogenicity of VP6 has been exploited for diagnostic purposes, with recombinant VP6 proteins used to generate polyclonal antibodies in rabbits for detection assays [8, 1].

The enterotoxin NSP4 plays a central role in the secretory component of rotavirus diarrhea. Early studies using rabbit brush-border membrane (BBM) vesicles demonstrated that the NSP4₁₁₄₋₁₃₅ peptide inhibits Cl⁻/H⁺ symport activity, but this effect was found to be nonspecific and equally produced by a norovirus peptide [17]. Importantly, in vivo infection of young rabbits accelerated both Cl⁻ influx and efflux rates across villus BBM without stimulating Cl⁻ transport in crypt BBM, leading to the conclusion that NSP4 does not exert a direct, specific effect on chloride transport [17]. Instead, current evidence supports a model wherein NSP4 triggers intracellular signal transduction pathways, likely involving phospholipase C and inositol trisphosphate-mediated calcium mobilization, that ultimately enhance net chloride secretion through activation of calcium-dependent chloride channels. This disruption of ion homeostasis, combined with virus-induced enterocyte apoptosis and villus atrophy, results in the malabsorptive and secretory diarrhea characteristic of LRV infection.

Antigenic Variation and Immune Evasion

The molecular pathogenesis of LRV is further modulated by antigenic variation in the neutralizing epitopes of VP7 and VP4. Cross-neutralization studies using rabbit antisera have revealed that the G genotype (defined by VP7) plays a more dominant role in determining neutralization specificity than previously appreciated [18]. Rabbit antisera raised against simian SA11 G3P[4] and equine G3P[19] showed robust cross-neutralization, whereas antisera against equine G14P[19] exhibited less cross-neutralization capacity against heterologous strains [18]. Interestingly, rabbit antisera to equine G14P[19] provided more robust cross-protection against SA11 G3P[4] than the reverse, suggesting that the immunodominance of VP7 epitopes can vary asymmetrically between strains [18]. Structural analyses identified multiple highly solvent-exposed amino acid residues conserved in VP7 and VP8* among these viruses that may serve as novel B-cell epitopes contributing to cross-neutralization [18]. These findings have direct implications for vaccine development, as they suggest that inclusion of diverse G genotypes may be necessary to achieve broad protection against LRV, a consideration relevant to both veterinary vaccine strategies and human rotavirus vaccine efficacy in regions where lapine-like strains circulate.

Co-infection Dynamics and Synergistic Pathogenesis

In commercial rabbitries, LRV rarely acts as a sole pathogen. Epidemiological studies from the Iberian Peninsula have demonstrated that in growing rabbits (post-weaning), co-infections between C. spiroforme, Eimeria spp., EPEC, and rotavirus are far more frequent than monoinfections [11]. The molecular basis for this synergism likely involves rotavirus-induced disruption of the intestinal epithelial barrier and mucin layer, facilitating bacterial adhesion and toxin-mediated damage. Conversely, pre-existing coccidial infection may immunosuppress the host, enhancing rotavirus replication. This polymicrobial pathogenesis complicates the attribution of clinical disease to LRV alone and necessitates comprehensive diagnostic approaches, such as the Luminex xMAP and x-TAG assays that have been developed for simultaneous detection of rabbit hemorrhagic disease virus, Sendai virus, and rabbit rotavirus [12, 13]. The high-throughput nature of these assays, with detection limits of 100 copies/μL and 100% concordance with conventional PCR, makes them invaluable for molecular epidemiological surveillance [13].

Zoonotic Potential and Public Health Implications

The molecular evidence for rabbit-to-human transmission of rotavirus is now unequivocal. The isolation of lapine-like G3P[1] strains from children with gastroenteritis in Belgium, with complete genome constellations indicative of lapine origin, demonstrates that LRV can cause clinical disease in humans [10]. Furthermore, the detection of bat-like G3 rotavirus genes in children from the Dominican Republic, with VP7 sequences showing ≥97% nucleotide identity to bat rotaviruses, highlights the role of diverse mammalian reservoirs in rotavirus evolution and the potential for spillover into human populations [9]. The close genetic relationship between lapine and bat rotavirus strains, as evidenced by the Korean Rab1404 strain sharing VP1-3 and VP7 genes with bat strain LZHP2 [7], suggests that rabbits may serve as intermediate or amplifying hosts for rotaviruses with zoonotic potential. This interconnected evolutionary network, involving rabbits, humans, bats, cattle, dogs, cats, and non-human primates, underscores the need for a One Health approach to rotavirus surveillance, as advocated by the WHO, WOAH, and the Food and Agriculture Organization (FAO). The molecular pathogenesis of rabbit rotavirus cannot be understood in isolation; it is a reflection of the broader ecology of rotaviruses in which rabbits serve both as reservoirs for lapine-adapted strains and as potential mixing vessels for reassortment events that generate novel viruses with unpredictable pathogenic properties.

Epidemiology and Inter-Species Transmission of Rabbit Rotavirus

Rotavirus species A (RVA) represents a significant etiological agent of acute gastroenteritis in both human infants and a wide array of animal species, with rabbits (Oryctolagus cuniculus) serving as a particularly instructive host for understanding viral evolution and cross-species dynamics. The epidemiology of lapine rotavirus (LRV) is characterized by high prevalence in commercial rabbitries, the circulation of distinct genotype constellations, and compelling evidence for both zoonotic and reverse-zoonotic transmission events. The segmented nature of the rotavirus genome, comprising 11 double-stranded RNA segments that encode six structural proteins (VP1–VP4, VP6, VP7) and six non-structural proteins (NSP1–NSP6), provides a molecular substrate for reassortment, a process that drives the emergence of novel strains capable of breaching species barriers. The World Organization for Animal Health (WOAH) has recognized rotavirus as a significant pathogen in livestock, and the European Centre for Disease Prevention and Control (ECDC) maintains surveillance for zoonotic rotavirus strains, underscoring the public health and agricultural importance of these transmission events.

Global Distribution and Prevalence in Rabbit Populations

Surveillance data from commercial rabbit farms consistently demonstrate that LRV is a widespread pathogen with substantial economic impact. A comprehensive diagnostic survey conducted across the Iberian Peninsula during 2018–2019 analyzed 757 clinical cases from rabbit enterprises, employing real-time PCR (qPCR) alongside bacteriological culture to identify enteropathogens [11]. This investigation revealed that rotavirus was frequently detected in growing rabbits (post-weaning animals) and was predominantly found in the context of polymicrobial infections, with coinfections between Clostridium spiroforme, Eimeria spp., enteropathogenic Escherichia coli (EPEC), and rotavirus being far more common than monoinfections [11]. The study documented that digestive disorders are the primary cause of economic damage in rabbit farming, and the frequent involvement of rotavirus in these multifactorial disease complexes necessitates complete laboratory diagnostics to inform targeted control measures [11]. Seroprevalence data from laboratory rabbit populations further corroborate the ubiquity of the virus; a high-throughput Luminex xMAP assay developed for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus, and rabbit rotavirus demonstrated 100% concordance with commercial ELISA kits across 52 clinical samples, establishing robust serological tools for ongoing surveillance [12]. A parallel Luminex x-TAG assay, targeting the same three pathogens, achieved a detection limit of 100 copies/μL for rotavirus and identified 18 positive specimens out of 40 clinical samples, with complete concordance to conventional PCR [13]. These findings collectively indicate that rotavirus circulation in both commercial and laboratory rabbit populations is extensive and that latent or subclinical infections may be more common than overt disease, a phenomenon with profound implications for biosecurity in research facilities.

Genotypic Diversity and Emerging Constellation Patterns

The genetic characterization of LRV strains has historically focused on the two outer capsid proteins, VP7 (defining the G genotype) and VP4 (defining the P genotype), which together determine serotype specificity and are primary targets for neutralizing antibodies. For decades, the most commonly reported combinations in rabbits were G3P[1] and G3P[2], but the application of complete genome sequencing has revealed far greater complexity [3, 10]. A landmark study from Italy characterized a G3P[1] LRV strain from a rabbit breeding farm experiencing recurring enteric disease in young animals; the complete genome analysis demonstrated a genotype constellation that was shared not only with other lapine G3P[1] strains but also with human G3P[1] isolates, suggesting a common ancestral lineage that has undergone cross-species transmission [3]. The G3 VP7 genotype is notably promiscuous, having been documented in humans, rabbits, pigs, rats, monkeys, and bats, which positions it as a critical node in the network of inter-species rotavirus transmission [5, 9].

The first report of a G3P[2] strain in China, isolate Z3171 from diarrheic rabbits, revealed a genotype constellation of G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3 that differed substantially from previously characterized LRV strains in terms of both gene content and sequence [4]. Notably, the genetic divergence between Z3171 and other lapine strains such as N5 and Rab1404 was so pronounced that the authors proposed either a reassortment event between human and rabbit rotaviruses or the circulation of undetected genotypes within rabbit populations [4]. Similarly, a Korean isolate, Rab1404 (G3-P[2]-I2-R3-C3-M3-A9-N2-T3-E3-H3), provided further evidence for complex evolutionary histories; phylogenetic analysis demonstrated that its VP1, VP3, and VP7 genes were closely related to those of a bat strain (LZHP2), while its NSP1–NSP4 genes clustered with the simian strain RRV, and its VP4, VP6, and NSP5 genes were most similar to other rabbit isolates [7]. The fact that eight of the eleven genome segments of Rab1404 showed a close relationship to the simian RVA strain RVA/Simian-tc/USA/RRV/1975/G3P[12], itself believed to be a reassortant between bovine-like and canine/feline strains, underscores the intricate web of host-switching events that shape LRV evolution [7]. Furthermore, the VP6 (I2), NSP1 (N2), and NSP5 (H3) segments of Rab1404 were closely related to those of bovine RVAs, indicating that the rabbit genome can incorporate segments from multiple divergent host species through successive reassortment events [7].

Direct Evidence for Zoonotic Transmission: Rabbit-to-Human Spillover

The most compelling evidence for zoonotic transmission of LRV comes from the isolation of a lapine-like G3P[1] strain from a two-year-old boy hospitalized with acute gastroenteritis in Belgium during the 2012–2013 rotavirus season [10]. Complete genome sequencing of this strain, designated BE5028, revealed a genotype constellation of G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3, which is entirely consistent with a lapine origin [10]. Phylogenetic analyses demonstrated that BE5028 was closely related to another lapine-like human strain, B4106, isolated from a Belgian child twelve years earlier, suggesting that a rabbit-derived rotavirus lineage had been circulating undetected in the human population for over a decade [10]. Several molecular features of BE5028 reinforced its lapine provenance: the NSP5 segment contained a head-to-tail partial duplication combined with two short insertions and a deletion, a genetic signature that is indicative of continuous circulation within rabbit populations and that was absent from contemporary human rotavirus strains [10]. Additionally, the NSP3 segment of BE5028 exhibited a bovine-like origin (genotype T6), likely introduced into the lapine RVA population through reassortment within the past twelve years, further demonstrating the fluid exchange of genome segments among species [10]. This case provides unequivocal evidence that rabbits can serve as a reservoir for human rotavirus disease and that the resulting infections can cause clinically significant gastroenteritis in immunocompetent children.

Reverse Zoonosis and the Detection of Human Genotypes in Rabbits

The bidirectional nature of inter-species transmission is exemplified by the identification of a G3P[9] rotavirus strain from an outbreak of enteritis in a commercial rabbitry in Mexico [5]. This genotype combination is remarkable because P[9] has historically been considered exclusively human; it is the most common P genotype circulating in human populations globally and is a component of all licensed rotavirus vaccines, including RotaTeq and Rotarix [5, 20]. The Mexican strain, C-3/15, was confirmed by sequence and phylogenetic analysis to possess VP7 and VP4 genes that were closely related to human rotaviruses rather than to established lapine strains [5]. This finding represents the first report of a P[9] genotype in rabbits and strongly suggests a human-to-rabbit transmission event, followed by adaptation and subsequent spread within the rabbit population to cause an outbreak of enteric disease [5]. This scenario is epidemiologically plausible given the close contact between humans and domestic rabbits in commercial settings and highlights the potential for human rotavirus vaccine strains or circulating wild-type strains to establish themselves in animal reservoirs, with implications for vaccine efficacy and viral evolution.

Cross-Species Infectivity and Host Range Determinants

Experimental evidence from controlled infection studies has provided additional confirmation of the broad host range of rotavirus and the susceptibility of rabbits to heterologous strains. In a comprehensive investigation of neonatal calf diarrhea, Castrucci and colleagues demonstrated that conventionally reared calves were fully susceptible to experimental infection induced by rotaviruses originating from monkeys (simian), pigs (porcine), and rabbits (lapine) [2]. Cross-neutralization tests performed in this study revealed that the simian and porcine strains were antigenically indistinguishable from each other and both shared antigenic relatedness with the bovine strain, while a reciprocal antigenic correlation was found between bovine and rabbit isolates [2]. These findings establish that rabbits can be experimentally infected with rotaviruses from multiple other species and that the immune responses generated by such infections are cross-reactive across host boundaries. Furthermore, the study demonstrated that feeding newborn calves with colostrum from dams vaccinated with an inactivated rotavirus vaccine could prevent neonatal diarrhea, indicating that passive immunity can protect against heterologous challenge [2]. This has practical implications for rabbit farming, suggesting that vaccination strategies might be developed to protect young kits from circulating lapine and potentially human-derived rotavirus strains.

The structural basis for cross-neutralization was explored in a detailed study using rabbit and equine monospecific antisera against simian SA11 G3P[4] and equine G3P[19] and G14P[19] rotaviruses [18]. This work demonstrated that rabbit antisera raised against equine G3P[19] could cross-neutralize simian G3P[4] and, interestingly, that rabbit antisera to equine G14P[19] provided more robust cross-protection against SA11 than the reverse [18]. The study identified multiple highly solvent-exposed amino acid residues conserved in VP7 and VP8* among these three viruses, suggesting that these residues serve as novel B cell epitopes driving cross-neutralization [18]. Importantly, the breadth and magnitude of cross-neutralization were broader and stronger in equine antisera than in rabbit antisera, indicating that the immunological context of the host species modulates the neutralization response [18]. These findings emphasize that the determinants of cross-species neutralization are complex and context-dependent, involving both the specific viral context and the animal species generating the immune response.

Rabbits as Reservoirs and the Role of Reassortment in Viral Emergence

The detection of bat-like rotavirus genes in children hospitalized with gastroenteritis in the Dominican Republic adds another dimension to the inter-species transmission network in which rabbits may participate. Among fifteen stool samples from children under one year of age, three strains possessed VP7 genes that were ≥97% identical to those of G3 bat rotaviruses detected in Bulgaria in 2008, but were more distantly related (≤92%) to G3 rotaviruses from humans, rabbits, pigs, rats, and monkeys [9]. Four of ten available VP6 sequences, including the three from the G3 bat-like strains, showed >97% nucleotide identity with I2 genotype VP6 from a Kenyan bat rotavirus [9]. This study suggests that bats serve as a reservoir for rotavirus genes that can ultimately appear in human infections, and the close genetic relationship of certain bat G3 sequences to rabbit G3 sequences raises the possibility that rabbits could act as intermediate hosts bridging bat and human rotavirus populations [9]. The rabbit population may thus function as a mixing vessel in which reassortment events occur, generating novel strains with pandemic potential.

The capacity for reassortment is profoundly influenced by the biomechanics of rotavirus replication. The RNA-binding protein NSP2 (NS35) forms 10S multimers that interact with the viral RNA polymerase VP1 in the cytoplasm, coordinating RNA packaging and core assembly [16]. These NSP2 multimers exhibit RNA-binding activity and form complexes with VP1 that are likely essential for incorporating the 11 distinct genomic segments into progeny virions [16]. The efficient assembly of VP6 into trimeric units, a process that requires a domain between amino acid residues 105 and 328 for trimerization and a C-terminal domain for binding to single-shelled particles, ensures that newly synthesized viral proteins can be rapidly incorporated into infectious particles [15]. This structural flexibility at the protein level may facilitate the accommodation of heterologous genome segments during reassortment, allowing viruses to quickly adapt to new hosts. The demonstration that rabbit reticulocyte lysates can support the in vitro translation and assembly of rotavirus proteins from multiple strains further confirms that the molecular machinery within rabbit cells is permissive for rotavirus replication across species boundaries [14, 16, 15].

Perspectives on Surveillance and One Health Implications

The cumulative evidence from molecular epidemiology, experimental infection studies, and serological surveys positions rabbit rotavirus as a dynamic and ecologically significant pathogen. The repeated documentation of lapine-like human strains (G3P[1]), human-like lapine strains (G3P[9]), and strains with gene segments from bovine, simine, canine, feline, and bat origins indicates that rabbits are not merely passive recipients of infection but active participants in the global rotavirus gene pool. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) have emphasized the importance of integrated human-animal-environment surveillance for emerging zoonotic pathogens, and the rotavirus system exemplifies the need for such a One Health approach. Serological assays based on recombinant VP6 antigen, which is highly conserved across group A rotaviruses, have proven effective for detection across species and can be deployed in large-scale surveillance efforts [8, 1]. The availability of rapid, high-throughput multiplex assays for detecting rotavirus antibodies and nucleic acids in rabbit populations now makes routine monitoring feasible, even in resource-limited settings [12, 13]. Given the economic importance of rabbit farming, particularly in Mediterranean countries, China, and Korea, and the potential for zoonotic spillover, the implementation of systematic rotavirus surveillance in rabbitries should be considered a priority for both agricultural sustainability and public health.

Diagnostic Approaches for Rabbit Rotavirus

The accurate and timely diagnosis of rabbit rotavirus (LRV) infection is a multifaceted endeavor, necessitating a spectrum of techniques ranging from classical virology to advanced molecular and serological platforms. Given the virus’s capacity for interspecies transmission, its frequent role in complex enteric disease syndromes, and the economic implications for commercial rabbitries, a robust diagnostic framework is paramount. The diagnostic toolkit for LRV is not monolithic; rather, it must be strategically deployed based on the clinical question, whether the goal is rapid pathogen detection in an outbreak, comprehensive genotyping for epidemiological surveillance, or serological screening for herd health management in laboratory animal facilities. This section provides an exhaustive analysis of the diagnostic approaches currently available, critically evaluating their mechanisms, applications, and limitations within the context of lapine rotavirology.

Molecular Diagnostics: The Cornerstone of Detection and Genotyping

The advent of molecular biology has revolutionized the detection of LRV, offering unparalleled sensitivity and specificity compared to traditional methods. Reverse transcription polymerase chain reaction (RT-PCR) in its various formats has become the gold standard for identifying rotavirus RNA in fecal samples and intestinal contents.

Conventional and Real-Time RT-PCR

The detection of rotavirus Group A (RVA) by RT-PCR typically targets highly conserved regions of the genome, most commonly the VP6 gene, which encodes the inner capsid protein and serves as the group-specific antigen [8, 1]. The VP6 protein is a robust target due to its abundant expression and antigenic stability, making it ideal for both nucleic acid and immunological detection [8]. The use of real-time quantitative PCR (qPCR) further enhances this approach, allowing for quantification of viral load and providing data on the severity of infection. The incorporation of multiplex qPCR panels has proven essential in rabbit medicine, as enteric disease is rarely monomicrobial. Solans et al. demonstrated this effectively in a large-scale epidemiological study across the Iberian Peninsula, employing a qPCR panel to simultaneously detect enteropathogenic Escherichia coli (EPEC), Clostridium spiroforme, C. perfringens, rotavirus A, Bacteroides fragilis, and Eimeria spp. [11]. This study revealed that coinfections involving rotavirus and bacterial or parasitic agents are the norm rather than the exception in growing rabbits, underscoring the absolute necessity of a comprehensive diagnostic panel rather than single-pathogen testing [11]. Such molecular surveillance, as advocated by the World Organisation for Animal Health (WOAH), is critical for implementing effective on-farm biosecurity and treatment protocols.

Genotyping and Full-Genome Sequencing

Beyond mere detection, molecular techniques are indispensable for characterizing the genetic diversity and evolutionary dynamics of LRV. Genotyping of the outer capsid proteins VP7 (G-types) and VP4 (P-types) is fundamental to understanding strain epidemiology and zoonotic potential. Historically, the predominant lapine genotypes have been G3P[1] and G3P[2] [3, 10, 7]. However, molecular diagnostics have unveiled a far more complex picture, identifying novel and reassortant strains that challenge our understanding of host range and transmission. Studies have now identified G3P[9] strains in Mexican rabbits that are closely related to human viruses, suggesting direct interspecies transmission [5]. Similarly, a G3P[2] strain from Korea (Rab1404) exhibited a mosaic genome, with gene segments closely related to simian, bovine, and canine/feline rotaviruses, indicating multiple reassortment events [7]. Full-genome sequencing, as performed by Omar et al. on an Italian G3P[1] strain and Zhao et al. on a Chinese G3P[2] strain (Z3171), provides the ultimate resolution, revealing the complete genotype constellation (e.g., G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3 for Z3171) [3, 4]. These analyses are not merely academic; they are critical for tracing the origins of outbreaks, assessing the risk of zoonotic spillover, a concern highlighted by the detection of a lapine-origin G3P[1] strain causing gastroenteritis in a human infant in Belgium [10], and monitoring the emergence of vaccine escape mutants. The complexity of LRV genetics, with evidence of reassortment involving bat-like rotaviruses [9], necessitates that reference laboratories employ advanced sequencing techniques for comprehensive surveillance.

Immunological and Serological Assays

While molecular methods excel at detecting active infection, serological and immunological assays provide critical information on past exposure, immune status, and antigen presence, offering complementary diagnostic capabilities.

Antigen Detection: ELISA and Lateral Flow

Enzyme-linked immunosorbent assays (ELISAs) remain a mainstay for the direct detection of rotavirus antigens in fecal samples. The target antigen is typically the highly immunogenic VP6 protein. The production of high-quality antibodies is central to this approach. Nga demonstrated that recombinant VP6 protein expressed in E. coli is highly immunogenic in rabbits, yielding polyclonal antibodies of sufficient purity and concentration (3.94 mg/ml serum) for diagnostic kit development [8]. Similarly, Zhu et al. developed a discrimination ELISA using rabbit anti-VP6 polyclonal antiserum to successfully differentiate porcine rotavirus from other swine enteric viruses, a principle directly applicable to the development of specific LRV diagnostic tests [1]. More recently, a double-antibody sandwich quantitative ELISA (DAS-qELISA), using rabbit polyclonal antibodies as capture agents, has been established for detecting porcine epidemic diarrhea virus, with a detection limit of 0.05 ng/mL [21]. This format could be readily adapted for LRV VP6 detection, offering a sensitive and quantitative antigen test that circumvents the need for cell culture.

Lateral flow immunoassays (rapid immunochromatographic strips) offer a point-of-care format for antigen detection. The polyclonal antibodies against VP6 developed by Nga were explicitly noted to be suitable for such rapid test development [8]. These tests are invaluable for field diagnostics in commercial rabbitries where laboratory infrastructure is limited.

Serological Surveillance: ELISA and High-Throughput Platforms

Monitoring antibody levels in rabbit populations is crucial for assessing herd immunity and confirming freedom from infection in specific pathogen-free (SPF) colonies. Traditional serological methods like ELISA are effective, but recent innovations have enabled high-throughput, multiplexed serology. Wu et al. developed a Luminex xMAP assay that simultaneously detects antibodies against rabbit hemorrhagic disease virus, Sendai virus, and rabbit rotavirus in a single serum sample [12]. This assay uses recombinant VP6 protein as the coating antigen for rotavirus detection and demonstrates high specificity, with no cross-reactivity, and excellent reproducibility (intra-assay CV less than 3%). When validated against 52 clinical samples, the Luminex assay showed 100% coincidence with commercial ELISA kits [12]. While the xMAP assay had a slightly lower limit of detection for rotavirus compared to the ELISA, its ability to process hundreds of samples rapidly and cost-effectively makes it an ideal tool for routine monitoring in large laboratory animal facilities, aligning with international standards for laboratory animal health.

Hypothetically, given the importance of cross-neutralization in vaccine design and the demonstration that rabbit antisera can be used to characterize rotavirus neutralizing epitopes [18], neutralization assays remain a key serological tool for functional antibody assessment. However, these are labor-intensive and not suitable for high-throughput screening, highlighting the role of binding assays like xMAP for routine surveillance and neutralization assays for specialized research.

Classical Virological and Ultrastructural Techniques

Before the molecular era, classical techniques like electron microscopy (EM) and virus isolation were the primary diagnostic tools.

Immune Electron Microscopy

Direct examination of fecal samples by negative-stain EM can reveal the characteristic 55-70 nm wheel-like morphology of rotavirus particles [6]. Immune electron microscopy (IEM) significantly enhances sensitivity and specificity. In a classic study, Saif et al. showed that incubating fecal samples with convalescent antiserum caused virus particles to aggregate into clumps, which were far easier to detect. The sensitivity could be increased ten-fold by using an indirect IEM approach, where rabbit anti-porcine IgG was added to further aggregate the primary virus-antibody complexes [6]. While this technique provides definitive visual confirmation of the virus and allows for assessment of particle integrity, it requires expensive equipment, highly skilled personnel, and is not practical for large-scale surveillance. Its role today is largely confined to research settings.

Virus Isolation and Cell Culture

Isolation of LRV in cell culture remains a challenging but important technique for obtaining high-titer virus stocks for research, vaccine development, and phenotypic characterization. The gold standard cell line is MA104, a line derived from fetal rhesus monkey kidney cells. However, not all field strains of LRV adapt readily to culture. The development of reverse genetics systems, which are still in their infancy for rotaviruses, aims to overcome such limitations. Richards et al. attempted to recover infectious rotavirus by co-transfecting synthetic full-length single-stranded RNAs in permissive cells but found that these RNAs were not infectious, highlighting the technical hurdles in manipulating the rotavirus genome [14]. Despite these challenges, successful isolation and adaptation of strains like the Korean Rab1404 and Chinese Z3171 are crucial for detailed laboratory characterization [4, 7]. Virus isolation, combined with cross-neutralization tests using polyclonal antisera raised in rabbits, has been used to demonstrate antigenic relationships between isolates from different host species, confirming the potential for cross-species transmission [2].

Diagnostic Algorithm and Contextual Considerations

Given the complexities of rabbit enteric disease, a tiered diagnostic approach is recommended. For initial clinical diagnosis of an outbreak, rapid antigen detection via commercial ELISA or lateral flow strips, combined with a qPCR panel for other major enteric pathogens (e.g., C. spiroforme, Eimeria spp., EPEC) [11], provides the fastest route to a diagnosis. For epidemiological surveillance, especially in breeding herds or SPF colonies, high-throughput serological screening using platforms like Luminex xMAP is optimal [12, 13]. For research and for investigating the origin of novel strains causing severe disease or suspected zoonotic events, full-genome sequencing is mandatory [3, 4, 10, 7]. The detection of unusual genotypes such as G3P[9] or those with bat-like gene segments [5, 9] in both human and lapine populations dictates that diagnostic laboratories remain vigilant and extend their genotyping efforts beyond the historical G3P[1] and G3P[2] stereotypes. The continuous evolution of LRV through reassortment, as documented globally from Italy to Korea, demands a dynamic and molecularly sophisticated diagnostic infrastructure to safeguard both animal and public health.

Clinical Significance and Disease Management

The Clinical Spectrum of Rabbit Rotavirus Infection

The clinical significance of rabbit rotavirus (LRV) infection extends far beyond a simple case of neonatal diarrhea. In commercial rabbitries, LRV is a primary driver of economic losses due to mortality, reduced weight gain, and the considerable costs associated with therapeutic interventions and biosecurity measures. The disease primarily manifests as acute gastroenteritis in young rabbits, typically between 15 and 35 days of age, a period coinciding with the waning of maternal antibody protection and the stresses of weaning [11]. Infections are rarely monomorbid; rather, LRV is most frequently encountered as a component of a complex, multifactorial enteropathy. Epidemiological surveys from the Iberian Peninsula, a major European rabbit-producing region, have demonstrated that co-infections involving Clostridium spiroforme, enteropathogenic Escherichia coli (EPEC), Eimeria spp., and rotavirus are the rule rather than the exception in growing rabbits [11]. This syndromic complexity is the defining clinical challenge. A rabbit presenting with diarrhea due to LRV may simultaneously be harboring a clostridial enterotoxemia and a coccidial burden, creating a synergistic pathology that is far more severe than any single agent could produce alone. The virus itself causes villous atrophy in the small intestine, disrupting absorptive capacity and leading to osmotic diarrhea. The mechanistic underpinning of this diarrhea is partially attributed to the non-structural protein NSP4, which has been demonstrated to act as an enterotoxin. While early in vitro work suggested that the NSP4[114-135] peptide could directly inhibit chloride transport in brush-border membranes, more nuanced investigations using rabbit models have shown that this peptide does not exert a direct, specific effect on chloride absorption or secretion in isolated villus cells [17]. Instead, the current evidence strongly supports a model whereby NSP4 triggers signal transduction cascades, likely involving intracellular calcium mobilization, that ultimately drive net chloride secretion and fluid loss, a mechanism that aligns with the pathophysiology observed in human and other mammalian rotavirus infections [17].

Diagnostic Challenges and the Imperative for Differentiated Detection

Accurate diagnosis of LRV is non-negotiable for effective disease management, yet it is fraught with pitfalls. Clinical signs are non-specific; a diarrheic rabbit with LRV is clinically indistinguishable from one with EPEC, coccidiosis, or clostridial enteritis. Reliance on clinical presentation alone is a recipe for therapeutic failure. Therefore, laboratory confirmation is essential. The World Organisation for Animal Health (WOAH) recognizes rotavirus as a significant pathogen in livestock, and standardized diagnostic approaches are critical. Traditional methods include electron microscopy, which is now largely superseded by molecular techniques. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) offer high sensitivity and specificity for detecting viral RNA in fecal samples or intestinal contents [11]. However, the landscape of diagnostic technology is evolving. High-throughput serological surveillance, once confined to ELISA, has been revolutionized by multiplex platforms. The Luminex xMAP assay, for instance, allows for the simultaneous detection of antibodies against rabbit hemorrhagic disease virus (RHDV), Sendai virus, and rotavirus from a single serum sample, offering a coefficient of variation for intra-assay and inter-assay comparisons of less than 3% and 4%, respectively, and demonstrating 100% concordance with commercial ELISA kits [12]. Furthermore, the Luminex x-TAG assay, based on multiplex PCR and fluorescent microsphere hybridization, provides a rapid, high-throughput method for detecting the viral nucleic acids of these same three pathogens directly from clinical specimens, with a detection limit of 100 copies/μL and excellent agreement with conventional PCR [13]. For laboratories seeking cost-effective, point-of-care solutions, the development of immunological tests targeting the VP6 protein, the highly conserved inner capsid antigen, represents a promising avenue. Polyclonal antibodies generated against recombinant VP6 have proven to be highly efficient and specific, and are suitable for incorporation into lateral flow immunoassay (rapid test) formats [8]. The clinical significance of robust diagnostics cannot be overstated: only by definitively identifying LRV and its co-pathogens can a veterinarian move from empiric, shotgun therapy to targeted, rational disease management.

The Zoonotic Dimension: A Growing Concern

Perhaps the most clinically alarming aspect of rabbit rotavirus is its documented zoonotic potential. The classification of LRV is no longer a simple matter of lapine strains circulating solely within Oryctolagus cuniculus. Genomic surveillance has revealed a tangled web of interspecies transmission, reassortment, and adaptation that directly impacts public health. The most common and historically appreciated lapine genotypes are G3P[1] and G3P[2] [3, 10, 7]. However, the emergence of strains such as the Mexican C-3/15 isolate, which possesses a G3 P[9] genotype, a combination previously exclusive to human rotaviruses, is a stark indicator of the fluidity of the species barrier [5]. The P[9] genotype is the most prevalent human VP4 type globally, and its detection in a rabbit with enteric disease strongly suggests a human-to-rabbit transmission event [5]. Conversely, there is unequivocal evidence of rabbit-to-human transmission causing clinical disease. The Belgian BE5028 strain, isolated from a two-year-old boy with gastroenteritis, was found to possess a complete lapine genotype constellation (G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3), proving that a rabbit rotavirus can cause significant disease in a human infant [10]. This is not an isolated incident; the B4106 strain, also from a Belgian child and isolated 12 years prior, demonstrated a similar lapine origin, indicating that such zoonotic events may be recurring periodically [10]. Furthermore, complex reassortment events are the norm. The Korean Rab1404 strain (G3P[2]) exhibited a mosaic genome, with genes closely related to simian (RRV), bovine, and canine/feline rotaviruses, suggesting that the rabbit is a mixing vessel for rotavirus strains from multiple mammalian species [7]. Similarly, Chinese strains like Z3171 (G3P[2]) carry a constellation suggesting reassortment between human and rabbit strains [4]. The clinical significance of this for disease management is profound. Veterinarians and rabbit farmers must be educated on the zoonotic risk, particularly for immunocompromised individuals and young children who may have contact with infected rabbits. The CDC and WHO both emphasize the role of animal reservoirs in rotavirus ecology, and the presence of "bat-like" G3 and I2 genotypes in children, which share high identity with strains found in rabbits, further underscores the interconnectedness of rotavirus circulation across species [9]. Disease management, therefore, must integrate a One Health approach, where controlling LRV in rabbitries is not just an agricultural issue but a public health imperative.

Strategic Disease Management: Biosecurity, Supportive Care, and Vaccine Development

Managing rabbit rotavirus disease in a commercial setting requires a multi-pronged strategy that prioritizes prevention and early intervention. There is no licensed, specific antiviral therapy for LRV. Management, therefore, hinges on three pillars: robust biosecurity, aggressive supportive care, and the strategic development of vaccines. Biosecurity is the first and most critical line of defense. Because LRV is highly contagious and environmentally stable, all-in/all-out production systems are strongly recommended to break the cycle of infection. Thorough cleaning and disinfection of hutches and equipment between batches is essential; rotaviruses are resistant to many common disinfectants, requiring the use of virucidal agents such as accelerated hydrogen peroxide or chlorine-based compounds. Quarantine protocols for new stock and stringent visitor hygiene are non-negotiable.

When outbreaks occur, the clinical management of affected kits is primarily supportive. Dehydration is the primary cause of death. Affected rabbits should be provided with easy access to fresh, clean water. In severe cases, oral or subcutaneous fluid therapy with isotonic electrolyte solutions may be necessary, though this is logistically challenging in large rabbitries. Nutritional support with highly digestible, low-residue feeds can help reduce osmotic diarrhea while the intestinal epithelium regenerates. The use of antibiotics is generally contraindicated for a purely viral infection, but given the prevalence of co-infections with EPEC and C. spiroforme, a veterinarian may judiciously prescribe antimicrobials if bacterial involvement is confirmed by laboratory diagnostics [11]. Attempting to treat diarrhea empirically with antibiotics without a confirmed bacterial etiology risks exacerbating dysbiosis and selecting for antimicrobial resistance. The World Health Organization (WHO) has long warned against the overuse of antibiotics in food animal production, and rabbitries are no exception.

Vaccination represents the most promising long-term strategy for control, but it remains a work in progress. No commercial rabbit rotavirus vaccine is currently widely available. However, the path forward is illuminated by research in other species and on the molecular biology of the virus. The simian rotavirus SA11 strain (G3P[4]) has been used in equine vaccines to protect foals against G3P[19] and G14P[19] equine rotaviruses, demonstrating that cross-neutralization across G genotypes is achievable [18]. Crucially, rabbit antisera raised against an equine G14P[19] rotavirus provided more robust cross-protection against SA11 than the reverse, indicating that the VP7 protein (which defines the G genotype) may be a more important determinant of broad neutralization than previously thought [18]. This finding suggests that a vaccine targeting a single, broadly representative G3 lapine strain might confer protection against a range of G3 variants. However, the discovery of P[2] genotypes [7] and the emergence of human-like P[9] strains in rabbits [5] complicate this picture. An effective vaccine may need to be multivalent, incorporating both G3 and multiple P types. The structural and functional understanding of VP6, the inner capsid protein, also offers a tantalizing target. VP6 is highly conserved and is the target of the immune electron microscopy and ELISA diagnostics developed for swine and rabbit rotaviruses [1, 6]. Anti-VP6 antibodies are not typically neutralizing, but they are highly immunogenic and could be leveraged for passive immunization strategies. For example, feeding newborn rabbits with colostrum or milk from does vaccinated with an inactivated rotavirus vaccine, a strategy proven effective in preventing neonatal calf diarrhea [2], could provide immediate, passive protection to kits during the critical first weeks of life. The development of a reverse genetics system for rotaviruses, while still technically challenging [14], holds the eventual promise of creating rationally attenuated, stable vaccine strains tailored specifically to the complex lapine rotavirus landscape. Until such vaccines are realized, the cornerstone of clinical management remains a combination of rigorous biosecurity, accurate molecular diagnosis, and supportive care that addresses the multifactorial nature of rabbit enteritis.

Evolutionary Dynamics and Host Range

The evolutionary trajectory of rabbit rotavirus (RRV) is a paradigm of the complex interplay between host adaptation, interspecies transmission, and genomic reassortment that characterizes group A rotaviruses (RVA) globally. As a pathogen of Oryctolagus cuniculus, RRV occupies a unique ecological niche, yet its genetic architecture reveals a history of repeated incursions from and into other mammalian reservoirs, including humans, bovines, simians, canines, felines, and potentially bats. Understanding these dynamics is not merely an academic exercise; it is fundamental to predicting emergent zoonotic risks, designing effective veterinary vaccines, and interpreting the evolutionary pressures that shape rotavirus diversity in agricultural and laboratory settings. The World Organisation for Animal Health (WOAH) recognizes rotavirus as a significant cause of neonatal diarrhea in livestock, and the Centers for Disease Control and Prevention (CDC) has highlighted the role of animal reservoirs in the global ecology of rotavirus disease, underscoring the public health and agricultural importance of these evolutionary processes.

Genotypic Diversity and the Lapine Core Constellation

The foundation of RRV evolutionary studies lies in the characterization of its genotype constellation, a full-genome classification system that assigns a specific number to each of the 11 gene segments (Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx). For decades, the canonical lapine genotype constellation was considered to be G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3, as exemplified by strains such as B4106 and the Belgian human isolate BE5028 [10]. However, comprehensive genomic surveillance has revealed a far more heterogeneous landscape. The identification of a G3P[2] strain (Z3171) in China with the constellation G3-P[2]-I2-R3-C3-M3-A9-N2-T1-E3-H3 demonstrated that the lapine backbone is not fixed [4]. This constellation differed from the prototypical lapine pattern in six of the 11 segments, including the VP1 (R3 vs. R2), VP2 (C3 vs. C2), VP3 (M3 conserved), NSP2 (T1 vs. T6), NSP3 (E3 vs. E5), and NSP4 (H3 conserved) genes [4]. Similarly, the Korean strain Rab1404 exhibited a constellation of G3-P[2]-I2-R3-C3-M3-A9-N2-T3-E3-H3, which shared only the VP6 (I2), NSP1 (A9), and NSP5 (H3) segments with the classic lapine pattern, while the remaining eight segments showed close phylogenetic affinity to simian, bovine, or canine/feline strains [7]. These data indicate that the "lapine genotype" is not a monophyletic entity but rather a dynamic assemblage of segments that can be exchanged through reassortment, creating novel constellations that may possess altered host range or virulence properties.

The P[1] and P[2] genotypes are considered the archetypal lapine VP4 types, yet their evolutionary origins are distinct. P[1] is a relatively rare genotype in the global rotavirus population, found predominantly in rabbits and humans, suggesting a history of cross-species transmission and subsequent adaptation [3, 10]. In contrast, P[2] appears to be more strictly associated with rabbits, although its detection in China and Korea indicates a wide geographic distribution within the lapine host [4, 7]. The VP7 gene, almost universally G3 in rabbits, is itself a promiscuous genotype found in humans, pigs, monkeys, cats, dogs, and bats [5, 9]. This ubiquity of G3 facilitates reassortment, as the outer capsid protein VP7 is a major target of neutralizing antibodies, and its exchange can allow a virus to escape pre-existing immunity in a new host population. The presence of the I2 VP6 genotype in virtually all lapine strains is a notable constant, as I2 is also common in bovine and some human rotaviruses, suggesting a deep evolutionary link between these host species at the level of the inner capsid [10, 7].

Interspecies Transmission: A Two-Way Street

The most compelling evidence for the dynamic host range of RRV comes from documented interspecies transmission events, which have occurred in both directions: from rabbits to humans and from humans (or other mammals) to rabbits. The isolation of a G3P[1] strain (BE5028) from a two-year-old Belgian child with gastroenteritis in 2012 represents a clear case of rabbit-to-human transmission [10]. Phylogenetic analysis of all 11 gene segments of BE5028 revealed a genotype constellation (G3-P[1]-I2-R2-C2-M3-A9-N2-T6-E5-H3) that was nearly identical to that of a lapine-like strain (B4106) isolated from a Belgian child 12 years earlier, indicating that this particular lapine lineage had been circulating in the human population for over a decade [10]. Crucially, the NSP3 segment of BE5028 was of bovine-like origin (T6), while the NSP5 segment contained a head-to-tail partial duplication with two short insertions and a deletion, a genetic hallmark of continuous circulation within the rabbit population [10]. This suggests that the virus had undergone reassortment with a bovine rotavirus after its initial introduction into rabbits, and then subsequently transmitted to a human child. This finding has profound implications for public health, as it demonstrates that lapine rotaviruses can cause clinical disease in humans and that the rabbit population can serve as a mixing vessel for rotavirus genes from different host species, potentially generating novel human-pathogenic strains.

The converse scenario, human-to-rabbit transmission, has been documented with even greater clarity. The identification of a G3P[9] rotavirus (strain C-3/15) in a diarrheic rabbit from a Mexican commercial rabbitry is a landmark finding, as P[9] is the most common VP4 genotype in human rotaviruses worldwide but had never before been reported in rabbits [5]. Both the VP7 and VP4 genes of C-3/15 were closely related to human, not lapine, rotaviruses, strongly suggesting a direct interspecies transmission event from a human to a rabbit [5]. The fact that this virus was associated with an outbreak of enteritis in the rabbitry indicates that the human-derived strain was capable of replicating efficiently in the lapine host and causing disease, a prerequisite for establishment in a new reservoir. This event is particularly concerning from a One Health perspective, as it implies that human rotavirus strains, which are under strong selective pressure from widespread vaccination programs (e.g., RotaTeq and Rotarix, recommended by the WHO for universal infant immunization), could potentially be introduced into rabbit populations and establish new enzootic cycles. The close contact between humans and domestic rabbits in both commercial and pet settings provides ample opportunity for such spillover events.

Reassortment as a Driver of Genomic Plasticity

Reassortment, the exchange of entire gene segments during co-infection of a single cell with two different rotavirus strains, is the primary engine of rotavirus evolution, and RRV is no exception. The genomic analyses of lapine strains from diverse geographic regions reveal a patchwork of segment origins that can only be explained by multiple, sequential reassortment events. The Korean strain Rab1404 is a striking example: its VP1, VP2, VP3, and VP7 genes were closely related to those of a bat rotavirus (LZHP2); its NSP1, NSP2, NSP3, and NSP4 genes were related to the simian strain RRV; and its VP4, VP6, and NSP5 genes were of lapine origin [7]. This mosaic genome suggests that Rab1404 is the product of at least two independent reassortment events: one involving a bat-like rotavirus and another involving a simian-like rotavirus, both superimposed on a lapine backbone. The simian strain RRV itself is believed to be a reassortant between bovine-like and canine/feline rotaviruses, further complicating the evolutionary history [7]. This degree of genomic chimerism indicates that rabbits are not isolated evolutionary dead-ends for rotaviruses but rather active participants in a broader network of gene flow that connects bats, primates, bovines, and carnivores.

The Chinese strain Z3171 provides another illustration of reassortment-driven evolution. While its VP7 (G3) and VP4 (P[2]) were typical of lapine strains, its internal gene segments (R3, C3, M3, A9, N2, T1, E3, H3) were more closely related to human rotavirus strains than to other lapine strains [4]. The authors of that study proposed two possible explanations: either a reassortment event occurred between a human and a rabbit rotavirus, or there are undetected genotypes circulating in the rabbit population that are more closely related to human strains [4]. The former hypothesis is more parsimonious and is supported by the known propensity for rotaviruses to reassort across species barriers. The detection of a G3P[1] strain in an Italian rabbit farm with a genotype constellation shared with other G3P[1] strains from both rabbits and humans further reinforces the notion that the lapine and human rotavirus gene pools are not completely segregated [3]. The World Health Organization (WHO) has emphasized the importance of monitoring such reassortant strains, as they can lead to the emergence of viruses with pandemic potential.

The Role of Bats and Other Reservoirs in the Evolutionary Network

The discovery of bat-like rotavirus genes in lapine strains has opened a new frontier in understanding the evolutionary origins of RRV. Bats are increasingly recognized as major reservoirs for a wide variety of zoonotic viruses, and rotaviruses are no exception. The VP7 genes of three rotavirus strains from hospitalized children in the Dominican Republic showed ≥97% nucleotide identity to bat rotaviruses of the G3 genotype detected in Bulgaria, and these same strains also possessed VP6 genes of the I2 genotype that were highly similar to bat rotaviruses from Kenya [9]. While this study focused on human infections, the presence of bat-like G3 and I2 genes in human rotaviruses suggests that bats may be a source of genetic material that can eventually find its way into rabbits. The Korean Rab1404 strain, with its bat-like VP1-3 and VP7 genes, provides a direct link between the bat rotavirus reservoir and the lapine host [7]. It is plausible that an intermediate host, such as a rodent or a non-human primate, served as a bridge between bats and rabbits, but the exact pathway remains to be elucidated. The Food and Agriculture Organization of the United Nations (FAO) has called for enhanced surveillance of rotaviruses in wildlife, particularly bats, to better understand the zoonotic risks they pose to both livestock and humans.

Cross-Species Serological Relationships and Host Barriers

The ability of RRV to cross species barriers is not merely a genetic phenomenon but is also reflected in serological cross-reactivity and cross-protection studies. Early work using cross-neutralization tests demonstrated a reciprocal antigenic correlation between bovine and rabbit rotavirus isolates, indicating that these two host species share conserved neutralizing epitopes [2]. Furthermore, conventionally reared calves were fully susceptible to experimental infection with rotaviruses of rabbit origin, confirming that lapine strains can replicate and cause disease in a heterologous host [2]. More recent studies using monospecific rabbit antisera have revealed that cross-neutralization between different G and P genotypes is complex and context-dependent. For example, rabbit antisera raised against simian rotavirus SA11 (G3P[4]) and equine rotavirus G3P[19] neutralized each other well, but showed less cross-neutralization against equine G14P[19] [18]. Interestingly, rabbit antisera to equine G14P[19] provided more robust cross-protection against SA11 than the reverse, suggesting that the G genotype (VP7) may play a more dominant role in determining neutralization specificity than previously appreciated [18]. These findings have direct implications for vaccine development, as they suggest that a vaccine based on a single G3 genotype may not provide adequate protection against all lapine rotavirus strains, particularly if they have acquired novel VP7 genes through reassortment.

The host range of RRV is also constrained by intrinsic factors such as the availability of specific cellular receptors and the compatibility of viral replication machinery with host cell factors. The VP4 protein, which mediates attachment to sialic acid receptors and subsequent entry, is a key determinant of host range. The P[1] and P[2] genotypes likely recognize specific glycan receptors on rabbit intestinal epithelial cells that are distinct from those used by human P[9] or bovine P[3] strains. However, the successful infection of a rabbit with a human P[9] strain in Mexico [5] indicates that these barriers are not absolute and can be overcome, perhaps through mutations in the VP8* domain of VP4 that alter receptor specificity. The NSP4 protein, which functions as an enterotoxin and is involved in the pathogenesis of diarrhea, may also contribute to host adaptation. The NSP4 of lapine strains, such as those used in studies of chloride transport in rabbit brush-border membranes, has been shown to have specific effects on ion transport that may be optimized for the rabbit intestine [17]. The acquisition of a bovine-like NSP3 segment in the Belgian human lapine strain BE5028 [10] suggests that even non-structural proteins can be exchanged across species boundaries, potentially altering the efficiency of viral replication or translation in a new host.

Implications for Surveillance and Control

The evolutionary dynamics of RRV underscore the need for continuous, genome-level surveillance of rotaviruses in rabbit populations. The emergence of novel G/P combinations, such as G3P[9] in Mexico and G3P[2] in China and Korea, indicates that the genotypic landscape of lapine rotaviruses is far from static. The use of high-throughput diagnostic tools, such as the Luminex xMAP and x-TAG assays developed for simultaneous detection of rabbit rotavirus, rabbit hemorrhagic disease virus, and Sendai virus, can facilitate large-scale monitoring efforts [12, 13]. However, these assays must be complemented by full-genome sequencing to detect reassortment events and identify the origins of novel segments. The detection of bat-like genes in lapine strains [7, 9] highlights the importance of expanding surveillance to include wildlife reservoirs, particularly bats and rodents, which may serve as sources of genetic diversity that can spill over into domestic rabbits.

From a control perspective, the evidence for interspecies transmission of RRV to humans [10] and from humans to rabbits [5] argues for a One Health approach to rotavirus vaccination. The widespread use of human rotavirus vaccines, which are highly effective against G1P[9], G2P[5], G3P[9], G4P[9], and G9P[9] strains, may exert selective pressure that favors the emergence of vaccine-escape reassortants. If such strains are introduced into rabbit populations, they could establish enzootic cycles that pose a continued risk of re-infection of humans, particularly immunocompromised individuals or those in close contact with rabbits. Conversely, the development of effective lapine rotavirus vaccines for use in commercial rabbitries could reduce the viral load in the environment and decrease the risk of spillover to humans. The demonstration that feeding newborn calves with colostrum from vaccinated dams can prevent rotavirus-induced diarrhea [2] suggests that passive immunization strategies could be

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