Rabbit Hemorrhagic Disease Virus 2

Overview and Taxonomy of Rabbit Hemorrhagic Disease Virus 2

Rabbit Hemorrhagic Disease Virus 2 (RHDV2), formally classified as Lagovirus europaeus genotype GI.2, represents a highly pathogenic, emergent calicivirus that has fundamentally altered the global landscape of lagomorph viral disease [1, 2]. As a member of the family Caliciviridae, genus Lagovirus, RHDV2 is a non-enveloped, single-stranded, positive-sense RNA virus approximately 32–40 nm in diameter [12, 34]. The virion is characterized by a distinctive icosahedral capsid composed of 90 dimers of the major structural protein VP60, organized into arch-like capsomers with a characteristic cup-shaped morphology typical of caliciviruses [27, 34]. The viral genome is approximately 7.4–7.7 kb in length, organized into two or three open reading frames (ORFs), with ORF1 encoding a polyprotein processed into nonstructural proteins (including the helicase, protease, and RNA-dependent RNA polymerase [RdRp]) and the major capsid protein VP60, while ORF2 encodes the minor structural protein VP10 [22, 28, 39]. This bipartite genomic organization, along with the presence of a VPg protein covalently linked to the 5' end of the genome, is a hallmark of the Caliciviridae family and is central to the virus's replication strategy [28, 34].

Taxonomy and Genotypic Classification

The taxonomic framework for pathogenic lagoviruses has undergone substantial revision since the emergence of RHDV2. Historically, the causative agent of rabbit hemorrhagic disease (RHD) was attributed to a single species, Lagovirus europaeus, with the classical genotype designated GI.1 (formerly RHDV) [1, 16, 31]. The GI.1 genotype itself encompasses several subtypes, including GI.1a (RHDVa), GI.1b, and GI.1c, which have circulated globally since the initial recognition of RHD in China in 1984 [29, 31]. However, the detection in 2010 of a genetically and antigenically distinct virus in France, one capable of causing disease in rabbits previously immunized against GI.1 and in juvenile animals that were historically refractory to classical RHD, necessitated the establishment of a second pathogenic genotype, GI.2, widely referred to as RHDV2 or RHDVb [2, 15, 16]. The International Committee on Taxonomy of Viruses (ICTV) and the World Organisation for Animal Health (WOAH) now recognize GI.1 and GI.2 as the two primary genogroups of pathogenic lagoviruses affecting lagomorphs, with GI.2 having rapidly supplanted GI.1 as the dominant circulating genotype in many regions of the world [2, 5, 15].

Critically, RHDV2 is not merely a variant of the classical virus; it represents a distinct viral entity with demonstrable differences in antigenicity, host range, and pathogenicity [7, 15, 27]. The capsid protein VP60 of GI.2 shares only approximately 80–85% nucleotide identity with GI.1 strains, a divergence that is sufficient to abrogate cross-protection afforded by classical RHDV vaccines [1, 7, 25]. This antigenic distance has been mapped to specific surface-exposed loops (loops 1, 4, and 5) in the P2 subdomain of VP60, which constitute a discontinuous neutralizing epitope recognized by monoclonal antibodies such as 2D9 [21, 27]. The structural basis for the diagnostic differentiation between GI.1 and GI.2 lies in a small number of amino acid substitutions within these loops that restrict antibody cross-reactivity, a finding that has driven the development of genotype-specific diagnostic assays [21, 22, 27].

Genomic Architecture and the Centrality of Recombination

A defining feature of RHDV2 biology is its chimeric genomic origin, which underscores the profound role of recombination in the emergence and ongoing evolution of this pathogen. The earliest characterized RHDV2 strains, including the prototype French isolate from 2010, were demonstrated to be recombinants bearing a capsid gene (VP60) derived from a pathogenic lagovirus lineage (GI.2) and nonstructural genes (including the RdRp-encoding region) from a non-pathogenic, enterotropic rabbit calicivirus classified as GI.3P (formerly RCV-E1) [1, 5, 16]. This recombination event is widely hypothesized to have been the key evolutionary leap that enabled the acquisition of enhanced virulence and a broader host range relative to the parental non-pathogenic strains [1, 38]. The GI.3P–GI.2 recombinant lineage thus represents the archetypal RHDV2, and strains of this composition have been detected across Europe, Africa, Asia, and North America [5, 24, 38].

However, the evolutionary narrative of RHDV2 does not end with a single recombination event. Extensive genomic surveillance, particularly in regions where the virus is endemic, has revealed a dynamic landscape of successive recombination events that have generated a constellation of distinct lineages [5, 9, 17, 40]. In Europe, the initial GI.3P–GI.2 strains were progressively replaced by GI.1bP–GI.2 recombinants, which dominated until approximately 2019, after which GI.4-related recombinants, specifically GI.4P–GI.2 and the more complex GI.4(p16)–GI.1bP–GI.2, became prevalent [5]. Generalised additive modeling of temporal data from northeastern Spain has demonstrated a statistically significant turnover of these recombinant lineages over the decade from 2014 to 2024, suggesting that variation in the nonstructural genomic region confers differences in viral fitness that are subject to natural selection [5]. In China, the scenario is equally complex: following the first detection of RHDV2 in April 2020, novel GI.1aP–GI.2 recombinants emerged and circulated within 12 months, exhibiting altered pathogenicity characterized by a longer clinical course and lower mortality but comparable viral loads at the moribund stage [9, 17]. These recombinants were even capable of causing lethal disease in rabbits that had been vaccinated against the parental GI.2 strain, highlighting the potential for recombination to drive immune evasion [17]. Similarly, in Singapore, the 2020 outbreak strain was identified as a GI.4P–GI.2 recombinant with high homology to contemporary Australian variants, demonstrating the global dissemination of distinct recombinant forms [40]. The nonstructural region of RHDV2 strains, therefore, is not fixed but is instead a mosaic of fragments derived from both pathogenic (GI.1, GI.2) and non-pathogenic (GI.3, GI.4) lagoviruses, a phenomenon that underscores the plasticity and adaptive capacity of this RNA virus [5, 9, 38].

Emergence and Global Dissemination

The emergence of RHDV2 in France in 2010, followed by its rapid spread across Europe, Africa, Oceania, Asia, and North America within a single decade, constitutes one of the most remarkable epizootic events in recent veterinary history [2, 15, 26]. The original GI.3P–GI.2 recombinant was first identified in French rabbitries, and within months, outbreaks were reported in Italy, Spain, and Portugal [2, 15]. By 2015, the virus had reached Australia, where it was detected in wild rabbits in May of that year, marking a significant shift from the deliberately introduced classical RHDV that had been used for biocontrol since the 1990s [15, 35]. The Australian incursion was particularly concerning because RHDV2 was found to partially overcome immunity to classical RHDV strains and to cause fatal disease in kittens as young as 30 days of age, an age group that is highly resistant to GI.1 infection [15, 35].

The global trajectory of RHDV2 continued unabated. In Africa, the virus was confirmed in Tunisia in 2015 and subsequently detected in Morocco, Algeria, and several Sub-Saharan nations, with phylogenetic evidence supporting multiple independent introductions from Europe [24, 30, 38]. The first confirmed outbreak in Asia occurred in China in May 2020, although retrospective analyses suggest that undetected circulation may have preceded this date [16]. The Chinese isolate, designated SC20-01, was a GI.3P–GI.2 recombinant closely related to European strains, implying a transcontinental introduction most likely via the movement of infected rabbits or rabbit products [16]. Shortly thereafter, RHDV2 was detected in Japan (2019–2020) and Singapore (2020), further cementing its pan-Asian distribution [12, 36, 40].

North America experienced a particularly dramatic and well-documented incursion. Following an initial and apparently contained detection in Quebec, Canada in 2016, and subsequent outbreaks on Vancouver Island (2018–2019) and in Washington State (2019), a massive epizootic erupted in the southwestern United States in March 2020 [2, 6, 14, 18, 37]. This outbreak, which began in New Mexico and swept through Arizona, California, Texas, and beyond, represented a paradigm shift in the perception of RHDV2 as a foreign animal disease in the U.S. [6, 37]. The virus spread with astonishing speed among both domestic and wild lagomorphs, infecting multiple native North American species for the first time and prompting emergency declarations and vaccination campaigns [2, 3, 6]. Genomic sequencing of the 2020 U.S. outbreak strains revealed that they were closely related to the 2018 British Columbia isolates, suggesting a single, large-scale viral incursion that had been circulating undetected or had been repeatedly introduced [32, 37]. A second, genetically distinct incursion was later identified in Washington State in 2023, with sequences showing less than 82% identity to previous North American strains, indicating that transcontinental introductions are an ongoing threat [23].

Host Range and Species Susceptibility

Perhaps the most biologically significant distinction between RHDV2 and its classical predecessor is its dramatically expanded host range. Classical RHDV (GI.1) is predominantly restricted to the European rabbit (Oryctolagus cuniculus), with only rare and sporadic infections reported in other leporids [2, 33]. In stark contrast, RHDV2 has been documented to cause fatal disease in an ever-growing list of lagomorph species spanning multiple genera. In addition to European rabbits, RHDV2 has been confirmed in several hare species (Lepus spp.), including the European brown hare (L. europaeus), the mountain hare (L. timidus), the Iberian hare (L. granatensis), and the black-tailed jackrabbit (L. californicus) [5, 11, 13, 20]. Among the cottontail rabbits and brush rabbits of the genus Sylvilagus, RHDV2 has been detected in the desert cottontail (S. audubonii), mountain cottontail (S. nuttallii), eastern cottontail (S. floridanus), and the endangered riparian brush rabbit (S. bachmani riparius) [3, 4, 11, 33]. Notably, experimental inoculation studies have confirmed that eastern cottontails are susceptible to RHDV2 and can shed viral RNA in urine, oral secretions, and feces, implicating this widespread species as a potential reservoir and bridging host in the epidemiology of the virus in North America [33].

The host range extends even further. RHDV2 has been documented in pygmy rabbits (Brachylagus idahoensis), a sagebrush obligate of conservation concern in the Intermountain West of the United States, and in red rock rabbits (Pronolagus spp.) in Africa [2, 3, 8]. Perhaps most startlingly, spillover events have been reported in non-lagomorph species. In Portugal, RHDV2 RNA and evidence of viral replication were detected in Eurasian badgers (Meles meles), indicating that the virus can cross the species barrier into a carnivore host [10]. The badgers were found to have high viral loads in multiple organs and to excrete virus in feces, raising the possibility that they could serve as mechanical or biological vectors for transmission to rabbits [10]. This expansion of host range is intimately linked to the virus's recombinant origin and ongoing evolution; the acquisition of novel genetic material in the nonstructural region may have conferred the ability to utilize different cellular receptors or evade host immune responses in a broader array of species [2, 13, 20]. The implications for conservation are profound, as RHDV2 now poses a direct threat to multiple threatened and endangered lagomorph species, including the riparian brush rabbit and the pygmy rabbit, populations of which are already under pressure from habitat loss and fragmentation [3, 4, 8, 19].

Molecular Pathogenesis of RHDV2: Mechanisms of Hepatic Necrosis and Disseminated Intravascular Coagulation

The molecular pathogenesis of Rabbit Hemorrhagic Disease Virus 2 (RHDV2, genotype GI.2) represents a paradigm of viral-induced fulminant hepatic failure culminating in a catastrophic hemorrhagic diathesis. Unlike its predecessor, classical RHDV (GI.1), RHDV2 exhibits a broader host range, infecting multiple leporid genera including Oryctolagus, Lepus, Sylvilagus, and Brachylagus [2, 3, 11, 20], and has demonstrated the capacity for spillover into non-lagomorph species such as the Eurasian badger (Meles meles) [10]. The disease is characterized by two interconnected pathological pillars: massive hepatocellular necrosis and disseminated intravascular coagulation (DIC). Understanding the molecular underpinnings of these processes requires a detailed examination of viral entry, replication kinetics, host cellular response, and the dysregulation of the coagulation cascade.

Viral Tropism and Initial Replication: The Gateway to Hepatic Catastrophe

The pathogenesis of RHDV2 begins with oral or nasal inoculation, followed by primary replication within the gastrointestinal tract. Using RNAscope in situ hybridization, O'Toole et al. [46] demonstrated that viral genomic RNA is detectable in the gastrointestinal tract as early as 12 hours post-inoculation (hpi), preceding the involvement of the liver. This initial replication phase is critical, as it establishes a viremic state that delivers the virus to its primary target organ: the liver. By 36 hpi, viral RNA is consistently detected within hepatocytes, marking the onset of the hepatic phase of infection [46].

The viral capsid, composed of the major structural protein VP60, mediates attachment to host cells. The VP60 protein of RHDV2, while antigenically distinct from that of GI.1, retains the ability to bind histo-blood group antigens (HBGAs), which serve as attachment factors on the surface of hepatocytes and biliary epithelial cells [27]. The P2 subdomain of VP60, particularly external loops 1, 4, and 5, contains critical neutralizing epitopes and is under significant selective pressure, as demonstrated by epitope mapping studies using the GI.2-specific monoclonal antibody 2D9 [21, 27]. The interaction between VP60 and host cellular receptors triggers clathrin-mediated endocytosis, delivering the viral genome into the cytoplasm.

Once internalized, the positive-sense single-stranded RNA genome is translated to produce the viral replicase complex, which includes the RNA-dependent RNA polymerase (RdRp), a 3C-like protease (3CLpro), and other nonstructural proteins (p16, p23). The 3CLpro is essential for processing the viral polyprotein into functional subunits, making it a prime target for antiviral intervention [50, 51]. The host protein nucleolin (NCL) has been identified as a critical physical link, mediating interactions between the viral RdRp and other host factors necessary for replication complex assembly [28]. Conversely, the host protein hemoglobin subunit beta (HBB) acts as a restriction factor, interacting with VP60, RdRp, and VPg to antagonize viral replication, though its expression is significantly downregulated during infection [39].

Mechanisms of Hepatic Necrosis: A Triad of Direct Cytotoxicity, Apoptosis, and Immune-Mediated Injury

The hallmark of RHDV2 infection is massive hepatocellular dissociation and necrosis. Lankton et al. [11] reported that 100% of examined North American lagomorphs (desert cottontails and black-tailed jackrabbits) exhibited massive hepatocellular dissociation and necrosis or apoptosis. This finding is consistent across species, including domestic rabbits, hares, and pygmy rabbits [12, 13, 36, 45]. The necrosis is typically random, multifocal, or centrilobular, progressing rapidly to panlobular involvement [45, 48].

Direct Viral Cytotoxicity: The virus replicates to extraordinarily high titers within hepatocytes. Viral loads in the liver are typically 4-5 logs higher than those detected in rectal swabs, with median cycle threshold (Ct) values of 12.69 in liver compared to 27.03 in rectal samples [44]. This massive viral burden directly disrupts cellular homeostasis. The viral replicase complex hijacks host machinery, leading to the formation of membranous replication complexes and the depletion of cellular energy stores. Ultrastructural studies have revealed the presence of calicivirus-compatible virions (approximately 32 nm in diameter) within the nucleus of hepatocytes, a finding that challenges the conventional paradigm of an exclusively cytoplasmic replication cycle for caliciviruses [10, 12].

Induction of Apoptosis: RHDV2 infection triggers both extrinsic and intrinsic apoptotic pathways. The rapid onset of hepatocellular dissociation, characterized by the loss of intercellular adhesion and rounding of hepatocytes, is a hallmark of apoptosis rather than simple lytic necrosis [11]. The virus likely activates caspase cascades through the upregulation of death receptors (e.g., Fas, TNF receptor) and the release of cytochrome c from mitochondria. The profound decrease in serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) observed in terminal cases, rather than the expected increase, is a paradoxical finding that reflects the massive, acute loss of functional hepatocyte mass and the inability of the dying liver to release these enzymes into the circulation [48].

Dysregulated Innate Immune Response and Cytokine Storm: The host's innate immune response, while intended to control viral replication, paradoxically contributes to the severity of hepatic necrosis. Transcriptomic analysis of the spleen, a critical component of the mononuclear phagocyte system (MPS), reveals a profound inflammatory disorder following RHDV2 infection. Yu et al. [42] identified 3,148 differentially expressed genes (DEGs) in the spleens of infected rabbits, with 100 upregulated DEGs encoding pro-inflammatory factors including IL-1α, IL-6, and IL-8. KEGG pathway analysis demonstrated significant enrichment of the cytokine-cytokine receptor interaction signaling pathway [42].

This systemic inflammatory response is corroborated by in situ hybridization studies. O'Toole et al. [46] demonstrated increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1β within the liver and spleen through 48 hpi. The cytokine storm, driven by these mediators, amplifies hepatocellular damage through the recruitment and activation of neutrophils and macrophages, which release reactive oxygen species and proteolytic enzymes. The role of microRNA in this process is emerging; miR-155-5p, a key regulator of inflammatory responses, shows a 2.4-fold increase in expression in the liver of infected rabbits, suggesting its potential as a tissue biomarker of inflammation [49].

Disseminated Intravascular Coagulation: The Hemorrhagic Catastrophe

The transition from hepatic necrosis to DIC is the terminal event in RHDV2 pathogenesis. The liver is the primary site of synthesis for most coagulation factors (fibrinogen, prothrombin, factors V, VII, IX, X, XI, XII) and anticoagulant proteins (protein C, protein S, antithrombin). Massive hepatic necrosis leads to a precipitous decline in the production of these factors, creating a profound hypocoagulable state. Bonvehí et al. [48] documented markedly decreased fibrinogen concentrations, prolonged prothrombin time (PT), and activated partial thromboplastin time (aPTT) in naturally infected pet rabbits.

Consumption Coagulopathy: Simultaneously, the widespread endothelial damage and exposure of subendothelial tissue factor trigger the extrinsic coagulation cascade. The virus infects endothelial cells throughout the body, as demonstrated by in situ hybridization showing significant endothelial staining within blood vessels of almost all organs [47]. This endothelial activation leads to the expression of tissue factor, which complexes with factor VIIa to activate factor X, ultimately generating thrombin. The resulting widespread microvascular thrombosis consumes platelets and coagulation factors, exacerbating the hemorrhagic tendency. Fibrin thrombi in small vessels are a consistent histologic finding, particularly in the glomerular capillaries of the kidney and pulmonary vasculature [12, 36].

Thrombocytopenia and Platelet Dysfunction: Thrombocytopenia is a consistent clinicopathologic finding in RHDV2 infection [48]. The mechanisms include: (1) consumption of platelets in widespread microthrombi; (2) direct viral interaction with megakaryocytes or platelets; and (3) immune-mediated destruction. The profound thrombocytopenia, combined with the consumption of coagulation factors, results in the characteristic hemorrhagic diathesis observed in terminal cases.

Clinical and Pathologic Manifestations of DIC: The clinical consequences of DIC are dramatic. Epistaxis is observed in over 90% of cases [11]. Pulmonary congestion, edema, and hemorrhage are present in 92-100% of affected animals [11]. Peritoneal petechiae, icteric and hemorrhagic abdominal fat, pericardial effusion, and pulmonary hemorrhages are common gross findings [48]. The combination of hepatic failure and DIC leads to a rapidly fatal outcome, with death typically occurring within 22-30 hours of clinical presentation in pet rabbits [48].

The Role of Recombination in Pathogenesis

The molecular pathogenesis of RHDV2 is further complicated by its propensity for recombination, which can alter virulence and host range. The emergence of RHDV2 itself is believed to have resulted from a recombination event between a non-pathogenic lagovirus (GI.3P) and a pathogenic strain [1, 5]. Subsequent recombination events have generated a diversity of circulating strains with distinct non-structural genomic profiles, including GI.3P-GI.2, GI.1bP-GI.2, GI.4(p16)-GI.1bP-GI.2, and GI.4P-GI.2 [5, 9, 17, 31, 40].

These recombinant strains exhibit differential pathogenicity. Hu et al. [9] reported that a novel recombinant strain circulating in China showed a longer infected time and lower mortality rate compared to the original RHDV2 strain, but maintained almost the same viral load at the moribund stage, potentially facilitating environmental contamination and transmission. Conversely, Li et al. [17] identified a GI.1aP-GI.2 recombinant that moderately enhanced virulence, even for rabbits vaccinated against parental GI.2. The non-structural genomic region, which varies among recombinants, likely contributes to differences in viral fitness, replication efficiency, and the ability to modulate the host innate immune response [5]. This dynamic evolution underscores the need for continuous molecular surveillance to anticipate shifts in pathogenic potential.

Comparative Pathogenesis Across Host Species

The molecular pathogenesis of RHDV2 is not uniform across all susceptible species. While European rabbits (Oryctolagus cuniculus) are exquisitely susceptible, with mortality rates approaching 100% in naive populations [33, 41, 43], other species show variable susceptibility. Mohamed et al. [33] demonstrated that eastern cottontails (Sylvilagus floridanus) are susceptible to RHDV2 but not to classical RHDV, with 3 of 5 inoculated animals developing fatal disease. Critically, surviving cottontails seroconverted and shed viral RNA in urine, oral swabs, and rectal swabs, suggesting a potential role as reservoir hosts [33].

The ability of RHDV2 to infect and cause disease in hares (Lepus spp.) represents a significant host range expansion compared to classical RHDV [13, 20]. Neimanis et al. [20] documented an outbreak in mountain hares (Lepus timidus) on an island where rabbits were absent, demonstrating that the virus can circulate and persist within hare populations alone for at least 4.5 months. The molecular basis for this expanded tropism likely resides in the VP60 capsid protein, which has evolved to recognize HBGAs or other receptors present on the cells of diverse leporid species [27]. The detection of RHDV2 in pygmy rabbits (Brachylagus idahoensis), a species of conservation concern, further underscores the ecological impact of this pathogen [3, 8].

1. Global Epidemiology and Spatiotemporal Spread of RHDV2 (2010–2023)

The emergence of rabbit hemorrhagic disease virus 2 (RHDV2; genotype GI.2) in 2010 in France [1, 2, 5] initiated a panzootic event of a magnitude unprecedented for a lagovirus. This strain, a recombinant between a non-pathogenic ancestral virus (GI.3P) and a pathogenic GI.2 capsid backbone [1], possessed a unique epidemiological profile: it was capable of causing fatal disease in both young and adult rabbits, broadened its host range beyond Oryctolagus cuniculus to include multiple Lepus and Sylvilagus species, and partially overcame immunity to classical RHDV (GI.1) [2, 13, 15, 20]. Over the subsequent thirteen years, RHDV2 demonstrated a relentless spatiotemporal expansion across five continents, establishing itself as the dominant pathogenic lagovirus globally [2, 15, 26]. This section provides a detailed analysis of the virus's global dissemination, the key epidemiological drivers of its spread, the ecological impact on wild lagomorph populations, and the molecular evolutionary patterns observed across different temporal and geographic scales.

1.1 Emergence and European Dominance (2010–2015)

The index cases of RHDV2 were identified in France in 2010 [2, 15]. Within a single year, the virus was detected in Italy and Spain (2011) [15], Portugal (2012) [15], and subsequently across the continent [5, 13]. The early epidemiology in Europe was characterized by a rapid shift in dominance. Classical RHDV (GI.1) was progressively and, in many regions, completely replaced by RHDV2 [15, 29]. This replacement was driven by the superior fitness of RHDV2, including its ability to cause lethal disease in kittens as young as 30 days old, which are naturally resistant to GI.1 strains, and its capacity for antigenic variation that allowed it to circumvent pre-existing immunity in vaccinated or convalescent adult populations [2, 15]. In the Iberian Peninsula, a keystone region for the European rabbit (O. cuniculus), the impact was profound. By 2011, the virus was declared endemic [52], leading to a classification of O. cuniculus as Endangered by the International Union for Conservation of Nature (IUCN) [52]. Analysis of hunting yield data from Castilla-La Mancha, Spain (2009–2022) revealed significant, sustained population declines in both wild rabbit subspecies (O. c. cuniculus and O. c. algirus) following the 2011 outbreak [52]. The epidemiological dynamics within Europe also revealed a complex pattern of recombinant strain turnover. Long-term surveillance in northeastern Spain (Catalonia, 2014–2024) identified four distinct recombinant lineages circulating in wild European rabbits and European brown hares (Lepus europaeus): GI.3P–GI.2, GI.1bP–GI.2, GI.4(p16)–GI.1bP–GI.2, and GI.4P–GI.2 [5]. This temporal monitoring detailed a clear succession: GI.3P–GI.2 appeared transiently in 2014, was replaced by a dominance of GI.1bP–GI.2 strains until 2019, and finally, from 2020 onward, GI.4P–GI.2 became the prevailing lineage [5]. This repeated and rapid replacement of non-structural genomic segments in wild populations strongly supports recombination as a primary driver of viral fitness and long-term persistence [5, 9]. Concurrently, spillover from rabbits to sympatric hare populations was confirmed. In Germany, captive mountain hares (Lepus timidus) succumbed to an RHDV2 strain, "Bremerhaven-17," with necrotizing hepatitis consistent with RHD [13]. In Sweden, a mortality event in a spatially isolated population of mountain hares on the island of Hallands Väderö, where rabbits were absent, provided unequivocal evidence of RHDV2's capacity for sustained transmission and circulation within a novel leporid host, independent of the original rabbit reservoir [20]. This outbreak, which lasted for at least 4.5 months, indicated that RHDV2 could persist solely within hare populations [20]. By 2016, the virus had effectively reached all corners of Europe [55].

1.2 Transcontinental Spread and Evolution in Oceania and Asia

The epidemiological trajectory of RHDV2 outside Europe was marked by multiple, independent introductions followed by rapid evolution via recombination. The first detection in Oceania occurred in May 2015 in a wild rabbit in Australia [15]. Given the prior use of RHDV as a biological control agent for invasive rabbit populations, the arrival of RHDV2 was met with significant concern. The virus established itself quickly and, echoing the pattern in Europe, began to undergo recombination. A landmark event was the emergence of a GI.4P–GI.2 recombinant variant, which by 2017 had become prevalent in lagomorph populations in Australia [40]. The epidemiological significance of this was later demonstrated by the outbreak in Singapore in September 2020. The etiological agent of the first confirmed RHDV2 cases in Singapore was identified through time-structured and phylogeographic analyses as an Australian GI.4P–GI.2 recombinant variant [40]. This finding strongly implicated the intercontinental movement of infected animals or contaminated fomites, likely via the pet trade, as a major pathway for long-distance viral dispersal [40].

The introduction of RHDV2 into Asia presents a compelling case study of rapid viral evolution following a discrete incursion. The virus was first reported in China in April 2020 [9, 16]. The initial strain (SC20-01) was phylogenetically related to European isolates [16]. However, within less than a year, a novel recombinant was detected. Characterized by a capsid gene closely related to North American GI.2 strains (97.9% identity) and non-structural genes from a local GI.1a lineage, this new variant emerged and circulated across at least three Chinese provinces by 2021 [1, 9]. This Chinese recombinant exhibited a paradoxical phenotype: it showed a longer infection period and lower mortality rate (70–80%) compared to the original strain, yet maintained a similar viral load at the moribund stage [9]. This created a longer window for viral excretion and environmental contamination, thereby facilitating enhanced transmission [9]. The ability of this virus to then recombine with local GI.1a strains, generating GI.1aP–GI.2 variants, underscores the high genomic plasticity of RHDV2 and the risk of rapid, unpredicted changes in virulence and transmissibility upon entering a naive ecosystem [17]. Multiple diagnostic investigations in China from 2020 to 2024 confirmed RHDV2 as the dominant (and sole) pathogenic lagovirus, with no classical RHDV detected in clinical submissions [57]. Broader Asian incursions included Japan, where outbreaks were confirmed in Ehime Prefecture in 2019 and Chiba Prefecture in 2020, the latter causing 55% mortality in a single colony [12, 36].

1.3 The North American Panzootic (2016–2023)

The epidemiology of RHDV2 in North America is characterized by multiple, genetically distinct incursions before a continent-wide epizootic. The first confirmed detection was in Quebec, Canada, in 2016 [14]. This isolate was genetically similar (97% identity) to the 2011 Spanish isolate (RHDV2-N11) [14]. A subsequent, independent incursion occurred in British Columbia (BC), Canada, between 2018 and 2019, with a virus sharing only 93% identity with the Quebec isolate [14]. This BC outbreak spread rapidly among feral and domestic European rabbit populations on Vancouver Island and the mainland, with epidemiological evidence strongly suggesting that anthropogenic translocation of infected animals played a crucial role [14, 18]. A third distinct incursion was detected in pet rabbits in an apartment in Vancouver in June 2019, further confirming that the virus had been introduced on at least two separate occasions into BC alone [14].

The most significant and widespread North American outbreak commenced in March 2020 in the southwestern United States, first detected in New Mexico [3, 6, 11, 37]. This event was distinct from the prior Canadian introductions. Full-genome sequencing of isolates from the 2020 outbreak in the US revealed they were closely related to the 2018 BC strain (98.6–98.7% identity) but only 92.4–92.6% identical to the 2016 Quebec strain, suggesting a southern route of introduction potentially linked to the earlier BC event or a common European ancestor [32]. The virus spread with alarming speed across the western states. It was detected in California on May 11, 2020, in a black-tailed jackrabbit (Lepus californicus), and quickly spilled over into domestic and other wild lagomorphs, including desert cottontail rabbits (Sylvilagus audubonii) [6, 32]. By March 2024, over 900 wild lagomorphs from 14 western states had been submitted for testing, with 313 (34.2%) positive for RHDV2 RNA by RT-qPCR [3]. This epizootic affected a remarkably broad range of native species: desert and mountain cottontails (S. audubonii, S. nuttallii), black-tailed and antelope jackrabbits (L. californicus, L. alleni), and, most concerningly, the pygmy rabbit (Brachylagus idahoensis) and the endangered riparian brush rabbit (Sylvilagus bachmani riparius) [3, 4, 8, 11]. The pathology in these novel North American species was consistent with that seen in European rabbits: massive hepatocellular dissociation, necrosis, pulmonary congestion, and edema [11]. The disease dynamics were further clarified by genetic evidence; in 2023, a highly divergent RHDV2 strain (<82% identity to previous North American sequences) was detected in domestic and wild rabbits in Washington State, representing yet another independent viral incursion into the continent [23]. This suggests that North America is subject to ongoing, low-level introductions of novel RHDV2 variants, complicating control efforts. Modeling studies have identified developed land cover and the presence of lagomorph (domestic) cases as significant predictors of infection counts in wild populations, underscoring the role of the wildlife-domestic animal interface and anthropogenic factors in the virus's spatial ecology [53].

1.4 African Incursions and Global Risk Factors

The epidemiological picture in Africa is one of repeated introductions, primarily from Europe, followed by local establishment. The first confirmed detection of GI.2 in Africa was in Tunisia in 2015 [38]. Phylogenetic analyses of Tunisian GI.2 strains collected between 2018 and 2020 identified them as GI.3P–GI.2 recombinants, most likely imported from Europe [38]. Further evidence of multiple introductions comes from Algeria, where a 2018 isolate grouped with older Tunisian strains, while later isolates were more closely related to North American strains [24]. The virus also reached Sub-Saharan Africa, with an outbreak in Ghana in September 2019 linked to isolates from the Netherlands (2015–2017), highlighting the role of the international rabbit trade in viral spread [54]. In Morocco, despite systematic vaccination against classical RHDV, the virus was detected in vaccinated rabbitries, confirming that the available vaccines were ineffective against the new genotype [30]. These findings across North and West Africa emphasize that RHDV2 is now entrenched on the continent and that vaccine strategies must be updated to include GI.2 antigens [30].

From a global risk perspective, spatial modeling using MaxEnt has identified key environmental and anthropogenic variables that predict RHDV2 outbreaks. For domestic rabbits, high road density, isothermality, and human population density were the most important factors [26]. For wild lagomorphs, road density, normalized difference vegetation index (NDVI), and mean annual solar radiation were key [26]. These models, which produced high AUC values (0.960 and 0.974), highlight that RHDV2 outbreaks are driven by a complex interplay of habitat suitability, human-mediated transport, and host ecology [26]. The seasonal pattern of outbreaks also shows a pronounced spring peak in the Northern Hemisphere and a late-autumn peak in the Southern Hemisphere, suggesting that environmental conditions and host behavior (e.g., breeding seasons) modulate transmission risk [26]. The global spread of this fast-evolving RNA virus into new geographic areas, combined with the presence of a wide array of non-pathogenic lagoviruses (GI.3, GI.4) in many of these same populations, creates a fertile ground for continued recombination [2, 56]. The potential for further host range expansion is high, as evidenced by spillover events into non-lagomorph species, such as the detection of replicating RHDV2 (GI.4P–GI.2) in Eurasian badgers (Meles meles) in Portugal, a finding of significant ecological and epidemiological consequence [10].

Diagnostic Approaches for RHDV2: Molecular, Serological, and Pathological Methods

The accurate and timely diagnosis of Rabbit Hemorrhagic Disease Virus 2 (RHDV2; Lagovirus europaeus GI.2) is paramount for effective disease surveillance, outbreak containment, and the implementation of appropriate biosecurity and vaccination strategies. The global spread of this highly contagious, often fatal pathogen across five continents, affecting domestic rabbits, wild lagomorphs, and even non-lagomorph species [1, 2, 10], has necessitated the development and refinement of a diverse array of diagnostic platforms. These range from rapid field-deployable antigen detection tests to exquisitely sensitive molecular assays and definitive histopathological examinations. The diagnostic landscape is complicated by the virus's inability to propagate in standard continuous cell lines, the existence of genetically and antigenically distinct genotypes (GI.1/GI.2 and their recombinants), and the need to discriminate between pathogenic and non-pathogenic lagoviruses circulating in the same populations [34, 56]. This section provides an exhaustive analysis of the three principal pillars of RHDV2 diagnosis: molecular detection, serological profiling, and pathological assessment, drawing upon a wealth of recent research to delineate the strengths, limitations, and specific applications of each methodological approach.

Molecular Detection: The Gold Standard and Beyond

Molecular methods, particularly real-time reverse transcription polymerase chain reaction (RT-qPCR), constitute the cornerstone of RHDV2 diagnostics due to their unparalleled sensitivity, specificity, and rapid turnaround time. The liver is unequivocally considered the gold-standard sample for post-mortem diagnosis, harboring the highest viral loads; median cycle threshold (Ct) values as low as 12.69 have been reported in liver samples from infected carcasses, reflecting the massive viral replication that characterizes the terminal stages of disease [44]. The World Organization for Animal Health (WOAH) recommends RT-qPCR as a primary diagnostic tool, and numerous validated assays targeting the highly conserved VP60 capsid gene have been developed. A seminal real-time Taqman RT-PCR designed by Duarte et al. [64], for example, targets a 127-nucleotide region within the VP60 gene, demonstrating the capacity to detect as few as nine RNA molecules with 99.4% efficiency and coefficients of variation below 2.40%. This assay was rigorously validated against a panel of common rabbit pathogens and showed high specificity for RHDV2, proving invaluable for diagnosing circulating strains and monitoring viral loads to assess disease progression and vaccine efficacy [64].

The molecular toolkit has expanded significantly to accommodate the emergence of recombinant strains and the need for genotypic differentiation. A recent multiplex RT-qPCR assay targeting the minor capsid protein VP10 (ORF2) has been developed to simultaneously detect and discriminate between GI.1 and GI.2 genotypes [22]. This assay, using FAM-labeled probes for GI.1 and HEX-labeled probes for GI.2, achieved a limit of detection of 1.0 × 10² RNA copies per reaction with excellent linearity and intra-assay variability below 3.5%, demonstrating no cross-reactivity with non-target pathogens such as Myxoma virus or Pasteurella multocida [22]. Similarly, SYBR Green I-based RT-qPCR assays have been developed, targeting a 435bp conserved fragment of the VP60 gene, providing a cost-effective alternative for specific RHDV2 detection, though with a slightly higher detection limit of approximately 128 copies/μL [65]. The ability to differentiate genotypes is critical not just for outbreak investigations but for understanding the evolutionary dynamics of RHDV2, particularly given the frequent recombination with non-pathogenic GI.3 and GI.4 strains which can alter pathogenicity and host range [5, 17]. For instance, novel GI.1aP-GI.2 recombinants identified in China have been shown to partially overcome immunity induced by parental GI.2 vaccines, underscoring the necessity for genotyping in diagnostic workflows [17].

Alternative Sample Types: Expanding Surveillance Scope

While liver is the ideal specimen, its collection requires carcass submission, which is often impractical for large-scale surveillance, particularly in wild populations. This limitation has driven the development and validation of alternative, less invasive sample types. A landmark study by Asín et al. [44] validated an RT-qPCR assay on rectal swabs collected from leporid carcasses. Compared to the liver gold standard, the rectal swab assay demonstrated a sensitivity of 88% and a specificity of 100%, despite having significantly lower viral loads (median Ct 27.03 versus 12.69). The authors concluded that while rectal swab RT-qPCR is approximately 4 logs less sensitive, it remains a highly effective screening tool for carcass surveillance, particularly valuable when liver tissue is degraded or unavailable, and crucially, it offers a non-lethal sampling method for live animals [44]. This approach is directly applicable to monitoring the health of endangered species like the riparian brush rabbit (Sylvilagus bachmani riparius) and pygmy rabbits (Brachylagus idahoensis), where lethal sampling is ethically and conservationally problematic [3, 4].

Further expanding field surveillance capabilities, studies have validated the use of dried blood on filter paper and ear punch samples for RT-qPCR detection [62]. In a study by Jennings-Gaines et al. [62], both sample types showed 100% specificity and sensitivity compared to concurrent liver samples from the same animals. The filter paper method demonstrated remarkable stability, maintaining >96% sensitivity over six weeks of storage, while ear punch sensitivity remained at 100% for up to seven weeks [62]. These findings offer wildlife biologists and field veterinarians flexible, low-biosecurity-risk options for sample collection that can be easily stored and shipped without cold chains. Similarly, non-invasive monitoring of fecal pellets for viral RNA has been explored as a tool to survey wild rabbit populations. However, a study by Calvete et al. [69] using duplex real-time PCR on fecal pellets revealed a weak concordance with observed mortality events, likely due to low viral RNA concentrations in feces and prolonged excretion periods. The authors concluded that while fecal pellet analysis is a complementary non-invasive method, it has low performance for monitoring acute infection incidence and should not replace direct sampling of carcasses or suspect animals [69].

The choice of RNA extraction methodology can influence assay sensitivity. A comparison of a magnetic bead-based extraction method with a rapid, one-tube heat-block method (SwiftX Swabs Viral RNA Extraction Reagent) found that while RHDV2 was detected in all samples by both methods, the rapid method resulted in a mean Ct increase of 3.79, representing approximately a 1-log₁₀ reduction in sensitivity [61]. This trade-off between speed, simplicity, and sensitivity must be carefully considered when designing surveillance programs, particularly in resource-limited settings.

Advanced Molecular and Emerging Techniques

Beyond conventional RT-qPCR, several advanced molecular and biosensing technologies are being refined for RHDV2 detection. Digital PCR (dPCR) offers absolute quantification of nucleic acids without the need for standard curves. This technique has been successfully applied to quantify ocu-miR-155-5p, a microRNA identified as a potential tissue biomarker of inflammation in RHDV-infected rabbits [49]. The study demonstrated significantly upregulated expression of this miRNA in the liver, lung, and kidney of infected animals using dPCR, opening a new avenue for molecular diagnostics that assess host-pathogen interaction dynamics rather than direct viral detection [49]. In situ hybridization (ISH) using RNAscope technology provides high-resolution spatial localization of viral RNA within formalin-fixed, paraffin-embedded (FFPE) tissues. This technique, validated by O'Toole et al. [47, 63], is exquisitely sensitive and specific, detecting viral mRNA (genomic and replicative) and negative-sense replicative intermediates. Notably, ISH revealed previously unrecognized sites of viral replication, including significant glomerular staining in the kidneys and endothelial staining within blood vessels of almost all organs, providing crucial insights into the pathogenesis of disseminated intravascular coagulation (DIC) and multi-organ failure [47]. The use of probes targeting the replicative intermediate RNA specifically identifies cells actively supporting viral replication, distinguishing them from cells that have merely phagocytosed viral debris, thereby refining our understanding of tissue tropism [63].

Biosensor technologies, particularly Surface Plasmon Resonance (SPR), represent a frontier in rapid, real-time virus detection. Urbinati et al. [66] developed an SPR biosensor using intact RHDV2 captured by a specific monoclonal antibody covalently immobilized to a sensor surface. This biosensor demonstrated excellent analytical performance for detecting RHDV2 in biological materials and could be used for screening antiviral monoclonal antibodies, offering a platform that combines high sensitivity with the potential for near-real-time results without nucleic acid amplification [66]. Next-generation sequencing (NGS) and metagenomics have become indispensable tools for characterizing the complete coding genome of circulating strains, detecting novel recombinants, and tracking viral incursions across geographic boundaries. Whole-genome sequencing of RHDV2 isolates from the 2020 outbreak in the United States revealed that sequences from California were closely related to each other (98.9-99.95% identity) and to a strain from British Columbia (2018), suggesting a related incursion event, while being distinct from a separate incursion in New York [32]. This molecular epidemiology is essential for understanding transmission pathways, such as the suspected anthropogenic translocation of infected animals implicated in outbreaks in British Columbia, Canada, and the introduction of an Australian recombinant variant into Singapore in 2020 [18, 40].

Serological Diagnostics: Profiling the Humoral Response

Serological assays are fundamental for determining population exposure, evaluating vaccine efficacy, and monitoring the duration of protective immunity. The primary tools employed are the Hemagglutination Inhibition (HI) test and Enzyme-Linked Immunosorbent Assays (ELISA), each offering distinct advantages and applications.

Hemagglutination Inhibition (HI) Test

The HI test remains a widely used, cost-effective method for quantifying antibodies that block the ability of RHDV2 to hemagglutinate human type O erythrocytes. The assay relies on the VP60 capsid protein's intrinsic hemagglutination activity, a property shared with classical RHDV [67]. HI titers are a well-established correlate of protection; titers of ≥1:8 to 1:16 are generally considered protective, though absolute thresholds can vary by vaccine and challenge model. In vaccine efficacy studies, HI is the primary serological endpoint. For instance, in a study evaluating a bivalent RHDV2-Pasteurellosis vaccine, HI testing revealed peak antibody titers of log₂ 11.5 at 12 weeks post-vaccination, which correlated with 100% protection against virulent RHDV2 challenge [58]. Similarly, a comparative study of adjuvants found that Montanide ISA70 oil-adjuvanted vaccines induced significantly higher and more durable HI titers (peaking at log₂ 10.67 at 5 months post-vaccination) compared to aluminum hydroxide gel-adjuvanted vaccines (peaking at log₂ 9.33 at 3 months) [60]. The HI test is also used to assess cross-protection between different RHDV genotypes and recombinant strains, an increasingly critical concern as GI.1aP-GI.2 recombinants emerge [17]. However, the HI test has limitations; it is relatively labor-intensive, requires fresh human O-type erythrocytes, and may not detect all subclasses of neutralizing antibodies.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA platforms, particularly indirect and competitive formats, offer higher throughput, objective endpoints, and the ability to detect different antibody isotypes. Various ELISAs have been developed using VP60 virus-like particles (VLPs) or specific peptide antigens. For example, a peptide-generated antibody-based capture ELISA was developed for immunohistochemistry, but the principle is directly applicable to serological detection of viral antigen [59]. In vaccine trials, antigen-specific antibody titers measured by ELISA can be several orders of magnitude higher than HI titers, reflecting total antibody binding rather than just functional inhibition; one study reported that SLA-adjuvanted VP60 induced geomean ELISA titers of 7,393, compared to 117 for antigen alone [68]. Competitive ELISAs, such as the AG-ELISA (Agar Gel Immunodiffusion) mentioned for detection of GI.2 in mountain hares [13], are less common but useful for detecting antibodies in a species-independent manner. The choice between HI and ELISA often depends on the laboratory's capacity and the specific question being asked, HI for functional antibody assessment and ELISA for high-throughput screening or total antibody measurement.

Immunohistochemistry (IHC) and Antigen Detection

For direct detection of viral antigen in tissues, immunohistochemistry (IHC) is an indispensable adjunct to histopathology. A major advancement in this field is the development of a peptide-generated, chicken egg-derived antibody specific to the RHDV2 VP60 capsid protein [59]. This antibody, validated on FFPE tissues from naturally infected domestic rabbits, provides a readily available, inexpensive tool for diagnostic laboratories. Viral antigen was prominently detected in the cytoplasm and nuclei of hepatocytes and macrophages in the liver, as well as in macrophages within the spleen and cecal lymphoid tissue [59]. Notably, immunolabeling was also observed in intravascular mononuclear cells in the lung, renal tubular epithelium, and biliary epithelium, corroborating the broad tissue tropism revealed by ISH [47, 59]. This IHC method is ideally suited for retrospective studies on archived FFPE samples and for confirming the presence of RHDV2 in cases where RT-qPCR is unavailable or has given ambiguous results.

Monoclonal antibodies (MAbs) have been pivotal in refining serological diagnostics and antigen detection. The MAb 2D9, for instance, is specific for RHDV GI.2 and has been used to discriminate between GI.1 and GI.2 in diagnostic tests [21]. Structural studies have mapped the binding epitope of 2D9 to the P2 subdomain of VP60, revealing that it interacts with external loops that are critical for virus neutralization and histo-blood group antigen (HBGA) attachment [21, 27]. The structural basis for its specificity, where amino acid substitutions on the GI.1b P domain restrict binding, provides a molecular explanation for the antigenic differences between genotypes and underscores the necessity for genotype-specific diagnostic reagents [27]. This MAb has even demonstrated neutralizing capacity in vivo, preventing GI.2 infection in experimental challenges, highlighting its potential therapeutic and prophylactic utility [21].

Pathological Examination: The Foundation of Diagnosis

Despite the sophistication of molecular and serological tools, gross and histopathological examination remains the essential first step in diagnosing RHDV2, often providing the initial suspicion that triggers confirmatory testing. The characteristic lesion profile is remarkably consistent across susceptible species, including European rabbits (Oryctolagus cuniculus), black-tailed jackrabbits (Lepus californicus), and desert cottontails (Sylvilagus audubonii) [11, 45].

Gross Pathology

The most distinctive finding on necropsy is acute hepatic necrosis, manifesting as a pale, swollen, and friable liver with a pronounced accentuated lobular pattern, often described as "nutmeg liver" [36, 48]. Hemorrhagic diathesis is a hallmark, reflected by epistaxis (reported in 92% of cases in one study), pulmonary hemorrhage and edema, pericardial effusion, and widespread petechiae and ecchymoses on serosal surfaces and abdominal fat [11, 48]. The lungs are frequently congested, edematous, and hemorrhagic, while the spleen may be enlarged and congested. Incoagulability of blood is often noted upon collection, a direct consequence of DIC. An unusual presentation documented in an outbreak in Washington State highlighted a random, multifocal pattern of hepatic necrosis in contrast to the more typical diffuse involvement, illustrating subtle but important variations in disease manifestation [45].

Histopathology

Histologically, the pathognomonic lesion is massive, acute hepatocellular dissociation and necrosis/apoptosis, often most severe in the periportal to midzonal regions [11, 36]. Discrete, round, deeply eosinophilic, shrunken hepatocytes undergoing apoptosis, sometimes with pyknotic or karyorrhectic nuclei, are scattered throughout the liver parenchyma. These changes are accompanied by a striking paucity of inflammatory cell infiltration, which is a key feature distinguishing RHDV2-induced necrosis from bacterial or toxic hepatopathies. Fibrin thrombi are frequently observed in the small vessels of the lungs, kidneys, and liver, confirming the presence of DIC [12, 36]. Renal changes include acute tubular injury, and in some cases, intraglomerular capillary hyaline thrombi [36]. Splenic necrosis, often multifocal to diffuse, is a common concurrent finding, reflecting the involvement of the mononuclear phagocyte system [45]. Transmission electron microscopy (TEM) confirms the presence of non-enveloped, icosahedral viral particles approximately 32-40 nm in diameter within the cytoplasm of degenerated hepatocytes [12]. Remarkably, a study by Santos et al. [10] reported the presence of calicivirus-compatible virions in the nucleus of hepatocytes from infected Eurasian badgers (Meles meles), a finding that challenges the conventional understanding of the exclusively cytoplasmic replication cycle of caliciviruses and warrants further investigation.

Diagnostic Specificity and the Role of Ancillary Testing

The clinical pathology of RHDV2 infection is equally striking. Antemortem bloodwork in pet rabbits reveals profound biochemical abnormalities: marked increases in alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and bilirubin indicate severe hepatic dysfunction and cholestasis, while paradoxically, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) may be decreased due to severe, acute hepatic necrosis depleting hepatocellular enzymes [48]. Severe thrombocytopenia, prolonged prothrombin and activated partial thromboplastin times, and profound hypofibrinogenemia are consistent with DIC. Hypoglycemia reflects end-stage hepatic failure [48]. While these clinicopathologic findings are highly suggestive, they are not pathognomonic and must be interpreted in conjunction with histopathology and molecular testing.

Given that the gross and histologic lesions of RHDV2 overlap significantly with those of other acute hepatitides, including classical RHDV (GI.1), European Brown Hare Syndrome (EBHSV), and toxicities (e.g., aflatoxicosis, acetaminophen poisoning), confirmatory testing is essential. This is where the integration of molecular, serological, and pathological methods becomes critical. For instance, IHC or ISH can confirm the presence of viral antigen or nucleic acid within the characteristic hepatic lesions, linking the pathology to the infectious agent [47, 59]. The development of reliable IHC methods using the validated chicken egg antibody [59] now provides a powerful tool, enabling diagnostic laboratories without access to RT-qPCR to make a definitive diagnosis on FFPE tissues. In the context of surveillance, careful post-mortem examination of all suspect lagomorph deaths, whether in commercial rabbitries, backyard flocks, or wildlife, remains the first line of defense. The detection of RHDV2 in a captive mountain hare (Lepus timidus) in Germany and in endangered pygmy rabbits in Nevada underscores the critical need for pathologists to be vigilant for RHDV2 in non-traditional host species, especially as the virus continues to expand its host range [8, 13].

Genetic Diversity, Recombination Events, and Emergence of Recombinant RHDV2 Strains

The emergence of Rabbit Hemorrhagic Disease Virus 2 (RHDV2; genotype GI.2) represents one of the most significant evolutionary events in lagovirus history, fundamentally altering the epidemiological landscape of rabbit hemorrhagic disease (RHD) worldwide. Unlike classical RHDV (GI.1), which was considered a genetically stable pathogen for decades following its emergence in the 1980s, GI.2 has demonstrated a remarkable propensity for genetic diversification, primarily driven by recombination, a mechanism that has been central to its origin, its ongoing evolution, and its capacity for host range expansion [1, 5, 16]. The genetic architecture of circulating GI.2 strains is now recognized as a complex mosaic of genomic segments derived from both pathogenic and non-pathogenic lagoviruses, underscoring the dynamic nature of this RNA virus within the family Caliciviridae.

The Recombinant Origin of the RHDV2 Lineage

A foundational insight into the biology of RHDV2 is that the very first recognized GI.2 strain was itself a recombinant virus. Phylogenetic and recombination analyses have consistently demonstrated that the ancestral RHDV2 originated from a recombination event between a non-pathogenic lagovirus (GI.3P) and a pathogenic ancestor, resulting in a chimeric genome where the non-structural protein (NSP) region was derived from the benign GI.3 lineage (often referred to as rabbit calicivirus, RCV) and the structural capsid protein (VP60) region was acquired from a pathogenic strain [1, 5, 38]. This seminal recombination event, which likely occurred in Europe prior to 2010, conferred upon the emergent virus a unique biological profile: it retained the high pathogenicity and hepatotropism of pathogenic lagoviruses while acquiring a novel antigenic and structural configuration that allowed it to evade pre-existing immunity in rabbit populations and, critically, to infect and cause lethal disease in young rabbits and multiple Leporidae species [15, 20]. The initial GI.3P-GI.2 recombinant, first detected in France in 2010, thus represents a paradigm of how recombination between co-circulating benign and virulent caliciviruses can catalyze the emergence of a pandemic pathogen [1, 13, 16]. The World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization (FAO) have recognized the profound implications of this recombination-driven emergence for global rabbit health and food security.

A Mosaic Landscape: Cataloging the Principal Recombinant Profiles

Since the initial emergence, the genomic landscape of GI.2 has become increasingly complex, with multiple distinct recombinant lineages now described. Comprehensive genomic surveillance, particularly in Europe, has revealed that while all circulating GI.2 strains share a common GI.2-derived capsid backbone, they harbor markedly different non-structural genomic profiles [5, 31]. Estruch et al. [5], in a landmark temporal study of wild lagomorphs in northeastern Spain from 2014 to 2024, characterized four distinct recombinant lineages: GI.3P-GI.2, GI.1bP-GI.2, GI.4(p16)-GI.1bP-GI.2 (a triple recombinant), and GI.4P-GI.2. This diversity indicates that the NSP region of RHDV2 is a hot spot for recombination, with the virus repeatedly acquiring new polymerase and protease sequences from other circulating lagoviruses, both pathogenic (GI.1b) and non-pathogenic (GI.3, GI.4) [5, 31, 56].

In China, where RHDV2 was first detected in May 2020, the emergence of novel recombinants has been particularly rapid and concerning. Within just 12 months of the initial GI.2 incursion, a novel recombinant strain was identified that contained a GI.2 capsid gene but nonstructural genes from an unclassified or GI.1a genotype [9, 17]. Hu et al. [9] reported that this novel recombinant, which spread across multiple provinces, exhibited a longer infected time and lower mortality rate but maintained viral loads comparable to the original strain at the moribund stage. This paradoxical combination, lower acute lethality coupled with high shedding, may facilitate prolonged environmental contamination and more efficient transmission, potentially driving endemicity [9]. Li et al. [17] further characterized GI.1aP-GI.2 recombinants in China, demonstrating that this unique recombination pattern could moderately enhance virulence, including the ability to cause disease in rabbits vaccinated against the parental GI.2 strain, thereby posing a direct challenge to existing vaccine efficacy [17]. The detection of a GI.1aP-GI.2 recombinant in North America [33] and the identification of a recombinant GI.4P-GI.2 strain in domestic rabbits in Singapore that showed high homology to Australian variants [40] further illustrate the global reach of these mosaic genomes.

Global Emergence and Spatial-Temporal Dynamics of Recombinant Lineages

The epidemiological trajectory of these recombinant strains is not static; rather, they exhibit clear temporal turnover and spatial partitioning. In the Iberian Peninsula, a region with extensive wild rabbit populations, a striking pattern of sequential replacement has been documented. The initial GI.3P-GI.2 recombinant appeared transiently in 2014 but was rapidly displaced by GI.1bP-GI.2 strains, which dominated until approximately 2019 [5]. From 2020 onward, GI.4-related recombinants, particularly GI.4P-GI.2, became the prevailing lineage in both rabbits and hares [5, 10]. This turnover suggests that variations in the non-structural genomic region confer differential viral fitness, potentially influencing replication kinetics, host range, or transmissibility [5]. Intriguingly, in Poland, Fitzner et al. [31] documented the simultaneous circulation of GI.1a, GI.3P-GI.2 recombinants, and GI.2 strains in the same small geographical area, demonstrating that diverse lineages can co-exist without immediate competitive exclusion, yet follow distinct evolutionary trajectories.

In Africa, multiple introductions of GI.2 have been documented, with Tunisian strains characterized as GI.3P-GI.2 recombinants most likely introduced from Europe [38]. Sahraoui et al. [24] reported that Algerian strains also clustered with GI.3P-GI.2 recombinants, with older isolates grouping with a strain from neighboring Tunisia and more recent isolates showing close relation to North American strains, indicating multiple routes of introduction onto the African continent [24, 30]. These findings underscore the role of anthropogenic movement, likely via the international rabbit trade, in disseminating recombinant lineages across continents, a concern highlighted by the Centers for Disease Control and Prevention (CDC) and WOAH in their risk assessments for transboundary animal diseases.

Biological and Epidemiological Consequences of Recombination

The biological implications of this recombination-driven diversification are profound and extend beyond simple phylogenetic changes. Recombination can alter virulence, as demonstrated by the Chinese GI.1aP-GI.2 recombinants that exhibited enhanced pathogenicity in vaccinated animals [17]. It can also expand host range; the ability of GI.2 to infect and cause fatal disease in multiple Lepus species (e.g., mountain hares, European brown hares) [13, 20], Sylvilagus species (e.g., eastern cottontails, desert cottontails) [3, 11, 33], and even non-leporid species such as the Eurasian badger [10] is a direct consequence of its unique antigenic and structural configuration, which was forged through recombination. Mohamed et al. [33] demonstrated that eastern cottontails (Sylvilagus floridanus) were susceptible to a recombinant GI.1bP-GI.2 strain, shedding viral RNA and potentially serving as a bridge host in North America.

Furthermore, the emergence of novel recombinant strains has implications for diagnostic detection and vaccine design. The structural diversity in the VP60 capsid protein, particularly within the P2 subdomain which contains neutralizing epitopes, can lead to antigenic drift. Podadera et al. [21] and Leuthold et al. [27] mapped the binding epitope for the GI.2-specific monoclonal antibody 2D9, revealing that key residues in external loops 1, 4, and 5 are under selective pressure and can differentiate between GI.2 and other genotypes. This highlights the potential for immune escape, necessitating continuous molecular surveillance to ensure that diagnostic assays and vaccines remain effective against circulating recombinant strains [7, 25, 70]. The development of broad-spectrum chimeric vaccines, such as those described by Xiang et al. [70] using virus-like particles (VLPs) that replace surface loops to protect against both GI.1 and GI.2, represents an adaptive strategy to counter the antigenic diversity generated by recombination.

The non-pathogenic lagoviruses (RCV, genotypes GI.3 and GI.4) that serve as recombination donors continue to circulate widely in rabbit populations [56]. Cavadini et al. [56] demonstrated that RCV strains have been present in Italy for over two decades, with an estimated prevalence of 26% in farmed and wild rabbits, and have diversified into potential new genotypes (hypothetically GI.5 and GI.6). The continued circulation of these benign viruses ensures a reservoir of genetic material that can be exchanged with pathogenic GI.2 strains, perpetuating a cycle of recombination that will likely drive future viral emergence. This ongoing genetic churn is a hallmark of positive-sense RNA viruses and is particularly pronounced in the Caliciviridae, where recombination at the ORF1-ORF2 junction is a well-documented phenomenon.

Host Range and Interspecies Transmission of RHDV2 in Leporidae and Beyond

The emergence of Rabbit Hemorrhagic Disease Virus 2 (RHDV2, genotype GI.2) in 2010 in France marked a fundamental shift in the ecological and epidemiological landscape of lagovirus infections, primarily due to its unprecedented capacity for interspecies transmission. Unlike its predecessor, classic RHDV (genotype GI.1), which was largely restricted to the European rabbit (Oryctolagus cuniculus) with only sporadic, non-lethal infections in other species, RHDV2 has demonstrated a remarkable ability to infect, cause lethal disease, and propagate within a broad spectrum of leporid hosts and, perhaps more alarmingly, across taxonomic boundaries into non-leporid species [2, 10, 13]. This capability represents one of the most significant evolutionary developments in calicivirus biology and poses profound challenges for wildlife conservation, domestic animal health, and international trade. The expansion of the host range is not a static phenomenon; it is a dynamic process driven by the virus's high mutation rate, its propensity for recombination, and its ability to exploit ecological niches where sympatric host species overlap.

Expansion of the Leporid Host Spectrum

The host range of RHDV2 within the order Lagomorpha is now known to be extensive, encompassing multiple genera across diverse geographic regions. The initial detection and subsequent characterization of the virus in European rabbits quickly gave way to reports of mortality in hares (Lepus spp.), a finding that was biologically novel, as classic RHDV was not known to cause fatal disease in these animals. Early evidence from Europe demonstrated that RHDV2 could infect European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis), with subsequent studies confirming susceptibility in mountain hares (Lepus timidus) and black-tailed jackrabbits (Lepus californicus) in North America [6, 11, 13, 20]. The infection of mountain hares on the island of Hallands Väderö in Sweden was particularly illuminating; the outbreak occurred in a population where no rabbits were present, indicating that the virus could circulate exclusively within hare populations and persist in the absence of the primary reservoir host [20]. This finding challenged the notion that rabbits were essential for the maintenance of RHDV2 in wild populations.

The impact on North American leporids has been especially severe. Following its introduction into the southwestern United States in March 2020, RHDV2 was rapidly detected in desert cottontails (Sylvilagus audubonii), mountain cottontails (Sylvilagus nuttallii), antelope jackrabbits (Lepus alleni), and black-tailed jackrabbits [6, 11]. The pathological presentation in these native species mirrored that seen in European rabbits, with massive hepatocellular necrosis, pulmonary congestion, and disseminated intravascular coagulation being consistent findings [11]. Experimental infections have confirmed the susceptibility of the eastern cottontail (Sylvilagus floridanus), one of the most widely distributed lagomorphs in the United States, to RHDV2. In controlled studies, three of five eastern cottontails inoculated with RHDV2 developed fatal disease, and importantly, viral RNA was detected in urine, oral swabs, and rectal swabs, suggesting that this species could act as a competent reservoir and shed virus into the environment, thereby facilitating further transmission [33]. This experimental evidence translates into real-world epidemiological consequences, as RHDV2 has been detected in eight native North American species across 14 western states from 2020 to 2024, with 313 of 916 sampled wild lagomorphs testing positive by RT-qPCR [3].

Perhaps the most concerning aspect of this host range expansion is the documented infection of species of significant conservation concern. The pygmy rabbit (Brachylagus idahoensis), a sagebrush obligate and a species of special concern across the Intermountain West of the United States, has been confirmed positive for RHDV2 in Nevada [3, 8]. Given the ongoing habitat fragmentation and population declines already facing this species, the introduction of a highly fatal pathogen like RHDV2 could precipitate local extirpations. Similarly, the riparian brush rabbit (Sylvilagus bachmani riparius), a federally endangered subspecies in California, has been identified as highly susceptible to RHDV2, leading to emergency conservation measures, including targeted vaccination campaigns [3, 4, 19]. Modeling of this subspecies' population dynamics under RHDV2 pressure suggests that even with vaccination rates of 30-40%, the expected population size one year post-incursion would be only 53% of the expected size without virus introduction, highlighting the precarious state of these populations [19]. Furthermore, RHDV2 has been detected in red rock rabbits (Pronolagus spp.), expanding the known host range to yet another leporid genus [2].

Genetic and Recombination-Driven Mechanisms of Host Expansion

The biological basis for the expanded host range of RHDV2 is intrinsically linked to its genetic plasticity and, in particular, its capacity for recombination. The emergence of RHDV2 itself is widely believed to have been the result of a recombination event between a non-pathogenic rabbit calicivirus (RCV, GI.3) and a pathogenic lagovirus [1, 2, 5]. This genomic fluidity has continued, leading to the circulation of multiple recombinant lineages with distinct non-structural genomic profiles. Studies in northeastern Spain, a region with a high density of sympatric rabbits and hares, have identified at least four distinct recombinant strains circulating in hares and three in rabbits: GI.3P-GI.2, GI.1bP-GI.2, GI.4(p16)-GI.1bP-GI.2, and GI.4P-GI.2 [5]. The temporal dynamics of these recombinants are striking; GI.3P-GI.2 appeared transiently in 2014, was replaced by GI.1bP-GI.2 strains that dominated until 2019, and then GI.4-related recombinants progressively took over, with GI.4P-GI.2 prevailing from 2020 onward [5]. This sequential replacement suggests that variations in the non-structural genomic region confer differential fitness advantages, potentially in terms of replication efficiency, host cell tropism, or immune evasion, which in turn facilitate cross-species transmission.

The genomic organization of these recombinants is critical. The capsid (VP60) gene, which mediates host cell attachment via histo-blood group antigens (HBGAs), typically retains the GI.2 backbone, while the non-structural proteins, including the RNA-dependent RNA polymerase (RdRp) and the 3C-like protease (3CLpro), originate from various parental lineages [1, 5, 50]. This modular evolution allows the virus to "mix and match" genetic elements. For instance, the discovery of a novel recombinant in China, where the nonstructural portion clustered with GI.1a variants while the capsid gene shared 97.9% identity with North American GI.2 strains, demonstrates the potential for intercontinental genomic mixing [1]. Similarly, the first RHDV2 strain identified in China, SC20-01, was characterized as a G6/RHDV2 recombinant, and within a year of its introduction, novel recombinants carrying nonstructural genes from an unclassified lagovirus genotype were already circulating [9, 16]. The emergence of a GI.1aP-GI.2 recombinant in Chinese rabbit farms has been associated with moderately enhanced virulence, even breaking through immunity in rabbits vaccinated against parental GI.2 strains [17]. This ability to rapidly generate novel antigenic and pathogenic profiles through recombination is a primary driver of host range expansion, as it allows the virus to overcome species-specific barriers to infection.

Spillover Events Beyond Lagomorpha: A Paradigm Shift

Perhaps the most extraordinary aspect of RHDV2 epidemiology is its documentation in non-leporid hosts. The detection of RHDV2 in Eurasian badgers (Meles meles) in Portugal represents a significant paradigm shift, demonstrating that the virus is not strictly limited to the order Lagomorpha [10]. In this study, two of seven badgers found dead between 2017 and 2020 tested positive for a recombinant GI.4P-GI.2 strain of RHDV2. Histopathological examination revealed systemic infection, with viral antigen detected in multiple organs. Critically, transmission electron microscopy identified calicivirus-compatible virions in the nucleus of hepatocytes, a finding that challenges the conventional understanding of calicivirus replication cycles, which are predominantly cytoplasmic [10]. High viral loads were found in various tissues and fecal samples, indicating that infected badgers could potentially excrete the virus and contribute to environmental contamination and onward transmission to sympatric rabbit populations. This finding suggests that RHDV2 has the capacity to establish a multi-host system involving non-lagomorph carnivores, which could act as bridging hosts or even as maintenance hosts in certain ecological contexts.

Beyond direct infection of mammals, the role of arthropod vectors in mechanical transmission has been explored. Experimental studies have investigated the potential for RHDV2 transmission by hematophagous Diptera. While Aedes albopictus mosquitoes appeared unable to serve as direct mechanical vectors, the sand fly Phlebotomus papatasi was shown to be capable of transmitting the virus, as evidenced by seroconversion in two rabbits exposed to sand flies that had previously fed on a viral suspension [71]. Although no mortality or clinical disease occurred in the exposed rabbits, the demonstration of seroconversion indicates that infectious virus can be transferred. This suggests that sand flies could play a localized role in the mechanical spread of RHDV2, particularly in Mediterranean environments where these insects are abundant. While not a biological amplification mechanism, this route could contribute to the rapid, cryptic spread of the virus within and between rabbit populations.

Epidemiological Drivers of Interspecies Transmission

The dynamics of interspecies transmission are heavily influenced by ecological and anthropogenic factors. The spatial and temporal overlap between domestic and wild lagomorphs is a critical driver. Bayesian conditional autoregressive models analyzing RHDV2 case counts across 14 U.S. states from 2020 to 2024 have demonstrated a statistically significant positive relationship between RHDV2 cases in domestic lagomorphs and subsequent case counts in wild lagomorphs [53]. This finding underscores that domestic rabbit populations, including those in backyard hutches, commercial farms, and urban feral colonies, act as important sources of virus spillover into native wildlife. The study also identified developed land cover and lower annual precipitation as significant predictors of wild RHDV2 cases, suggesting that urban and peri-urban environments, where domestic and wild rabbits coexist at higher densities, represent high-risk interfaces for interspecies transmission [53]. This aligns with reports from California, where the initial detection of RHDV2 in a black-tailed jackrabbit on May 11, 2020, was rapidly followed by spillover into domestic rabbits in southern California, highlighting the porous boundary between wild and domestic populations in these landscapes [6].

Anthropogenic translocation of infected animals is another major, and often underappreciated, driver. The incursion of RHDV2 into Canada provides a clear example. The first detection in Quebec in 2016 was followed by independent incursions on Vancouver Island and the British Columbia mainland in 2018 and 2019 [14]. Whole-genome sequencing revealed that the Quebec isolate shared only 93% nucleotide identity with the Vancouver Island isolates, and a third incursion in Vancouver in 2019 shared only 97% identity with the 2018 isolates, indicating multiple, distinct introductions likely mediated by the movement of infected rabbits or contaminated fomites [14]. The outbreak scenario in Singapore in 2020 further illustrates the role of international trade and transport. Phylogenetic analyses of the Singapore GI.2 strain revealed it was a GI.4P-GI.2 recombinant with high homology to Australian variants, suggesting an introduction from Australia [40]. Similarly, the first detection of RHDV2 in China was linked to European strains, but subsequent recombinants showed a mosaic of European and North American genetic signatures, pointing to a complex network of global viral traffic [1, 9, 16, 17]. The detection of RHDV2 in Ghana in 2019, phylogenetically related to isolates from the Netherlands, and its subsequent identification in multiple North and Sub-Saharan African countries, underscores that the virus has become a globally distributed pathogen with frequent intercontinental movement [38, 54]. The World Organisation for Animal Health (WOAH) has classified RHD as a reportable disease, and the movement of live rabbits and rabbit products across borders remains the single most important factor in the long-distance dissemination of this virus.

Vaccine Development and Control Strategies: Virus-Like Particle and Recombinant Vaccines

The emergence and global dissemination of Rabbit Hemorrhagic Disease Virus 2 (RHDV2/GI.2) have necessitated a paradigm shift in vaccine development strategies. Unlike its predecessor, classical RHDV (GI.1), RHDV2 exhibits a broader host range, infecting multiple leporid species including Oryctolagus, Lepus, and Sylvilagus spp., and demonstrates significant antigenic divergence that renders traditional GI.1-based vaccines inadequately protective [2, 7, 15]. The inability to propagate RHDV2 efficiently in conventional cell culture systems has historically impeded the production of traditional inactivated or attenuated vaccines, driving innovation toward recombinant platforms, particularly virus-like particle (VLP) technologies [1, 34, 75]. These advanced platforms leverage the intrinsic immunogenicity of the VP60 major capsid protein, which self-assembles into structures morphologically and antigenically analogous to native virions, thereby eliciting robust humoral and cellular immune responses without the risks associated with live virus handling [25, 70, 75].

Molecular Basis of VLP Vaccine Design and Immunogenicity

The cornerstone of recombinant vaccine development against RHDV2 is the VP60 capsid protein, the primary antigen responsible for inducing protective neutralizing antibodies [7, 21, 27]. When expressed in heterologous systems, most commonly baculovirus-infected insect cells (e.g., Spodoptera frugiperda Sf9 or Hi-5 cells), the VP60 protein spontaneously assembles into non-infectious VLPs that are 30–40 nm in diameter, closely resembling the native calicivirus architecture [1, 25, 57]. This self-assembly is a critical feature, as the particulate nature of VLPs facilitates efficient uptake by antigen-presenting cells, leading to enhanced cross-presentation and activation of both B-cell and T-cell responses. Hu et al. (2025) demonstrated that cloning the vp60 gene from the Chinese GI.2 strain SC2020/0401 into a baculovirus expression vector yielded VLPs with potent hemagglutination activity, reaching titers of 1:2¹⁴ at 4 days post-infection, and that immunization with these VLPs conferred complete protection against homologous lethal challenge in rabbits [1, 57]. The structural integrity of these VLPs is paramount; the P2 subdomain of VP60, particularly its surface-exposed loops (loops 1, 4, and 5), contains critical neutralizing epitopes that are targeted by protective antibodies such as the GI.2-specific monoclonal antibody 2D9 [21, 27]. Leuthold et al. (2022) elucidated the atomic-level interaction of 2D9 with the VP60 P domain, revealing that this antibody binds with nanomolar affinity to a discontinuous epitope and may function by obstructing histo-blood group antigen (HBGA) attachment, a co-factor essential for viral entry [27]. This structural understanding underscores the importance of preserving native conformational epitopes in VLP-based vaccines, a feat readily achieved through recombinant expression but often compromised in traditional inactivated preparations.

Comparative Efficacy of VLP and Inactivated Platforms

While inactivated vaccines have historically been the mainstay of RHD control, their application to RHDV2 presents unique challenges. Traditional inactivated vaccines prepared from liver homogenates of infected rabbits carry inherent biosafety risks, batch-to-batch variability, and often require potent adjuvants to achieve adequate immunogenicity [60, 76]. In contrast, recombinant VLP vaccines offer a defined, scalable, and safer alternative. Wang et al. (2026) conducted a comprehensive evaluation of a VLP vaccine based on the prevalent Chinese YC05 strain, demonstrating that a single immunization with VLPs at a hemagglutination inhibition (HI) titer of 1:2⁸ induced specific antibodies that persisted at protective levels (HI ≥ 1:2⁵) for at least 180 days, with all vaccinated rabbits surviving challenge with heterologous RHDV2 strains [57]. This durability is particularly noteworthy given the rapid evolution of RHDV2 and the frequent emergence of recombinant variants [5, 9, 17].

Adjuvant selection remains a critical determinant of vaccine performance, even for highly immunogenic VLPs. Comparative studies have consistently shown that oil-emulsion adjuvants, such as Montanide ISA 70 or ISA 71 VG, elicit superior and more sustained antibody responses compared to traditional aluminum hydroxide gels [60, 76]. Ahmed et al. (2024) reported that inactivated RHDV2 vaccines formulated with Montanide ISA70 induced HI titers peaking at 10.67 log2 at 5 months post-vaccination, significantly higher than the 9.33 log2 peak observed with aluminum hydroxide-adjuvanted vaccines, and conferred 80–100% protection against homologous challenge for up to 6 months [60]. Similarly, Abodalal et al. (2022) demonstrated that a bivalent inactivated vaccine (targeting both RHDVa and RHDV2) adjuvanted with Montanide provided longer-lasting protective immunity than its aluminum hydroxide counterpart, reducing the need for frequent booster vaccinations [76]. The mechanism underlying this enhanced efficacy is attributed to the formation of a stable antigen depot at the injection site, promoting sustained antigen release and prolonged stimulation of the immune system. Novel adjuvant systems, such as sulfated lactosyl archaeol (SLA) archaeosomes, have also shown promise in experimental settings. Akache et al. (2023) demonstrated that SLA-adjuvanted recombinant VP60 formulations induced geometric mean antibody titers of 7,393 in rabbits, compared to just 117 with antigen alone, and achieved up to 87.5% survival following lethal RHDV2 challenge [68]. These findings highlight the potential of next-generation adjuvants to further optimize VLP vaccine performance.

Broad-Spectrum and Chimeric Vaccine Strategies

The co-circulation of both GI.1 and GI.2 genotypes, along with their recombinant derivatives, necessitates the development of broad-spectrum vaccines capable of conferring protection against multiple antigenic variants [25, 31, 70]. A landmark study by Xiang et al. (2025) addressed this challenge through a rational design approach, creating chimeric VLPs in which surface loops of the GI.1 VP60 protein were replaced with corresponding sequences from GI.2. These chimeric VLPs, expressed in baculovirus, assembled into particles of native size and morphology and, upon immunization, protected rabbits against lethal challenge with both GI.1 and GI.2 virulent strains, achieving efficacy comparable to a mixture of two wild-type VLPs [70]. This substitution strategy is particularly elegant as it preserves the overall capsid architecture while selectively presenting critical heterologous epitopes, thereby simplifying manufacturing and reducing costs. Mukhin et al. (2023) independently confirmed the necessity of bivalent or chimeric approaches, showing that monovalent GI.1 VLPs provided only 40% protection against GI.2 challenge, while monovalent GI.2 VLPs provided 30% protection against GI.1 challenge; however, a mixture of both VLPs achieved 100% protection against both genotypes [25]. These data underscore the antigenic distinctiveness of the two genotypes and the imperative for multivalent vaccine formulations in regions where both viruses circulate, such as parts of Europe, Africa, and Asia [24, 29, 31].

Alternative recombinant platforms have also been explored to enhance vaccine stability and delivery. Liu et al. (2022) developed a recombinant swinepox virus (rSWPV) expressing VP60, which self-assembled into VLPs within infected cells. A single immunization with 10⁶ PFU of this construct induced high levels of neutralizing antibodies within one week, persisting for at least 13 weeks, and provided complete protection against lethal RHDV challenge [75]. This live vector approach offers the advantage of intrinsic adjuvant properties and the potential for mucosal delivery, though concerns regarding pre-existing vector immunity in commercial rabbit populations may limit its widespread application. The Medgene Platform vaccine, a conditionally licensed recombinant product in the United States, has demonstrated remarkable durability, with a single vaccination series providing complete protection against lethal RHDV2 challenge for at least six months, while 18 of 19 unvaccinated controls succumbed to infection within 10 days [41]. Bosco-Lauth et al. (2022) further validated this platform, reporting 100% survival in vaccinated rabbits compared to 69% mortality in controls, with no clinical signs observed in the immunized group [43]. These findings are particularly significant given the absence of fully licensed RHDV2 vaccines in the United States and the urgent need for effective control measures following the 2020 outbreak [3, 6, 37].

Vaccination Strategies for Conservation and Endangered Species

The devastating impact of RHDV2 on wild lagomorph populations, particularly threatened and endangered species, has catalyzed the application of vaccination as a conservation tool. The riparian brush rabbit (Sylvilagus bachmani riparius), an endangered subspecies endemic to California, has been the focus of intensive vaccination efforts following the detection of RHDV2 in the state in May 2020 [4, 6, 19]. Moriarty et al. (2024) conducted a pivotal vaccine safety trial using Filavac VHD K C+V (Filavie), a commercially available inactivated bivalent vaccine, in 19 wild-caught riparian brush rabbits. Remarkably, all rabbits seroconverted within 7–10 days post-vaccination, with antibody titers ranging from 1:10 to 1:160, and no adverse effects were observed [4]. Antibody responses persisted for at least 60 days in 12 of 13 rabbits, and recapture data suggested longer-term immunity. This study established a critical proof-of-concept for emergency vaccination of free-ranging endangered lagomorphs and provided a model for protecting other vulnerable species, such as the pygmy rabbit (Brachylagus idahoensis), which has been severely impacted by RHDV2 in the western United States [3, 8].

Mathematical modeling has further refined vaccination strategies for conservation. Russell et al. (2024) developed a spatially explicit model for riparian brush rabbits on the San Joaquin River National Wildlife Refuge, demonstrating that even modest vaccination coverage (20–30%) could increase median population size by 34% compared to no intervention following an RHDV2 incursion [19]. Critically, the model projected that a 1% increase in vaccination rate was associated with a net increase of six rabbits in the remaining population, underscoring the dose-dependent benefit of vaccination. However, the model also highlighted that the success of vaccination campaigns is highly dependent on landscape connectivity, environmental transmission rates, and the need for booster doses given the relatively short lifespan of wild rabbits [19]. These insights are directly applicable to the management of other threatened leporids, including the mountain hare (Lepus timidus) in Scandinavia and the Iberian hare (Lepus granatensis) in Spain, both of which have experienced RHDV2-associated mortality [5, 13, 20].

Regulatory, Logistical, and Global Implementation Challenges

Despite the clear efficacy of VLP and recombinant vaccines, their global deployment faces significant regulatory and logistical hurdles. The World Organisation for Animal Health (WOAH) recognizes RHD as a notifiable disease, and the emergence of RHDV2 has prompted many countries to implement emergency use authorizations for vaccines that have not undergone full licensure [41, 43]. In the United States, the absence of a fully licensed RHDV2 vaccine has forced reliance on conditionally licensed products, which are subject to annual renewal and limited distribution [41, 72]. This regulatory patchwork creates uncertainty for rabbit owners, veterinarians, and wildlife managers. Shapiro et al. (2022) identified inconsistent jurisdiction across state and federal agencies, limited knowledge of domestic rabbit industry composition, and resource constraints as major barriers to effective RHDV2 management in the United States [72]. The study emphasized the need for federal leadership to coordinate cross-jurisdictional surveillance and vaccination campaigns, particularly at the wildlife-domestic animal interface where spillover events are most likely [53, 72].

In Europe, where RHDV2 has been endemic since 2010, vaccination strategies are more established but remain heterogeneous. Several EU member states have licensed bivalent vaccines targeting both GI.1 and GI.2, yet the emergence of novel recombinant strains, such as GI.3P-GI.2, GI.1bP-GI.2, and GI.4P-GI.2, raises concerns about vaccine escape [5, 9, 17]. Estruch et al. (2026) documented a progressive replacement of recombinant lineages in wild lagomorphs from northeastern Spain over a decade, with GI.4P-GI.2 strains becoming dominant after 2020 [5]. This dynamic evolution necessitates continuous antigenic surveillance and periodic vaccine strain updates, analogous to the annual reformulation of influenza vaccines. The detection of a GI.1aP-GI.2 recombinant in China that exhibited moderately enhanced virulence, even in rabbits vaccinated against parental GI.2, serves as a stark warning of the potential for vaccine breakthrough [17]. Li et al. (2023) reported that the novel recombinant SCNJ-2021 caused mortality in vaccinated rabbits, highlighting the urgent need for next-generation vaccines that target conserved epitopes or incorporate multiple antigenic variants [17].

Cost and accessibility remain formidable barriers, particularly in low- and middle-income countries where rabbit farming is a critical source of protein and income. In Africa, RHDV2 has been confirmed in multiple countries, including Algeria, Ghana, Morocco, and Tunisia, yet vaccine availability is often limited to imported, expensive products [24, 30, 38, 54]. The development of thermostable VLP vaccines that do not require cold chain storage, or the use of plant-based expression systems (e.g., molecular farming), could dramatically reduce production costs and improve access in resource-limited settings. Additionally, the use of alternative delivery methods, such as oral baits for wild lagomorphs, could revolutionize vaccination campaigns for conservation purposes, though significant research is needed to overcome challenges related to antigen stability in the gastrointestinal tract and the induction of mucosal immunity.

Future Directions: Antiviral Therapeutics and Integrated Control

While vaccination remains the cornerstone of RHDV2 control, the development of specific antiviral therapeutics offers a complementary strategy, particularly for outbreak containment and treatment of valuable genetic stock or endangered individuals. The 3C-like protease (3CLpro) of lagoviruses, which is essential for viral polyprotein processing, has emerged as a promising drug target [50, 51]. Perera et al. (2022) identified potent small-molecule inhibitors of RHDV2 3CLpro, including GC376, a protease inhibitor originally developed for feline infectious peritonitis, which demonstrated nanomolar potency in enzyme-based assays and efficacy in cell-based replicon systems [50]. Homology modeling revealed that the substrate-binding pocket of lagovirus 3CLpro shares structural similarities with other caliciviruses, suggesting that existing inhibitor libraries could be repurposed for RHDV2. The development of oral formulations of such inhibitors could enable rapid deployment during outbreaks, reducing viral shedding and environmental contamination. Furthermore, the identification of host factors essential for RHDV2 replication, such as nucleolin (NCL) and hemoglobin subunit beta (HBB), opens avenues for host-directed therapies [28, 39]. Zhu et al. (2022) demonstrated that NCL acts as a physical scaffold linking the viral RNA-dependent RNA polymerase (RdRp) to host proteins, and that NCL knockdown severely impaired RHDV replication in RK-13 cells [28]. Similarly, HBB was shown to interact with VP60, RdRp, and VPg, and its overexpression inhibited viral replication while activating interferon-γ expression [39]. These host-targeted approaches may offer a higher barrier to the development of viral resistance compared to direct-acting antivirals.

An integrated control strategy combining vaccination, antiviral therapy, enhanced biosecurity, and public education is essential for mitigating the global impact of RHDV2. The World Health Organization (WHO) and WOAH have emphasized the importance of One Health approaches that recognize the interconnectedness of human, animal, and environmental health. For RHDV2, this includes monitoring viral evolution through genomic surveillance, implementing rapid diagnostic testing using validated RT-qPCR assays on non-invasive samples such as rectal swabs or dried blood spots [44, 62], and engaging stakeholders, including hunters, pet owners, and commercial rabbit producers, in biosecurity practices [73, 74]. Shapiro et al. (2023) found that rabbit owners’ willingness to adopt biosecurity measures was strongly correlated with their knowledge of RHDV2 and trust in government agencies, underscoring the importance of targeted outreach and transparent communication [74]. As RHDV2 continues its relentless global spread, the development, licensure, and equitable distribution of safe, effective, and affordable VLP and recombinant vaccines will remain the most critical tool for protecting both domestic and wild lagomorph populations from this devastating pathogen.

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