Myxoma Virus
Overview and Taxonomy of Myxoma Virus
Taxonomic Classification and Phylogenetic Context
Myxoma virus (MYXV) is the prototypic member of the genus Leporipoxvirus within the subfamily Chordopoxvirinae of the family Poxviridae [10, 31]. This classification places MYXV among the large, double-stranded DNA (dsDNA) viruses that replicate exclusively within the cytoplasm of infected cells, a hallmark feature of the poxvirus family that necessitates the encoding of complete transcriptional and replicative machinery independent of the host cell nucleus [3, 19]. The Leporipoxvirus genus comprises viruses that naturally infect lagomorphs (rabbits and hares) and sciurids (squirrels), inducing cutaneous fibromas that facilitate mechanical transmission by biting arthropods [31]. MYXV, as the type species of this genus, has become a paradigmatic model for understanding host-pathogen coevolution, viral host range determinants, and the molecular basis of species jumping [3, 5, 31].
The genome of MYXV is a linear dsDNA molecule of approximately 161–163 kilobase pairs, encoding roughly 170–180 open reading frames (ORFs), depending on the strain [14, 17, 29]. Like all poxviruses, the genome is organized with a central conserved core region encoding essential replicative and structural proteins, flanked by variable terminal regions that contain genes involved in host range determination, immune evasion, and pathogenesis [3, 19, 31]. The terminal inverted repeats (TIRs) at each end of the genome contain identical sequences that are critical for genome replication and encapsidation. This genomic architecture underlies MYXV's remarkable ability to modulate host immune responses, a feature that has been refined through millions of years of coevolution with its natural hosts in the Americas [19, 31].
Natural Host Range and Historical Biogeography
In its ancestral ecological niche, MYXV is endemic in two species of Sylvilagus rabbits: the forest rabbit (Sylvilagus brasiliensis) in South America and the brush rabbit (Sylvilagus bachmani) in California [6, 31]. In these natural reservoir hosts, infection typically results in benign cutaneous fibromas localized at the site of arthropod inoculation, with systemic disease rarely observed [31]. The virus is transmitted mechanically by biting arthropods, particularly mosquitoes and fleas, which acquire the virus from the superficial fibroma lesions during blood feeding and subsequently transmit it to naive hosts [6, 32]. This vector-borne transmission strategy has profound implications for viral evolution, as the duration of host survival and the titer of virus in cutaneous lesions directly influence transmissibility, creating selective pressures that shape virulence phenotypes [7, 31].
The introduction of MYXV into European rabbit (Oryctolagus cuniculus) populations in the 1950s represents one of the most extensively documented cases of a host species jump and subsequent pathogen-host coevolution [12, 31]. In stark contrast to the benign infection in Sylvilagus species, MYXV causes a rapidly lethal disease in European rabbits known as myxomatosis, characterized by severe immunosuppression, disseminated cutaneous myxomas, conjunctival and periorbital edema, and bacterial superinfections leading to septicemia [7, 8, 29]. The mortality rate in naive European rabbit populations approached 99.8% upon initial introduction, yet within decades, a remarkable coevolutionary dynamic emerged: attenuated virus strains became dominant because they allowed infected rabbits to survive longer, thereby enhancing arthropod-mediated transmission, while rabbit populations evolved genetic resistance through selection on standing variation in immune-related genes [9, 26, 31].
Genomic Architecture and Host Range Determinants
The MYXV genome encodes a wealth of immunomodulatory proteins that collectively function to subvert host innate and adaptive immune responses [3, 19]. These viral immune evasion strategies are essential for productive infection in permissive hosts and, critically, represent the primary barriers to infection of novel host species [3, 5]. The host range of MYXV is remarkably narrow in nature, only leporids are permissive for productive viral replication, yet the virus exhibits a paradoxically broad cellular tropism in cultured cells from diverse mammalian species, including humans [10, 25]. This dichotomy arises because MYXV possesses multiple host range genes that function in a species-specific manner to counteract intrinsic antiviral mechanisms [4, 25].
Among the most critical host range determinants is the M062R gene, which encodes a member of the poxvirus C7L host range superfamily [4, 28]. The M062 protein functions by antagonizing the human sterile alpha motif domain-containing protein 9 (SAMD9), a potent antiviral factor that restricts poxvirus replication [4, 28]. In cells lacking functional SAMD9 or in the presence of M062, MYXV can replicate efficiently; however, deletion of M062 results in abortive infection in most mammalian cells due to SAMD9-mediated restriction [4, 28]. Similarly, the M029 protein, a double-stranded RNA (dsRNA) binding protein orthologous to vaccinia virus E3, plays a multifunctional role in inhibiting protein kinase R (PKR) and the type I interferon-induced antiviral state in a highly species-specific manner [19, 30]. M029 is essential for MYXV replication in rabbit cells but partially dispensable in human cells and completely unable to function in murine cells, illustrating the exquisite species specificity of these host range factors [30].
The MYXV genome also encodes the M013 protein, a viral pyrin domain-only protein that simultaneously antagonizes both the inflammasome and NF-κB signaling pathways through distinct structural motifs [22]. M013 interacts with ASC-1 to inhibit caspase-1 activation and with NF-κB1 to suppress pro-inflammatory cytokine secretion [22]. Additionally, the M-T7 protein functions as a chemokine-binding protein that disrupts chemokine-glycosaminoglycan interactions, attenuating inflammatory responses and promoting a pro-resolution environment during infection [18, 24]. The Serp-1 protein, a serine protease inhibitor, targets plasminogen activators and factors in the coagulation cascade, further modulating the inflammatory response [18]. These immunomodulatory proteins, among many others encoded by MYXV, collectively enable the virus to establish productive infection even in the face of robust host immune activation [3, 19].
The Species Jump to Iberian Hares: Emergence of Recombinant MYXV
In late 2018, a dramatic epidemiological event occurred in the Iberian Peninsula: MYXV was identified as the cause of widespread mortality in Iberian hares (Lepus granatensis), a species previously considered resistant to myxomatosis [13, 14, 17]. Genomic sequencing of viruses isolated from affected hares revealed a novel recombinant MYXV strain, designated MYXV-Toledo (MYXV-Tol) or hare-associated MYXV (ha-MYXV), that harbored a ~2.8 kilobase insertion within the M009L gene, disrupting it into two ORFs (M009L-a and M009L-b) [14, 17, 23]. This insertion region, absent in all previously characterized MYXV strains, contains four intact ORFs (M060L, M061L, M064L, and M065L) that are phylogenetically related to genes found in other poxviruses, including MYXV itself and possibly other chordopoxviruses [14, 17]. Critically, one of these inserted genes, designated M159, encodes a novel member of the poxvirus C7L host range family that was functionally demonstrated to be essential for MYXV replication in hare cells [5].
The acquisition of M159 enabled MYXV-Tol to productively infect and replicate in hare cells, whereas the parental MYXV-Lausanne strain could not [5]. Recombinant viruses engineered to lack M159 (vMyxTol-ΔM159) were completely unable to replicate in hare kidney cells, demonstrating that M159 is the key host range factor permitting this species leap [5]. Interestingly, M159 is expressed as an early/late gene and translocates to the nucleus at later time points, suggesting a nuclear function distinct from the cytoplasmic activities of other C7L family members [5]. This genomic recombination event transformed MYXV from a rabbit-specific pathogen into a dual-host pathogen capable of causing lethal disease in both European rabbits and Iberian hares [13, 17].
The emergence of ha-MYXV has had devastating consequences for Iberian hare populations. Epidemiological surveillance from 2018–2020 documented over 487 PCR-confirmed cases across 372 affected areas in Spain, with an apparent mean mortality rate of 55.4% and a median of 70% [13]. Retrospective analysis suggests the virus may have been circulating since June 2018, with outbreaks peaking in August and October before declining sharply in winter [13]. The disease presentation in Iberian hares differs markedly from classic myxomatosis in rabbits: affected hares exhibit bilateral blepharoconjunctivitis, epistaxis, intense congestion and edema of multiple organs, and internal hemorrhages, but notably lack the characteristic cutaneous myxomas seen in rabbits [8]. Histopathologically, ha-MYXV infection in hares is characterized by hyperplastic epidermis with hyperkeratosis, myxoid matrix deposition in the dermis, severe lymphoid depletion in the spleen, interstitial pneumonia, and hepatic necrosis [8]. The virus targets epithelial cells, myxoma cells, macrophages, lymphocytes, fibroblasts, endothelial cells, hepatocytes, and Leydig cells, indicating a broad cellular tropism in this novel host [8].
Ongoing Range Expansion and Implications
Since its initial detection in Iberian hares, ha-MYXV has continued to expand its geographic and host range. By 2020, the recombinant virus was detected in European rabbits (Oryctolagus cuniculus) in Portugal, demonstrating that ha-MYXV can also cause lethal disease in the original rabbit host [15, 16]. This finding shattered the initial assumption of species segregation, wherein classic MYXV strains circulated in rabbits and recombinant strains in hares, and raised urgent concerns about the safety of the rabbit farming industry and conservation of wild leporid populations [15, 16]. More recently, in 2023–2024, recombinant MYXV emerged in European brown hares (Lepus europaeus) along the Germany–Netherlands border, causing increased deaths associated with swollen eyelids, head edema, and dermatitis of the face, legs, and perineum [1]. Epidemiological data suggest the virus introduction may date back as early as September 2020, with radial spread from the border area [1]. Phylogenetic analyses of ha-MYXV genomes from these outbreaks indicate independent emergence events rather than direct dispersal from the Iberian Peninsula, suggesting that the recombination event enabling hare infection may have occurred multiple times or that the virus is undergoing rapid adaptive evolution in new geographic regions [1, 2].
Serological surveys of Iberian hares conducted before and after the 2018 outbreak have revealed critical insights into the pre-existing immunological landscape. Antibodies reactive to MYXV or antigenically related viruses were detected in hares as early as 1994–1999, with a seroprevalence of 5.4% in apparently healthy animals during that period, increasing to 13.0% in 2017–2019 [27]. This indicates that Iberian hares had experienced sporadic, likely subclinical, infections with MYXV or a related leporipoxvirus decades before the emergence of the highly virulent ha-MYXV [27]. These findings suggest that the species barrier between rabbits and hares was not absolute, but that the recombination event, particularly the acquisition of M159, allowed ha-MYXV to overcome the residual host restrictions that previously limited MYXV replication in hare cells [5, 27].
Evolutionary Paradigm and Taxonomic Implications
The MYXV–rabbit coevolutionary system has long served as a textbook example of pathogen adaptation to a novel host, demonstrating how selection for transmissibility drives attenuation of virulence while host populations evolve resistance through parallel genetic changes across geographically separated populations [7, 9, 31]. In Australia, the release of MYXV in 1950 initiated a coevolutionary arms race that has been meticulously documented: early highly virulent strains (grades 1–2) were rapidly replaced by moderately attenuated strains (grades 3–4) that enhanced transmission by prolonging host survival, while rabbit populations evolved resistance through selection on multiple immunity-related genes, particularly interferon proteins [7, 9, 26]. Remarkably, similar resistance alleles were selected in parallel across Australian, French, and British rabbit populations, demonstrating convergent evolution on standing genetic variation [9].
The emergence of ha-MYXV represents a new chapter in this evolutionary narrative. The acquisition of the 2.8 kb recombinant region and the functional characterization of M159 as a novel host range factor demonstrate that genomic recombination, rather than simple point mutation accumulation, can enable dramatic host range expansions [5, 14]. This finding has profound implications for understanding poxvirus evolution and the potential for future species jumps. The taxonomic classification of MYXV, while firmly established within the Leporipoxvirus genus, must now accommodate the recognition that the species boundary between MYXV and other poxviruses is not impermeable; recombination with unknown poxviruses in sympatric lagomorph populations could generate novel strains with altered host range, virulence, and epidemiological potential [3, 5, 20]. Indeed, coinfection of Iberian hares with MYXV-Tol, polyomaviruses, and anelloviruses has been documented, highlighting the potential for viral recombination in these naive hosts [20]. The continued surveillance of wild lagomorph populations for emerging MYXV strains, coupled with genomic characterization and functional studies of host range determinants, remains essential for predicting and mitigating future cross-species transmission events [2, 11, 13]. The development of differential diagnostic tools, such as the quadruplex qPCR capable of distinguishing classic MYXV from ha-MYXV strains [11], and serological assays for detecting exposure in novel host species [2, 21, 27], are critical components of this surveillance infrastructure.
Molecular Pathogenesis of Myxoma Virus
The molecular pathogenesis of myxoma virus (MYXV) represents a paradigm of host-pathogen coevolution, characterized by an intricate arsenal of immunomodulatory proteins that subvert host antiviral defenses, determine species tropism, and drive the clinical manifestations of myxomatosis. As the prototypic member of the Leporipoxvirus genus within the Poxviridae family, MYXV possesses a large, double-stranded DNA genome of approximately 161.8 kilobases, encoding over 170 open reading frames [10, 31]. A substantial proportion of these genes are dedicated to non-essential functions related to immune evasion, host range determination, and modulation of the cellular environment, reflecting the virus’s evolutionary adaptation to its natural leporid hosts [3, 19]. The molecular mechanisms underlying MYXV pathogenesis are not monolithic; they vary dramatically between the virus’s natural reservoir hosts (Sylvilagus spp.), its highly susceptible accidental host (the European rabbit, Oryctolagus cuniculus), and newly emergent hosts such as the Iberian hare (Lepus granatensis), where a recent species jump has rewritten the textbook understanding of poxvirus host range [5, 14, 17].
The Immunomodulatory Arsenal: Subversion of Innate and Adaptive Immunity
Central to MYXV pathogenesis is its capacity to disarm the host innate immune response, particularly the type I interferon (IFN) system, the inflammasome, and the complement cascade. The virus encodes a suite of proteins that function at virtually every level of the antiviral signaling pathway. The M029 protein, a double-stranded RNA (dsRNA) binding protein orthologous to vaccinia virus E3, is a critical host range and virulence determinant. M029 inhibits protein kinase R (PKR) and, importantly, antagonizes the type I IFN-induced antiviral state in a highly species-specific fashion. In rabbit cells, M029 completely overcomes the IFN-induced block to viral replication, whereas in human cells this inhibition is only partial, and in murine cells it is virtually absent [19, 30]. This species-specificity is a key molecular barrier that restricts MYXV’s natural host range but is also a factor that permits its use as an oncolytic agent in non-leporid species [10].
Another cornerstone of MYXV immune evasion is the M013 protein, a viral pyrin domain-only protein (vPOP) that dually antagonizes both the NF-κB and inflammasome pathways. M013 binds directly to the adaptor protein ASC-1 via its N-terminal pyrin domain, utilizing a distinctive negatively charged surface electrostatic region to inhibit caspase-1 activation and subsequent secretion of IL-1β and IL-18 [22]. Simultaneously, a unique, positively charged 33-residue C-terminal tail of M013 mediates interaction with NF-κB1, suppressing the transcription of pro-inflammatory cytokines [22]. This dual blockade is particularly effective in myeloid cells, which are key sentinels of the innate immune system. The M-T7 protein, a secreted glycoprotein, functions as a chemokine modulator by binding to and inhibiting the interaction of chemokines with glycosaminoglycans, thereby disrupting the chemokine gradient required for leukocyte recruitment to sites of infection [18, 24]. This broad-spectrum chemokine inhibition reduces damaging inflammation but also delays the adaptive immune response, allowing the virus to establish a foothold in the host.
The virus also targets the complement system and the apoptotic machinery. The Serp-1 protein, a serine protease inhibitor, targets the plasminogen activator and factor Xa pathways, reducing inflammation and tissue damage, a mechanism that has been exploited therapeutically in models of spinal cord injury [18]. Furthermore, MYXV encodes multiple proteins that inhibit apoptosis, including M-T5, which interacts with the cellular protein Akt to promote cell survival and viral replication [36]. The M-T5 protein also interacts with the SKP-1 component of the SCF ubiquitin ligase complex, linking viral manipulation of the cell cycle to the inhibition of apoptosis [36]. This anti-apoptotic function is essential for maintaining the cellular machinery required for viral DNA replication and assembly, which occurs exclusively in the cytoplasm within characteristic viral factories [39].
Host Range Determinants and the Molecular Basis of Species Jumping
The molecular pathogenesis of MYXV is inextricably linked to its host range. The virus is naturally maintained in Sylvilagus rabbits (e.g., S. brasiliensis, S. floridanus), where it causes only benign cutaneous fibromas. In the European rabbit, however, infection results in the lethal, systemic disease myxomatosis [31]. This differential pathogenesis is governed by a set of host range genes that allow the virus to overcome species-specific restriction factors. The M062R gene, a member of the poxvirus C7L host range superfamily, is the dominant and broad-spectrum host range determinant for MYXV. The M062 protein antagonizes the host restriction factor Sterile Alpha Motif Domain-containing 9 (SAMD9). In human cells, SAMD9 functions as an antiviral protein that restricts poxvirus replication; M062 binds to and inhibits SAMD9, allowing productive infection to proceed [4, 28]. The interaction between M062 and SAMD9 is highly specific, and the N-terminal domain of SAMD9 is critical for this antagonism [28]. When M062 is deleted (ΔM062R), MYXV infection becomes abortive in most mammalian cells, and the virus activates a cGAS-dependent type I interferon response, highlighting the critical role of this gene in suppressing innate DNA sensing pathways [4].
The most dramatic demonstration of host range evolution occurred in 2018, when a recombinant MYXV (designated MYXV-Tol or ha-MYXV) emerged in the Iberian hare, causing high mortality in a species previously considered resistant to myxomatosis [13, 17, 33]. Genomic sequencing revealed that this new strain acquired a ~2.8 kb recombinant region inserted within the M009L gene, disrupting it into two open reading frames (M009L-a and M009L-b) [14, 16, 17]. This insertion contains four novel genes, including M159, which is an orthologue of the vaccinia virus C7L host range family [5]. Functional studies demonstrated that M159 is the critical factor enabling MYXV-Tol to replicate in hare cells. While wild-type MYXV (lacking M159) and a MYXV-Tol ΔM159 knockout virus were unable to productively infect hare fibroblasts, the parental MYXV-Tol underwent fully productive infection [5]. M159 is expressed as both an early and late gene, and interestingly, it translocates to the nucleus at later time points, suggesting a novel mechanism of action distinct from other C7L family members [5]. This acquisition of a new host range gene allowed the virus to leap a species barrier that had been stable for over 70 years, demonstrating the profound impact of recombination on poxvirus pathogenesis and emergence [3, 14].
Pathogenesis in the European Rabbit: From Cutaneous Nodules to Systemic Collapse
In the susceptible European rabbit, MYXV pathogenesis proceeds through a well-characterized sequence of events. Following inoculation by an arthropod vector (primarily mosquitoes and fleas), the virus replicates locally in dermal fibroblasts and epidermal keratinocytes [32, 34]. The primary lesion is a cutaneous myxoma, a gelatinous tumor-like swelling characterized by the proliferation of stellate “myxoma cells” embedded in a matrix of mucopolysaccharides [8]. The virus then spreads via the lymphatics to regional lymph nodes, where it replicates extensively, causing lymphoid depletion and immunosuppression [8]. This is followed by a cell-associated viremia, with virus carried by infected leukocytes (particularly monocytes and lymphocytes) to distant sites, including the skin (secondary myxomas), conjunctiva, respiratory tract, and genitalia [8, 34].
The hallmark clinical signs, bilateral blepharedema (swollen eyelids), anogenital edema, and severe conjunctivitis, are a direct consequence of viral replication in endothelial cells and the induction of vascular permeability [8, 34]. Histopathologically, infected keratinocytes exhibit hydropic degeneration and contain characteristic eosinophilic intracytoplasmic inclusion bodies (the “myxoma bodies”), which are sites of viral assembly [8, 34]. In peracute and acute cases, death can occur within 7-10 days due to a combination of factors: severe immunosuppression leading to secondary bacterial septicemia, pulmonary edema, and overwhelming viral burden [7, 8, 29]. In the Australian context, modern MYXV isolates have evolved to cause an “amyxomatous” phenotype, characterized by minimal cutaneous lesions but acute death with pulmonary edema and bacterial invasion, suggesting a shift in pathogenesis towards a more immunosuppressive and septicemic disease [7, 29]. This evolution is driven by selection for prolonged virus replication and transmission, as rabbits that survive longer with high viral titers in the skin are more likely to be fed upon by vectors [7].
Pathogenesis in the Iberian Hare: An Acute and Hyperacute Presentation
The molecular pathogenesis of ha-MYXV in the Iberian hare differs markedly from classic myxomatosis in rabbits. Infected hares present with an acute or hyperacute disease course, with a median mortality rate of 70% [13]. Gross lesions include bilateral blepharoconjunctivitis, epistaxis, intense congestion and edema of multiple organs, and internal hemorrhages, but notably, visible myxomas are absent [8]. Histologically, the skin shows hyperplastic epidermis with hyperkeratosis and a myxoid matrix in the dermis, but the characteristic myxoma cells are less prominent than in rabbits [8]. Viral antigen is detected in epithelial cells, macrophages, lymphocytes, fibroblasts, and endothelial cells in the skin, as well as in hepatocytes, Leydig cells, and within the spleen and lungs [8]. The dramatic lymphoid depletion in the spleen and necrosis in the liver and testis indicate a systemic, highly virulent infection that overwhelms the naive host before significant cutaneous pathology can develop [8]. This hyperacute pathogenesis is likely due to the absence of prior co-evolutionary pressure; the hare immune system has not been selected to recognize and control this specific viral strain, and the virus, armed with the novel M159 host range factor, replicates unchecked [5, 8].
Cellular Tropism and the Role of Host Factors in Restriction
The outcome of MYXV infection at the cellular level is determined by a complex interplay between viral host range proteins and cellular restriction factors. In human cancer cells, which are non-permissive for MYXV in normal tissues but permissive in many transformed lines, the virus has been extensively studied as an oncolytic agent [10]. The tropism for cancer cells is largely mediated by defects in antiviral signaling pathways, particularly the type I IFN response and the Akt signaling pathway [10, 41]. However, even within permissive cancer cells, MYXV replication is regulated by host DEAD-box RNA helicases. An siRNA screen identified five helicases (DDX3X, DDX5, DHX9, DHX37, DDX52) that inhibit MYXV replication (anti-viral), and three (DHX29, DHX35, RIG-I) that are required for optimal replication (pro-viral) [25]. DHX9, in particular, forms unique cytoplasmic “antiviral granules” during late MYXV infection in restrictive human cancer cells. These granules sequester viral proteins and dsRNA, reducing late protein synthesis and limiting progeny virus formation [39]. The formation of these granules is dependent on viral DNA replication and late gene expression, and their disruption (e.g., by DHX9 knockdown) significantly enhances MYXV replication [39]. This highlights a novel, poxvirus-specific antiviral mechanism that operates at the post-replicative stage.
Furthermore, the metabolic state of the host cell profoundly impacts MYXV pathogenesis. The virus lacks the gene for one subunit of ribonucleotide reductase (RR), an enzyme essential for DNA synthesis. Consequently, MYXV replication is dependent on the host cell’s RR activity. In soft tissue sarcomas, the oncolytic efficacy of MYXV directly correlates with the expression level of the RRM2 subunit; tumors with high RRM2 expression are more susceptible to viral killing [40]. Similarly, defects in arginine metabolism, specifically the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), create a functional arginine auxotrophy that limits MYXV replication in tumors [37]. This metabolic dependency can be overcome by engineering the virus to express exogenous ASS1, providing a strategy to enhance oncolytic efficacy in metabolically restrictive tumor microenvironments [37]. These findings underscore that molecular pathogenesis is not solely a function of viral immune evasion but is also critically dependent on the metabolic and signaling landscape of the host cell.
Molecular Mechanisms of Cell Death and Viral Dissemination
MYXV induces a multifaceted cell death program in infected cells, which is central to both its pathogenesis and its oncolytic potential. While the virus encodes anti-apoptotic factors to ensure productive replication, it also triggers cell death pathways, particularly in the context of oncolytic virotherapy. MYXV infection can induce apoptosis, autophagy, and a specialized form of cell death termed autosis [35, 36]. In multiple myeloma cells, MYXV upregulates autophagy markers such as ATG-5, Beclin-1, and LC3B, and autophagosomes are observed by transmission electron microscopy, indicating that autophagy contributes to the anti-myeloma effect [35]. In a remarkable synergy with T cells, MYXV-infected CAR-T cells induce tumor cell autosis, a type of cell death characterized by the accumulation of autophagosomes and endoplasmic reticulum stress. This is driven by a dual signaling mechanism: T cell-derived IFNγ activates Akt signaling, while the viral M-T5 protein interacts with SKP-1 and VPS34 to trigger robust autosis [36]. This mechanism provides a potent bystander killing effect that can overcome tumor antigen escape, a major limitation of conventional immunotherapy [36].
The virus also manipulates the nuclear export machinery to facilitate its replication. In restrictive human cancer cells, the nuclear export pathway, mediated by XPO-1 (exportin 1), limits MYXV replication by retaining antiviral restriction factors in the cytoplasm. Inhibition of XPO-1 with drugs like selinexor traps these restriction factors in the nucleus, dramatically enhancing viral replication and killing of cancer cells [38]. This demonstrates that the subcellular localization of host proteins is a critical determinant of MYXV permissivity and represents a targetable vulnerability for enhancing oncolytic therapy [38]. In summary, the molecular pathogenesis of MYXV is a dynamic and multifaceted process, governed by a sophisticated viral immunomodulatory arsenal, a rapidly evolving host range repertoire, and a deep dependence on the metabolic and signaling state of the host cell. The recent species jump to Iberian hares, facilitated by a single recombinant host range gene, serves as a stark reminder of the evolutionary plasticity of poxviruses and the constant threat of emerging viral diseases in naive populations [5, 17].
Immune Modulation and Host Range Determinants
Myxoma virus (MYXV) represents a masterclass in viral immune evasion, encoding a sophisticated arsenal of immunomodulatory proteins that not only facilitate its replication in the natural leporid host but also dictate its capacity for cross-species transmission. The interplay between these viral immune modulators and the host’s innate and adaptive immune systems is the central determinant of MYXV pathogenesis, host range, and the remarkable evolutionary trajectory that has led to recent species jumps. Understanding these molecular mechanisms is critical for predicting future spillover events and for rationally designing oncolytic MYXV-based therapeutics.
The Immunomodulatory Arsenal of MYXV
MYXV, like all poxviruses, dedicates a substantial portion of its large dsDNA genome to encoding proteins that subvert host antiviral responses. These immune modulators function at virtually every level of the host defense system, from pattern recognition and interferon signaling to inflammasome activation and apoptosis. The M013 protein is a particularly illustrative example of viral multifunctionality. M013 is a viral homologue of the pyrin domain-only protein (vPOP) family and acts as a dual antagonist of both the NF-κB and inflammasome signaling pathways. Structural studies have revealed that M013 employs distinct structural motifs for each function: a negatively charged surface on its pyrin domain (PYD) mediates binding to ASC-1 to inhibit caspase-1 activation and subsequent IL-1β secretion, while a unique, positively charged 33-residue C-terminal tail is essential for interaction with NF-κB1, thereby suppressing pro-inflammatory cytokine production [22]. This dual targeting of two central pro-inflammatory hubs demonstrates the evolutionary pressure on MYXV to simultaneously disarm both the transcriptional and proteolytic arms of the innate immune response.
The type I interferon (IFN) system represents a critical barrier that MYXV must overcome. The viral M029 protein, an ortholog of the vaccinia virus E3 dsRNA-binding protein, is a key player in this arena. M029 functions as a host range factor by inhibiting protein kinase R (PKR), a sentinel enzyme that detects viral dsRNA and shuts down translation. However, the activity of M029 extends beyond PKR antagonism. Critically, M029 inhibits the type I IFN-induced antiviral state in a highly species-specific fashion: it is fully effective in rabbit cells, partially effective in human cells, and completely ineffective in mouse cells [30]. This species-specificity is a fundamental constraint on MYXV host range. In rabbit cells, M029 can completely overcome the antiviral state established by pre-treatment with type I IFN, whereas in mouse cells, the IFN-induced state remains fully restrictive even in the presence of M029. This observation suggests that M029 targets additional, as-yet-unidentified host proteins that are species-specific, and that the ability of MYXV to establish a productive infection in a new host species is contingent upon the compatibility of M029 (and other host range factors) with the host’s IFN signaling network.
Host Range Determinants and the C7L Superfamily
The poxvirus host range C7L superfamily is a conserved group of proteins that are essential for determining species and cell type tropism. In MYXV, the dominant and broad-spectrum host range determinant of this family is the M062R gene product. M062 is essential for MYXV infection in almost all mammalian cells tested, and its mechanism of action involves antagonizing the host restriction factor Sterile Alpha Motif Domain-containing 9 (SAMD9). The M062-SAMD9 interaction is a critical molecular battleground. In human cells, SAMD9 functions as an antiviral restriction factor, and its activity is suppressed by M062. Deletion of M062 (ΔM062R) results in an abortive infection that is dependent on SAMD9 function. Furthermore, ΔM062R MYXV activates the cGAS-dependent DNA sensing pathway, triggering a robust type I IFN response in human monocytes and macrophages, a response that is normally suppressed during wild-type MYXV infection [4]. This establishes a direct link between the host range function of M062, the suppression of DNA sensing, and the evasion of innate immunity. The interaction domain on human SAMD9 that is targeted by M062 has been mapped, revealing that exogenous expression of the SAMD9 N-terminus can sequester viral M062 and potently inhibit wild-type MYXV infection, underscoring the delicate stoichiometric balance required for successful viral replication [28].
The Species Leap: Acquisition of a Novel Host Range Factor
The most dramatic demonstration of the importance of host range determinants in MYXV biology is the recent species jump from European rabbits (Oryctolagus cuniculus) to Iberian hares (Lepus granatensis). For over 70 years, MYXV circulated in European rabbits without causing significant disease in sympatric hare species. However, in 2018, a novel recombinant virus, designated MYXV-Tol (or ha-MYXV), emerged in Spain and Portugal, causing a hyperacute, highly lethal myxomatosis-like disease in Iberian hares [13, 17]. Genomic characterization of MYXV-Tol revealed a critical ~2.8 kb insertion within the M009L gene, which disrupted it into two open reading frames (M009L-a and M009L-b) and introduced four novel poxviral genes [14, 17]. Among these newly acquired genes is M159, which has been identified as the key factor enabling the species leap.
M159 is an orthologous member of the vaccinia virus C7 host range family [5]. In a series of elegant experiments using recombinant viruses, it was demonstrated that while MYXV-Tol undergoes fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (which lacks M159) nor a MYXV-Tol ΔM159 knockout virus can replicate in these cells. This unequivocally demonstrates that the ability of MYXV-Tol to infect and replicate in hare cells is entirely dependent on the presence of M159 [5]. Interestingly, M159 does not contribute to increased replication in rabbit cells, but it does upregulate MYXV replication in non-permissive and semi-permissive human cancer cells, suggesting that the cellular pathway targeted by M159 is conserved across mammalian species. The acquisition of M159, therefore, represents a gain-of-function event that provided MYXV with the necessary molecular toolkit to overcome the innate immune barriers present in a previously resistant host species. This event has had devastating ecological consequences, with population declines of Iberian hares documented across the Iberian Peninsula [33]. The emergence of ha-MYXV underscores the potential for poxviruses, with their large, plastic genomes and extensive repertoires of host range genes, to undergo rapid adaptation and cross species barriers, particularly when recombination events introduce novel immunomodulatory genes.
Innate Immune Barriers and Cellular Tropism in Non-Leporid Hosts
Outside of its natural leporid hosts, MYXV exhibits a highly selective tropism for cancer cells, a property that has been exploited for oncolytic virotherapy. This tropism is largely dictated by the status of the host cell’s innate immune and signaling pathways. In normal, non-transformed human cells, MYXV infection is typically abortive, as these cells mount a robust antiviral response that restricts viral replication. However, many cancer cells harbor defects in antiviral signaling pathways, such as the type I IFN response or the PKR pathway, rendering them permissive to MYXV infection and oncolysis [10].
The cellular DEAD-box RNA helicases are a family of proteins that play critical roles in RNA metabolism and antiviral immunity. A systematic siRNA screen targeting all 58 human DEAD-box RNA helicases identified several that regulate MYXV tropism in human cancer cells. Five helicases (DDX3X, DDX5, DHX9, DHX37, DDX52) were found to be antiviral, inhibiting optimal MYXV replication, while three others (DHX29, DHX35, RIG-I) were identified as proviral [25]. Among these, DHX9 has been shown to form unique cytoplasmic antiviral granules during the late stages of MYXV replication in human cancer cells. These DHX9 antiviral granules sequester viral proteins and dsRNA, significantly reducing nascent protein synthesis and viral late gene transcription, thereby restricting progeny virus formation [39]. The formation of these granules is dependent on MYXV DNA replication and late protein synthesis, and their disruption through DHX9 knockdown significantly enhances MYXV replication in normally restrictive cancer cells. This reveals a novel, non-canonical antiviral mechanism that poxviruses must overcome to establish productive infection in human cells.
Further metabolic constraints also influence MYXV host range at the cellular level. The replication of oncolytic MYXV is dependent on the bioavailability of arginine. Many cancers are functionally auxotrophic for arginine due to the epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme required for arginine biosynthesis. Tumors formed from ASS1-negative cells display significantly reduced MYXV replication and poorer therapeutic responses, as the virus cannot access sufficient arginine for its own replication. This metabolic dependency can be partially rescued by engineering MYXV to express exogenous ASS1, demonstrating that intratumoral metabolic defects can serve as a novel barrier to viral host range and therapeutic efficacy [37].
Implications for Viral Evolution and Control
The ongoing evolution of MYXV, particularly the emergence of the ha-MYXV recombinant, highlights the dynamic nature of poxvirus host range. The acquisition of M159 is a stark reminder that recombination events can rapidly expand the host range of a virus, with profound consequences for wildlife conservation and potentially for domestic animal health. The detection of ha-MYXV in European rabbits and domestic rabbits in Portugal [15, 16] indicates that the species barrier is not absolute and that the virus can spill back into the original host, potentially with altered virulence. This necessitates enhanced surveillance efforts, such as the quadruplex qPCR developed to differentiate classic from recombinant MYXV strains [11], to monitor the spread and evolution of these viruses in wild and domestic lagomorph populations. The World Organisation for Animal Health (WOAH) recognizes myxomatosis as a reportable disease, and the emergence of these new strains underscores the need for continued vigilance and the development of effective vaccines that can protect both rabbits and hares against the expanding range of MYXV strains [42]. The interplay between viral immune modulators and host restriction factors will continue to shape the evolutionary arms race between MYXV and its leporid hosts, with the potential for further host range expansions remaining a distinct possibility.
Epidemiology and Geographic Spread in Leporids
The epidemiology of myxoma virus (MYXV) in leporids represents one of the most comprehensively documented examples of host-pathogen coevolution and cross-species transmission in virology. For over seven decades, MYXV has shaped and been shaped by the population dynamics of its leporid hosts, evolving from a relatively benign pathogen of South American Sylvilagus species into a highly virulent agent of epizootic mortality in European rabbits (Oryctolagus cuniculus) and, more recently, in hare species (Lepus spp.) [3, 31]. The geographic distribution of MYXV is now nearly global among rabbit populations, but the epidemiological landscape has been profoundly altered by the emergence of recombinant strains capable of leaping species barriers within the Leporidae family.
The Classical Epizootic in European Rabbits: A Textbook Paradigm
The introduction of MYXV into naive European rabbit populations in Australia (1950) and Europe (1952) initiated a classic natural experiment in virulence evolution. The released virus (Standard Laboratory Strain in Australia; Lausanne strain in Europe) initially caused >99% mortality, a virulence grade that proved evolutionarily unstable because it killed hosts before optimal transmission could occur [31]. Within a decade, field isolates became dominated by moderately attenuated strains (Grades II–III) that allowed infected rabbits to survive longer, thereby increasing the duration of viremia and exposure to arthropod vectors. This evolutionary trajectory was remarkably parallel across continents, despite different progenitor viruses and vector communities, underscoring the selective pressure for transmission efficiency over absolute virulence [31].
In Australia, the epidemiological picture has continued to evolve dramatically. Genome sequencing of isolates collected between 2008 and 2017 revealed a punctuated evolutionary event: one lineage (lineage c) exhibited a greatly elevated evolutionary rate and a breakdown of the clock-like structure that had characterized MYXV evolution for the previous five decades [26]. This punctuated change occurred between 1996 and 2012, coinciding with the introduction of rabbit hemorrhagic disease virus (RHDV) and prolonged drought in southeastern Australia. These environmental perturbations caused catastrophic declines in rabbit populations, potentially altering the selective landscape for MYXV transmission. The viruses from this lineage reversed a mutation in the M005L/R gene that had been fixed since the progenitor SLS strain, suggesting that rare reversion mutations can become epidemiologically significant when population bottlenecks alter transmission dynamics [26].
A similar pattern of complex evolution is evident in Europe. Analysis of UK isolates from 2008–2013 revealed three distinct lineages, indicative of long-term separation and independent evolution, with evolutionary rates nearly identical to those observed in Australia [29]. However, the genomic and phenotypic outcomes showed little convergence with Australian strains, demonstrating that multiple genetic pathways can produce successful field phenotypes. Notably, UK isolates exhibited two broad disease phenotypes: classical nodular myxomatosis and an amyxomatous form characterized by few or no secondary skin lesions but acute death with pulmonary edema and bacterial septicemia [29]. This amyxomatous phenotype is particularly concerning epidemiologically because it may escape early clinical detection, facilitating silent spread among rabbit populations.
In Finland, the first documented MYXV outbreak occurred in summer 2020, affecting feral rabbits. Whole genome sequencing revealed that all samples collected during the outbreak were identical, with highest nucleotide identity (99.97%) to the German field strain FLI-H, indicating a recent introduction from Central Europe [43]. The virus genome showed only minor changes during a one-year follow-up period, suggesting that the virus was well-adapted to the local host population upon introduction. Notably, coinfection with RHDV was observed in six cases, highlighting the complex multi-pathogen dynamics that characterize contemporary lagomorph epidemiology [43, 45].
The Species Jump to Hares: A New Epidemiological Era
The most dramatic epidemiological development in MYXV history is the confirmed host species jump from European rabbits to hares, a transition that shattered the long-held dogma that hares were resistant to myxomatosis. For over 70 years, from the original introduction of MYXV into Europe until 2018, European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were considered incidental, dead-end hosts at worst, with only sporadic, self-limiting cases reported [13, 17]. This epidemiological equilibrium was disrupted in 2018 when outbreaks of myxomatosis-like disease emerged in Iberian hares in Spain, with high mortality and rapid geographic spread [17].
The causative agent, designated MYXV-Tol or ha-MYXV, harbors a novel ~2.8 kb recombinant region inserted within the M009L gene, disrupting it into two open reading frames (M009L-a and M009L-b) [14, 17]. This insertion contains four novel genes (M060L, M061L, M064L, M065R) phylogenetically related to MYXV genes from classical strains, representing a natural recombination event between MYXV and an unknown poxvirus [14]. Critically, the inserted M064R ortholog (M159) functions as a host range factor that enables productive infection in hare cells [5]. Deletion of M159 from MYXV-Tol abrogates replication in hare cells both in vitro and in vivo, whereas wild-type MYXV (lacking M159) cannot infect hare cells, definitively establishing M159 as the key molecular determinant of the species leap [5].
The epidemiological consequences have been severe. Between 2018 and 2020, 487 Iberian hares from 372 affected areas across Spain were confirmed MYXV-positive by PCR, with most outbreaks concentrated in southern and central regions [13]. The spatial distribution was not homogeneous; clustering was observed, likely reflecting both hare population density and vector activity. Consecutive outbreaks over two years indicated that ha-MYXV had transitioned from epidemic to endemic circulation in Spain [13]. Retrospective analysis suggests that the virus may have been circulating since June 2018, with clinical cases first detected in the Toledo region [13]. The apparent mean mortality rate was 55.4% (median: 70%), with peaks in August and October, followed by a sharp decline in January, suggesting temperature-dependent vector activity [13].
The geographic spread is not static. As of August 2024, the disease has been spreading radially from the Germany–Netherlands border area into European brown hare (Lepus europaeus) populations [1]. Detection of recombinant MYXV in hares from this region, dating back potentially as early as September 2020, indicates that the virus has escaped the Iberian Peninsula and is now establishing itself in Central European hare populations. The clinical presentation in European brown hares mirrors that observed in Iberian hares: swollen eyelids, head edema, and dermatitis affecting the face, legs, and perineum [1]. This northward expansion raises urgent concerns about the potential for ha-MYXV to spread throughout Europe’s hare populations, which are immunologically naive and highly susceptible.
Spillback and Cross-Species Dynamics: The Rabbit-Hare Interface
The epidemiological picture is further complicated by bidirectional transmission at the rabbit-hare interface. Although early surveys after the 2018 hare outbreak suggested species segregation, with classic MYXV circulating in rabbits and recombinant ha-MYXV circulating in hares, this paradigm has since been challenged [13]. In 2020, recombinant ha-MYXV was detected in both domestic and wild rabbits in Portugal, causing high mortality and demonstrating that the recombinant virus can infect and cause disease in rabbits [15, 16]. This “spillback” event has profound implications: rabbits may serve as bridging hosts, maintaining ha-MYXV in sympatric populations and facilitating further geographic spread.
In the Iberian Peninsula, a comprehensive surveillance study of 114 European hares (L. europaeus) detected MYXV DNA in 1.8% of individuals, with one hare infected with classic MYXV and another with ha-MYXV [2]. Seroprevalence was 3.2%, indicating that sporadic, low-level transmission occurs from sympatric European rabbits (O. cuniculus) or Iberian hares (L. granatensis) into European hares [2]. This occasional spillover appears to be temporally and spatially scattered, predominantly in areas where lagomorph species overlap. The low seroprevalence suggests that European hares are immunologically naive and that each infection likely represents a separate spillover event rather than sustained hare-to-hare transmission [2]. This immunological naivete creates an epidemiological powder keg: should ha-MYXV acquire additional adaptations for hare-to-hare transmission, the consequences for European hare populations could be catastrophic, mirroring what occurred with Iberian hares in 2018 [2].
Retrospective serological and molecular surveys have extended the known temporal scale of MYXV-hare interactions. Analysis of 451 Iberian hare sera collected between 1994–1999 and 2017–2019 detected MYXV-related antibodies in hares as early as 1996, more than 20 years before the severe outbreaks of 2018. Four seronegative hares from 1997 were even MYXV-DNA positive, indicating that hares had been exposed to, and perhaps transiently infected by, classical MYXV strains long before the recombinant emerged [27]. Seroprevalence increased significantly from 5.4% in 1994–1999 to 13.0% in 2017–2019, with a notable peak of 24.7% in 2019, immediately following the epizootic. This suggests that the recombinant virus did not encounter a completely naive host population; rather, it may have exploited a host that had experienced sporadic, subclinical exposures but lacked immunity capable of preventing a highly virulent, sustained outbreak [27]. The implications are clear: even low-level historical exposure did not protect against the recombinant virus, raising questions about vaccine development and cross-protection strategies.
Vector Ecology and Transmission Dynamics in Leporids
The epidemiology of MYXV is inextricably linked to its arthropod vectors, primarily fleas and mosquitoes. The classic transmission cycle involves mechanical transfer of virus on the mouthparts of biting arthropods, with mosquitoes serving as the principal vectors in most temperate regions [31]. Recent work in the United Kingdom has identified Anopheles atroparvus as a particularly important vector. In a study at Elmley Nature Reserve, Kent, 33 of 36 blood-fed An. atroparvus had fed on rabbits, and 27% of those containing rabbit blood were positive for MYXV DNA [32]. This strong feeding preference for both healthy and myxoma-infected rabbits, combined with the species’ ubiquity, positions An. atroparvus as a significant transmission bridge in UK rabbit populations [32].
In Spain, the discovery of MYXV DNA in ixodid ticks suggests that the vector spectrum may be broader than previously appreciated. A survey of ticks collected from wild rabbits and Iberian hares in southern Spain detected MYXV DNA in 50.7% of pools of Rhipicephalus pusillus (nymphs and adults) and Hyalomma lusitanicum (nymphs) [44]. Antibodies to MYXV were found in 68% of wild rabbits and 67% of Iberian hares, confirming widespread exposure. Importantly, the partial viral envelope protein gene sequenced from ticks showed a mutation (G383A) in the MYXV_gp026 locus that distinguished the rabbit strain from the recently isolated Iberian hare strain, suggesting lineage-specific vector associations [44]. While the competence of ticks as biological vectors remains unconfirmed, the high prevalence of viral DNA in ticks parasitizing viremic hosts suggests that ticks could serve as mechanical vectors or perhaps as overwintering reservoirs, sustaining MYXV during periods when mosquito activity is low.
The Impact on Leporid Populations: A Conservation and Economic Crisis
The epidemiological consequences of the hare species jump extend beyond individual mortality to population-level declines. In the Iberian Peninsula, the emergence of ha-MYXV has been associated with significant reductions in Iberian hare abundance. Analysis of hunting bag data revealed a marked decline in hare populations following the 2018 outbreak, with the effect on average abundance index correlating with the timing and intensity of epizootics [33]. This population crash raises conservation concerns: the Iberian hare is a keystone species in Mediterranean ecosystems, serving as prey for endangered predators such as the Iberian lynx (Lynx pardinus) and the Spanish imperial eagle (Aquila adalberti). The loss of hare populations could trigger cascading ecological effects.
The economic impact on the rabbit industry is equally severe. The detection of recombinant ha-MYXV in farmed rabbits in Portugal, associated with rapid mortality (five of six rabbits died within 24–48 hours), highlights the vulnerability of commercial operations [15]. The fact that available commercial vaccines, developed against classic MYXV strains, may not confer complete protection against ha-MYXV in hares, though they do protect rabbits, compounds the problem [42]. In a pilot study, vaccination of Iberian hares with commercial doses of Mixohipra-FSA or Mixohipra-H failed to protect against ha-MYXV challenge, although higher doses of Mixohipra-FSA induced some protection [42]. In contrast, the same vaccines fully protected wild rabbits against ha-MYXV, confirming that the recombinant virus retains rabbit susceptibility while evolving strategies to circumvent hare immune defenses [42].
Evolutionary Epidemiology: Genomic Signatures of Spread and Adaptation
The genomic epidemiology of MYXV in leporids reveals ongoing adaptation and geographic structuring. The Finnish outbreak strain, identical across five samples from 2020, contained novel mutations not previously described, though the genome showed minimal change during a one-year follow-up, suggesting that the virus was already well-adapted for rabbit-to-rabbit transmission upon introduction [43]. Phylogenetic analysis placed the Finnish strain closest to the German FLI-H strain, indicating that the virus entered Finland from Central Europe, likely via human-mediated movement of infected rabbits or vectors [43].
In Australia, the punctuated evolution of lineage c viruses correlated with the collapse of rabbit populations following RHDV introduction [26]. This suggests that MYXV evolution may be highly sensitive to host population density, with periods of low transmission intensity favoring the fixation of mutations that might otherwise be purged by purifying selection. The fact that the rapidly evolving lineage c showed a reversal of a fixed M005L/R disruption underscores the dynamic nature of MYXV genomes and the potential for rapid phenotypic change when selection pressures shift [26].
The World Organisation for Animal Health (WOAH) recognizes myxomatosis as a notifiable disease in many member countries, and the US Department of Agriculture classifies it as a reportable pathogen [34]. The continued spread of recombinant ha-MYXV through Central Europe into new hare populations warrants heightened surveillance. The emergence of the recombinant virus in Finland, a country previously free of myxomatosis [43], and the radial spread from the Germany–Netherlands border [1], indicate that the virus is not constrained by the climate or ecological boundaries that historically limited MYXV distribution. This expansion into higher latitudes suggests that the recombinant virus may have a broader thermal tolerance or vector compatibility, enabling it to persist in regions where classic MYXV was previously unable to establish endemic cycles.
The molecular epidemiological toolbox has advanced to meet this challenge. A quadruplex qPCR technique now allows rapid differentiation between classic MYXV and recombinant ha-MYXV strains, targeting the M000.5L/R gene (conserved in all strains) plus strain-specific markers for the M009L disruption and the M060L insert [11]. This assay, capable of detecting as few as nine viral genome copies, is essential for monitoring the geographic spread and potential co-infections of both strains, providing the high-resolution surveillance data needed to track the evolving epidemiological landscape [11]. The emergence of ha-MYXV represents a paradigm shift in myxomatosis epidemiology, transforming a classic rabbit-specific disease into a multi-host epizootic with continent-spanning consequences.
Diagnostic Methods: Molecular, Serological, and Pathological
The accurate diagnosis of myxoma virus (MYXV) infection, whether in its classic rabbit-restricted form or in the context of the emergent recombinant strains (ha-MYXV/MYXV-Tol) infecting hares, requires a multi-pronged laboratory approach. The diagnostic armamentarium has evolved considerably, moving from traditional virological and serological assays to highly sensitive and specific molecular platforms that can differentiate between viral lineages. This section provides an exhaustive analysis of the three principal diagnostic pillars, molecular, serological, and pathological, detailing their biological principles, operational characteristics, and specific applications in the context of MYXV epidemiology and pathogenesis.
Molecular Diagnostics: PCR-Based Detection and Genomic Characterization
Nucleic acid detection represents the gold standard for active MYXV infection, offering unparalleled sensitivity and the capacity for strain differentiation. Real-time and conventional PCR assays target conserved genetic elements for pan-MYXV detection, while more sophisticated multiplex systems have been developed to address the critical need to distinguish classic MYXV from the emerging recombinant ha-MYXV strains [11]. The genetic basis for this differentiation lies in the unique ~2.8 kb insertion within the M009L gene of ha-MYXV, which disrupts this open reading frame into M009L-a and M009L-b and introduces novel host range determinants such as M159 [5, 14, 17]. This genomic signature has been exploited for diagnostic purposes, allowing for the rapid discrimination of viral strains in field samples.
A landmark development in this regard is the quadruplex qPCR system described by Santos et al., which simultaneously amplifies four targets: the m000.5L/R duplicated gene (conserved across all MYXV strains), a classic MYXV-specific junction spanning the M009L-a/M009L-b boundary, a ha-MYXV-specific fragment within the m060L gene, and a eukaryotic 18S rRNA housekeeping gene serving as an internal control for sample quality [11]. This assay was optimized for both TaqMan probe-based and EvaGreen dye-based detection, demonstrating a limit of detection as low as nine viral DNA copies per reaction with an efficiency exceeding 93% [11]. The quadruplex format is particularly powerful for epidemiological monitoring, as it can simultaneously detect co-infections with both classic and recombinant strains, an event that has significant implications for understanding recombination dynamics and viral evolution in sympatric lagomorph populations [11, 16].
For routine surveillance, simpler PCR approaches targeting the M071L gene have been validated with an internal amplification control (IAC) to mitigate false negatives due to PCR inhibition, a common issue in complex tissue samples [46]. This IAC-PCR, validated according to OIE (now WOAH) standards, demonstrated a diagnostic sensitivity (DSe) of 0.976 and a diagnostic specificity (DSp) of 0.955, with a limit of detection of 2 TCIU per 0.2 ml of tissue homogenate [46]. Similarly, real-time PCR assays targeting the M000.5L/R gene are widely employed in surveillance programs, as they detect both classic and recombinant strains due to the conserved nature of this region [16, 27]. The importance of PCR in field diagnostics cannot be overstated; it has been instrumental in confirming MYXV DNA in ticks (Rhipicephalus pusillus and Hyalomma lusitanicum), providing evidence for the potential vector competence of these arthropods, and in identifying viral DNA in European brown hares where classical clinical signs may be absent [1, 44].
Beyond diagnostic confirmation, molecular methods including whole genome sequencing (WGS) have become indispensable for evolutionary and epidemiological tracking. WGS of isolates from the first Finnish MYXV outbreak revealed 99.97% nucleotide identity to the German field strain FLI-H, with unique mutations that may influence viral properties, yet only minor changes were observed during one year of endemic circulation [43]. In Australia, genomic sequencing of modern isolates has documented a punctuated evolutionary event characterized by a breakdown in clock-like evolution and the emergence of a rapidly evolving lineage marked by nonsynonymous mutations and indels, including a reversal of a disruptive mutation in M005L/R [26, 29]. These data underscore that molecular diagnostics are not merely confirmatory but are central to understanding the ongoing coevolutionary arms race between MYXV and its lagomorph hosts.
Serological Diagnostics: Antibody Detection and Population Surveillance
Serological methods provide critical insights into past exposure, population immunity, and the spatiotemporal dynamics of MYXV circulation, particularly in wild lagomorph populations where active viral detection may be fleeting. The most commonly employed serological platform is the enzyme-linked immunosorbent assay (ELISA). A commercial indirect ELISA (iELISA), originally developed for rabbits, has been adapted for use in hares with adjustments to the cut-off threshold to minimize false positives, as determined by semiparametric finite mixture analysis [27]. Using this approach, MYXV-related antibodies were detected in 12.6% of Iberian hares (Lepus granatensis) sampled over two decades, with a significantly higher seroprevalence in the 2017–2019 period (13.0%) compared to 1994–1999 (5.4%) [27]. Crucially, this retrospective analysis detected antibodies in hares as early as 1996, demonstrating that exposure to MYXV or an antigenically related virus predated the severe outbreaks of 2018 by at least two decades [27]. This finding radically alters the epidemiological narrative, suggesting that the recombinant ha-MYXV did not simply encounter a naïve population but rather emerged in a context of low-level prior immunity.
Comparative studies evaluating the diagnostic performance of different serological assays have been conducted. The agar gel immunodiffusion (AGID) assay, a traditional method for detecting precipitating antibodies, was assessed against molecular methods for detecting the virus itself rather than host antibodies. One study evaluating AGID for direct viral antigen detection in tissue homogenates found a DSe of 0.65 and DSp of 1.00, with accuracy at 0.67, concluding that AGID is suitable only for samples with high viral loads (≥3,125 PCR units) and is far less sensitive than PCR-based methods [21]. This highlights the inherent limitations of antigen detection serology compared to nucleic acid amplification.
More sophisticated serological approaches include the indirect immunofluorescence test (IFT) and competitive ELISA (cELISA), which have been employed in parallel for cross-validation. In a survey of Iberian hares, IFT and cELISA were used to confirm iELISA results, with the cELISA offering the advantage of species-independent detection by using a monoclonal antibody that competes with host antibodies for a conserved viral epitope [27]. Serological surveys of sympatric lagomorph populations in Spain have detected antibodies in 68% of wild rabbits and 67% of Iberian hares, confirming widespread exposure in both species [44]. In European hares from the Iberian Peninsula, a commercial iELISA detected antibodies in 3.2% of animals, indicating sporadic, likely spillover, transmission events from sympatric rabbits or Iberian hares [2]. These serological data are biologically meaningful: they suggest that while European hares appear immunologically naïve, the barrier to infection is not absolute, raising the specter of further host range expansions or recombination events [2, 3].
Pathological Diagnostics: Gross Lesions, Histopathology, and Immunohistochemistry
Pathological examination, combined with histopathology and immunohistochemistry (IHC), remains foundational for characterizing disease presentation and confirming diagnosis, particularly in cases with atypical clinical signs or in novel hosts. The pathology of MYXV infection is profoundly influenced by the viral strain and the host species. In classic rabbit myxomatosis caused by the California MSW strain, the hallmark lesions include bilateral blepharedema, anogenital edema, and cutaneous myxomas [34]. Histological examination of eyelid biopsies from affected pet rabbits reveals keratinocytes containing eosinophilic intracytoplasmic viral inclusion bodies (so-called "myxoma cells") and extensive dermal edema with a myxoid matrix [34]. Splenic lymphocyte necrosis is a consistent finding (observed in 10/11 cases), and IHC demonstrates viral antigen in skin, heart, lung, ileum, spleen, and lymph nodes, underscoring the systemic nature of the infection [34].
The pathological presentation of the recombinant ha-MYXV in Iberian hares is strikingly different and more severe. Gross lesions are characterized by bilateral blepharoconjunctivitis, epistaxis, intense congestion and edema in multiple organs, and internal hemorrhages, but notably, visible cutaneous myxomas are absent [8]. This amyxomatous form reflects an acute or hyperacute disease course. Histologically, hares exhibit hyperplastic epidermis with hyperkeratosis, and dermal myxoid matrix with hydropic degeneration of keratinocytes containing intracytoplasmic inclusion bodies [8]. Importantly, the virus demonstrates broad cellular tropism in hares, infecting not only epithelial cells and myxoma cells but also macrophages, lymphocytes, fibroblasts, endothelial cells, hepatocytes, and Leydig cells [8]. This expanded tropism is associated with dramatic lymphoid depletion in the spleen, alveolar edema, interstitial pneumonia, and hepatic and testicular necrosis, findings consistent with a systemic, highly virulent infection [8]. Ultrastructural examination of tissues from infected wild rabbits has confirmed the presence of viral particles in lungs and eyelids, and virus isolation in RK13 cells has attested to the infectivity of the recombinant virus [16].
IHC is an indispensable tool for identifying viral antigen in tissues and for understanding viral pathogenesis. Using polyclonal or monoclonal antibodies against MYXV structural proteins, IHC can localize viral antigen to specific cell types. In hares infected with ha-MYXV, IHC has been instrumental in demonstrating infection of Leydig cells within the testis, a finding that may have implications for sexual transmission or persistence [8]. In rabbits, IHC has confirmed MYXV antigen in the heart and lungs of animals that succumbed suddenly, linking viral myocarditis or pneumonitis to peracute death [34]. The integration of histopathology and IHC provides a spatial and cellular context for molecular findings, confirming that PCR-positive tissues correspond to active viral replication and tissue damage rather than merely passive viral carriage.
Emerging Recombinant Strains and Cross-Species Transmission
The Paradigm Shift in Myxoma Virus Host Tropism
For over six decades following the introduction of myxoma virus (MYXV) into European rabbit populations, the host range of this leporipoxvirus was considered remarkably stable and well-defined. The European rabbit (Oryctolagus cuniculus) was the primary susceptible host for myxomatosis, while various hare species (Lepus spp.) were long regarded as resistant or, at most, occasional spillover hosts that rarely developed clinical disease [17, 31]. This textbook understanding of MYXV host restriction was fundamentally overturned in 2018, when a novel recombinant virus emerged on the Iberian Peninsula, causing catastrophic mortality in Iberian hares (Lepus granatensis) [13, 17]. This event, designated as the emergence of ha-MYXV (also termed MYXV-Tol or MYXV Toledo), represented not merely a host range expansion but a genuine cross-species transmission event, a species leap, that has reshaped our understanding of poxvirus evolutionary plasticity and the molecular determinants of host tropism [5, 14].
The significance of this event is underscored by the fact that hares and rabbits, while both belonging to the family Leporidae, diverged from a common ancestor approximately 12 million years ago [3]. Despite prolonged sympatry and frequent ecological overlap between these lagomorph lineages, MYXV had never been documented to cause significant disease in hares prior to 2018. Retrospective serological and molecular surveys of Iberian hare populations now indicate that hares had been exposed to MYXV or antigenically related viruses at least since the mid-1990s, without the development of epizootic disease [27]. This historical pattern of sporadic, subclinical infection in hares, contrasted with the explosive outbreak of lethal myxomatosis beginning in 2018, provides compelling evidence that the recombinant virus acquired fundamentally new biological properties enabling productive infection and pathogenesis in a previously resistant host species.
Genomic Architecture of the Recombinant Viruses
The defining genomic feature of the emergent recombinant MYXV strains is the acquisition of a ~2.8 kilobase insertion located within the M009L gene, effectively disrupting this open reading frame into two smaller ORFs (M009L-a and M009L-b) [14, 17]. Deep sequencing and comparative genomic analyses of MYXV-Tol, the prototypic recombinant strain isolated from an Iberian hare in the Toledo region of Spain, revealed that this insertion contains four novel poxviral open reading frames, M060L, M061L, M064L, and M065L, that are phylogenetically related to existing MYXV genes [14, 17]. Importantly, these inserted genes are not merely duplicates of existing host range determinants but appear to have originated from a broader poxvirus gene pool, suggesting that the recombination event that generated ha-MYXV involved the acquisition of genetic material from an as-yet-unidentified poxvirus donor [14, 20].
Among these newly acquired genes, the M159 open reading frame, an orthologue of the vaccinia virus C7 host range family, has emerged as a critical determinant of the expanded host tropism [5]. The C7L superfamily of poxvirus host range factors is highly conserved across mammalian poxviruses, and most sequenced members of the Chordopoxvirinae retain at least one homologue of this gene family [5]. However, the M159 protein encoded by ha-MYXV possesses unique structural and functional properties that distinguish it from the canonical MYXV host range factor M062R. Águeda-Pinto and colleagues demonstrated definitively that while wild-type MYXV (the Lausanne strain, which lacks M159) cannot productively infect hare-derived HN-R cells, and while a knockout mutant (vMyxTol-ΔM159) is similarly incapable of replication in these cells, the intact MYXV-Tol strain undergoes fully productive infection in hare cells [5]. This M159-dependent permissivity reveals that the acquisition of a single host range gene was sufficient to overcome the intrinsic antiviral barriers present in hare cells.
The functional characterization of M159 has yielded insights into the molecular mechanisms of species jumping. Like other C7L family members, M159 is expressed as both an early and a late gene, but it exhibits the unusual property of nuclear translocation at later time points post-infection [5]. This nuclear localization suggests that M159 may interact with host nuclear factors, potentially including transcription factors or DNA damage response proteins, to subvert hare-specific innate immune defenses. Furthermore, in human cancer cells that are normally nonpermissive or semipermissive for MYXV replication, the expression of M159 was shown to upregulate viral replication levels, indicating that the cellular pathway targeted by M159 is conserved across mammalian species and is not restricted to leporids [5]. This observation has important implications for the potential of MYXV-based oncolytic virotherapy, as it suggests that the host range determinants that enable species jumping in nature may also be harnessed to enhance therapeutic efficacy in human cancers [10, 25].
Epidemiological Patterns and Geographic Expansion
The emergence and dissemination of recombinant MYXV strains have followed distinct spatial and temporal trajectories that reflect the complex interplay between viral genetics, host ecology, and environmental factors. In the Iberian Peninsula, the initial detection of ha-MYXV occurred in the Toledo region of Spain in late 2018, with retrospective molecular analysis subsequently revealing that the virus may have been circulating in Iberian hare populations as early as June 2018 [13]. The ensuing epizootic spread with remarkable rapidity: between 2018 and 2020, García-Bocanegra and colleagues confirmed MYXV infection in 487 hares from 372 affected areas across most Spanish regions where the Iberian hare is present [13]. The spatial distribution was markedly heterogeneous, with outbreaks concentrated in southern and central Spain, and the temporal pattern showed pronounced peaks in August and October, coinciding with periods of high arthropod vector activity [13]. The apparent mean mortality rate during this initial epidemic period was estimated at 55.4%, with a median of 70%, underscoring the catastrophic impact on naïve hare populations [13].
Critically, the species leap of MYXV has not been unidirectional. While the initial emergence involved transmission from sympatric rabbit populations to Iberian hares, subsequent epidemiological data have documented the reverse phenomenon: the transmission of ha-MYXV from hares back into both domestic and wild rabbits. In 2020, Santos and colleagues reported the first confirmed cases of ha-MYXV infection in European rabbits (Oryctolagus cuniculus) in southern Portugal, involving a backyard rabbitry where unvaccinated animals developed classic signs of myxomatosis, eyelid edema, anogenital swelling, and cutaneous myxomas, and succumbed within 24–48 hours of symptom onset [15]. Molecular analysis confirmed the presence of the recombinant virus, with the 2.8 kb insert sequence demonstrating 100% similarity to the insert sequences previously described in Iberian hares from Spain [16]. This bidirectional transmission capability has profound implications for MYXV epidemiology, as it establishes a potential reservoir community wherein hares and rabbits can sustain viral circulation independently, thereby complicating control and vaccination strategies.
The geographic expansion of recombinant MYXV strains has extended well beyond the Iberian Peninsula. In the period spanning 2023–2024, Fischer and colleagues documented the emergence of a recombinant myxoma virus in European brown hares (Lepus europaeus) along the Germany–Netherlands border region, with evidence suggesting that viral introduction may have occurred as early as September 2020 [1]. The clinical presentation in affected European brown hares paralleled that seen in Iberian hares, swollen eyelids, head edema, and dermatitis affecting the face, legs, and perineum, although the disease course appeared somewhat more protracted in some cases [1]. As of August 2024, the virus has been spreading radially from this initial focus, raising concerns about its potential to establish endemicity in central European lagomorph populations. This northern expansion is particularly noteworthy because it demonstrates that the recombinant virus is not restricted to the unique ecological context of Mediterranean ecosystems but can establish transmission cycles in temperate regions with different vector communities and host densities.
Molecular Determinants of Host Range and Pathogenesis
The ability of recombinant MYXV to productively infect and cause lethal disease in hares represents a fundamental alteration in the virus-host interface, mediated by multiple interacting molecular factors. At the core of this adaptation is the M159 host range factor, which functions by antagonizing host antiviral responses that previously rendered hare cells nonpermissive for MYXV infection [5]. The precise cellular target of M159 in hare cells remains under active investigation, but the available evidence points toward the manipulation of pathways involving sterile alpha motif domain-containing protein 9 (SAMD9) and related antiviral restriction factors. In human cells, the essential MYXV host range determinant M062 is known to antagonize SAMD9 function, and the acquisition of M159 appears to provide an analogous, though not identical, function in hare cells [4, 28].
Beyond the host range factors, the recombinant MYXV strains also exhibit alterations in their immunomodulatory repertoire that likely contribute to their enhanced virulence in hares. The genetic disruption of M009L, which results from the 2.8 kb insertion, may itself have functional consequences, as M009L encodes a protein involved in modulation of host immune responses [14]. Additionally, the duplication and divergence of M064R and M065R within the recombinant region may provide the virus with expanded capacity to counteract innate immune defenses, including type I interferon responses and inflammasome activation [3, 19]. The M013 protein, a viral pyrin domain-only protein that inhibits both the inflammasome and NF-κB signaling pathways through distinct structural motifs, is present in both classic and recombinant MYXV strains but may function differently in the context of hare myeloid cells [22].
Pathological examination of hares naturally infected with ha-MYXV has revealed a disease presentation that is both similar to and distinct from classical rabbit myxomatosis. Agulló-Ros and colleagues conducted comprehensive histopathological and immunohistochemical analyses of 28 PCR-confirmed ha-MYXV-positive Iberian hares and documented several notable features [8]. Unlike classic rabbit myxomatosis, where visible cutaneous myxomas are a hallmark, affected hares developed bilateral blepharoconjunctivitis, epistaxis, and intense congestion and edema of multiple organs without the formation of discrete myxomatous masses [8]. Histopathologically, infected hares exhibited hyperplastic epidermis with prominent hyperkeratosis and myxoid matrix deposition in the dermis, along with alveolar edema, interstitial pneumonia, dramatic lymphoid depletion in the spleen, and necrosis in the liver and testis. The viral antigen was detected not only in epithelial cells and myxoma cells of the skin but also in macrophages, lymphocytes, fibroblasts, endothelial cells, hepatocytes, and Leydig cells, a pattern of cellular tropism that is broader than that typically observed in rabbit myxomatosis [8]. This expanded tissue tropism likely reflects the altered host range determinants of the recombinant virus and may contribute to the rapid, hyperacute disease course observed in many hares.
Ecological and Evolutionary Implications
The emergence of recombinant MYXV strains capable of cross-species transmission has created a new and dynamic epidemiological landscape that challenges existing paradigms of myxomatosis management. In the Iberian Peninsula, the impact on hare populations has been severe, with Cardoso and colleagues documenting significant population declines using hunting bag data as a proxy for abundance [33]. The decline in Iberian hare populations has cascading ecological consequences, as these lagomorphs serve as key prey species for the endangered Iberian lynx (Lynx pardinus) and the Spanish imperial eagle (Aquila adalberti). The World Organisation for Animal Health (WOAH) has recognized the emergence of ha-MYXV as a significant event in wildlife disease surveillance, and the Food and Agriculture Organization of the United Nations (FAO) has noted the potential implications for food security in regions where wild lagomorphs are hunted for subsistence or commerce.
The evolutionary trajectory of the recombinant MYXV strains remains an open question with critical implications for long-term disease dynamics. The Australian experience with MYXV, where the virus was released in 1950 as a biological control agent and subsequently underwent decades of co-evolution with European rabbits, provides a useful framework for understanding potential outcomes [7, 26]. In Australia, MYXV evolved from a highly virulent progenitor toward an intermediate virulence phenotype that optimizes transmission, while rabbit populations in turn evolved genetic resistance through selection on standing genetic variation in immunity-related genes, including interferon pathways [9]. Whether similar co-evolutionary dynamics will occur in hare populations exposed to ha-MYXV is uncertain, but the initial evidence suggests that hares are immunologically naïve and thus highly susceptible [2]. Cardoso and colleagues found that only 3.2% of European hares tested in the Iberian Peninsula had detectable antibodies against MYXV, indicating very low prior exposure and minimal pre-existing immunity [2].
The role of arthropod vectors in facilitating the cross-species transmission of MYXV deserves particular attention. Myxoma virus is mechanically transmitted by biting arthropods, primarily mosquitoes and fleas, and the efficiency of vector-mediated transmission is a key determinant of viral spread [32]. Studies of Anopheles maculipennis complex mosquitoes in the United Kingdom have demonstrated that these vectors readily feed on both rabbits and hares, with 27% of blood-fed Anopheles atroparvus that had fed on rabbits testing positive for MYXV DNA [32]. Similarly, García-Pereira and colleagues detected MYXV DNA in ticks (Rhipicephalus pusillus and Hyalomma lusitanicum) collected from lagomorphs in Spain, with the viral sequences showing genetic differences between rabbit-derived and hare-derived strains [44]. These findings suggest that ticks may serve as competent vectors for MYXV transmission, potentially facilitating cross-species transmission when host populations overlap ecologically.
The potential for further host range expansion remains a significant concern. The European hare (Lepus europaeus) has already been affected in central Europe [1], and the susceptibility of other leporid species, including the mountain hare (Lepus timidus) and various Sylvilagus species, remains unknown. Enow and colleagues have argued that the critical restrictions that MYXV would encounter in colonizing a potentially new host species stem from interactions with the host’s innate immune environment, and that the recombination event that generated ha-MYXV may have provided the virus with a "toolkit" of host range factors that could facilitate further species jumps [3]. The ongoing surveillance of MYXV in wild lagomorph populations, using the newly developed quadruplex qPCR assays that can differentiate classic from recombinant strains, will be essential for early detection of further host range expansion [11].
Vaccine Efficacy and Control Implications
The emergence of recombinant MYXV strains capable of causing disease in hares has necessitated a reevaluation of vaccination strategies for both domestic and wild lagomorphs. Current commercial myxomatosis vaccines were developed for use in domestic rabbits and were designed to protect against classic MYXV strains. Santos and colleagues conducted a pilot study to evaluate the efficacy of two commercial rabbit vaccines (Mixohipra-FSA and Mixohipra-H, both from Hipra) in Iberian hares challenged with ha-MYXV [42]. The results were sobering: when administered at the standard commercial dose, neither vaccine provided any protection against ha-MYXV challenge in hares. However, administration of a higher dose of Mixohipra-FSA induced partial protection, suggesting that dose optimization may be a feasible short-term strategy for protecting captive hare populations in genetic reserves or breeding programs [42]. Importantly, the same study demonstrated that two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) were fully protective against ha-MYXV challenge in wild rabbits, indicating that vaccine-induced immunity in the original host species is not compromised by the genetic changes in the recombinant virus [42].
The implications of these findings extend beyond veterinary medicine to wildlife conservation and agricultural economics. The rabbit industry in the Iberian Peninsula, which includes both meat production and hunting management, has been significantly affected by the emergence of ha-MYXV. The detection of ha-MYXV in domestic rabbitries [15] raises the possibility that unvaccinated or improperly vaccinated commercial rabbit populations could serve as amplification hosts, further fueling the epizootic in wild lagomorphs. The Centers for Disease Control and Prevention (CDC) and WOAH have emphasized the importance of biosecurity measures, including vector control, quarantine of newly introduced animals, and appropriate vaccination protocols, in preventing the introduction and spread of recombinant MYXV strains.
The development of vaccines specifically designed for use in hares is an urgent priority. The observation that high-dose vaccination with an existing rabbit vaccine can induce partial protection in hares [42] suggests that the immunological barriers to vaccine efficacy are quantitative rather than qualitative, and that appropriately formulated hare-specific vaccines could be developed. The detection of MYXV-specific antibodies in apparently healthy hares sampled before the 2018 outbreak [27] indicates that hares are capable of mounting a humoral immune response to MYXV infection, providing a basis for vaccine-mediated protection. However, the resources required for large-scale vaccination of wild hare populations are substantial, and the logistical challenges of delivering vaccines to free-ranging wildlife are considerable. Targeted vaccination of captive hare populations for genetic conservation, combined with enhanced surveillance of wild populations, represents a pragmatic approach to managing the impact of recombinant MYXV until more comprehensive control strategies can be developed.
Diagnostic Challenges and Surveillance Recommendations
The genetic divergence between classic and recombinant MYXV strains has created new challenges for diagnostic laboratories and surveillance programs. Traditional diagnostic methods for myxomatosis, including clinical examination and agar gel immunodiffusion (AGID) assays, are inadequate for distinguishing between classic and recombinant strains [21]. Molecular diagnostic tools that target conserved MYXV genes, such as the M071L gene used in validated PCR assays with internal amplification controls, can confirm MYXV infection but cannot differentiate between strains [46]. The development of the quadruplex qPCR technique by Santos and colleagues represents a significant advance, as this assay simultaneously targets the M000.5L/R gene (conserved in all MYXV strains), the M009L gene boundaries (specific to classic strains), and the M060L gene (specific to recombinant strains), with an internal control targeting the 18S rRNA gene [11]. This assay can detect as few as nine copies of viral DNA per sample with greater than 93% efficiency, making it suitable for both diagnostic and surveillance applications.
The implementation of systematic surveillance programs for MYXV in wild lagomorph populations is essential for monitoring the spread and evolution of recombinant strains. The retrospective study by Cardoso and colleagues in the Iberian Peninsula, which included sampling of 140 European hares over a six-year period, revealed that both classic and recombinant MYXV strains can infect European hares, albeit at low prevalence
Ecological and Evolutionary Implications of Myxoma Virus Infections
Myxoma virus (MYXV) represents one of the most extensively documented natural experiments in host-pathogen coevolution, a paradigm that has profoundly shaped our understanding of virulence evolution, host resistance, and cross-species transmission dynamics. The ecological and evolutionary implications of MYXV infections extend far beyond the canonical textbook narrative of rabbit-myxoma coevolution in Australia and Europe, encompassing recent and ongoing species jumps, the emergence of recombinant strains with altered host tropism, and complex interactions with arthropod vectors and coinfecting pathogens. This section synthesizes the available evidence to present a comprehensive analysis of the evolutionary forces shaping MYXV populations and the ecological consequences of infection across leporid hosts.
The Foundational Coevolutionary Paradigm: Virulence and Resistance
The introduction of MYXV into naïve European rabbit (Oryctolagus cuniculus) populations in Australia (1950) and Europe (1952) initiated a now-classic demonstration of natural selection acting on virulence. The progenitor virus, the Standard Laboratory Strain (SLS) derived from the Brazilian strain, exhibited case fatality rates approaching 99.9% in susceptible rabbits. However, within a decade, field isolates demonstrated a marked attenuation in virulence, with moderately attenuated strains (grades 2–4 on a 5-point scale) rapidly dominating circulating populations [7, 31]. This attenuation was not a random drift but a direct consequence of selection for transmission efficiency: highly virulent strains killed their hosts too rapidly to permit effective vector-mediated transmission, while overly attenuated strains were cleared by the host immune response before reaching transmissible titers. The stability of moderately attenuated phenotypes in both Australia and Europe, despite independent introductions and distinct viral lineages, underscores the convergent evolutionary pressures imposed by vector-borne transmission dynamics [31].
Parallel to viral attenuation, European rabbit populations experienced intense selection for genetic resistance to myxomatosis. Whole-exome sequencing of rabbits collected before and after the pandemic in Australia, France, and the United Kingdom revealed a striking pattern of parallel evolution acting on standing genetic variation [9]. Resistance alleles at multiple immunity-related loci, including interferon proteins and other innate immune determinants, were repeatedly selected across geographically isolated populations. This polygenic basis of resistance contrasts with the simpler monogenic resistance mechanisms sometimes observed in other host-pathogen systems and suggests that the evolutionary arms race between rabbits and MYXV operates on multiple fronts simultaneously [9]. Importantly, this selection for resistance did not eliminate the virus from rabbit populations; rather, it shifted the equilibrium, with continued circulation of attenuated viruses in increasingly resistant hosts, creating an ongoing coevolutionary dynamic [31].
Experimental infections using modern Australian field isolates collected between 2012 and 2015 have revealed further evolutionary complexity and divergence from the early attenuation paradigm [7]. These contemporary viruses exhibit a spectrum of disease phenotypes that differ qualitatively from those observed in the first decades post-release. Notably, modern isolates frequently cause acute death with features of neutropenic septicemia, characterized by pulmonary edema, bacterial invasions throughout internal organs, and a notable absence of inflammatory response, rather than the classic nodular myxomatosis [7]. The emergence of an "amyxomatous" phenotype, where infected rabbits develop greatly swollen cutaneous tissues with extremely high virus titers but few classical myxomas, may represent a novel adaptation favoring prolonged virus replication and enhanced mosquito transmission. Critically, the experimental demonstration that the same disease phenotype manifests in both susceptible and innately resistant rabbit lines, albeit with shifted virulence grades in resistant animals, indicates that host resistance modifies but does not fundamentally alter the evolutionary trajectory of the virus [7].
The Species Jump: Recombinant MYXV and the Iberian Hare Catastrophe
For seventy years following its introduction to Europe, MYXV was considered a pathogen strictly of European rabbits, with only sporadic, clinically insignificant infections reported in hare species. This long-held assumption was dramatically overturned in 2018–2019 when outbreaks of myxomatosis-like disease caused catastrophic mortality in Iberian hares (Lepus granatensis) on the Iberian Peninsula [13, 17, 33]. Genomic characterization of viruses isolated from affected hares revealed a novel recombinant MYXV strain, designated MYXV-Tol or ha-MYXV, harboring a ~2.8 kb insertion within the M009L gene that disrupted it into two open reading frames (M009L-a and M009L-b) and introduced four additional genes [14, 17, 23]. This recombinant region, acquired from an unknown poxvirus donor, includes orthologues of MYXV genes M060R, M061R, M064R, and M065R, with M064R being a member of the poxvirus C7 host range superfamily [5, 14].
The ecological and evolutionary significance of this recombination event cannot be overstated. The newly acquired host range determinant, designated M159, was demonstrated to be the critical factor enabling productive infection of hare cells [5]. In experimental studies, wild-type MYXV-Lau (lacking M159) failed to infect and replicate in hare HN-R cells, while MYXV-Tol underwent fully productive infection. Deletion of M159 from MYXV-Tol (vMyxTol-ΔM159) completely abrogated its ability to infect hare cells, confirming that M159 is necessary and sufficient for this species leap [5]. Remarkably, M159 functions in a similar fashion to other C7 family members in human cancer cells, upregulating MYXV replication in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species [5]. Mechanistically, the host range restriction MYXV would encounter in colonizing a new species stems fundamentally from interactions with the host's innate immune environment, particularly the complex interplay between viral immune modulators and host antiviral pathways [3].
The epidemiological consequences of this host jump have been devastating. Monitoring data from Spain between 2018 and 2020 confirmed 487 hares from 372 affected areas as MYXV-positive by PCR, with outbreaks detected across most regions where Iberian hares are present [13]. The apparent mean mortality rate was 55.4% (median 70%), and the spatial distribution indicated radial spread from initial foci in southern and central Spain [13]. Retrospective analysis revealed that the virus may have been circulating since June 2018, with outbreak peaks in August and October before declining sharply during winter months [13]. The impact on Iberian hare populations has been severe, with hunting bag analyses demonstrating significant population declines associated with myxomatosis outbreaks [33]. Population viability models suggest that continued circulation of ha-MYXV could lead to local extinctions and substantial reductions in hare abundance across the Iberian Peninsula [33]. Crucially, Iberian hares appear to be immunologically naïve to MYXV, with serological evidence indicating only 5.4% seroprevalence in samples from 1994–1999 and 13.0% in 2017–2019 among apparently healthy animals [27]. This low background immunity, combined with the high virulence of ha-MYXV, creates conditions for epidemic spread with devastating demographic consequences [27].
The host range of the recombinant virus has not remained restricted to Iberian hares. Subsequent surveillance documented ha-MYXV infection in European brown hares (Lepus europaeus) along the Germany-Netherlands border, with introduction potentially dating to September 2020 and radial spread observed through August 2024 [1]. The disease presentation in European brown hares mirrors that in Iberian hares: swollen eyelids, head edema, and dermatitis affecting the face, legs, and perineum, associated with significant mortality [1]. Furthermore, molecular evidence confirms that ha-MYXV has infected both domestic and wild rabbits (Oryctolagus cuniculus), including farmed rabbits in Portugal where six unvaccinated animals developed severe myxomatosis with 83% mortality within 24–48 hours of symptom onset [15, 16]. This host range expansion is not a laboratory artifact but a demonstrated epidemiological reality: routine surveillance shows that both classic MYXV and recombinant ha-MYXV strains co-circulate in sympatric rabbit and hare populations, with occasional spillover events [2, 16]. The recent detection of ha-MYXV in European hares from the Iberian Peninsula, where one of 114 hares analyzed (1.8%) tested positive for the recombinant strain, further confirms ongoing spillover [2].
Molecular Mechanisms Underlying Host Range and Species-Specific Virulence
The MYXV genome encodes a sophisticated arsenal of immunomodulatory proteins that determine host and cellular tropism, among which the poxvirus host range C7L superfamily plays a central role. In MYXV, the dominant and broad-spectrum host range determinant is the M062R gene product, which functions by antagonizing the host Sterile Alpha Motif Domain-containing 9 (SAMD9) protein [4, 28]. SAMD9 is a potent antiviral factor; its expression is associated with cancer suppression, inflammation regulation, and developmental processes. The M062 protein directly binds to and inhibits SAMD9, allowing productive MYXV infection in otherwise restrictive cells [28]. This interaction is highly species-specific, and the ability of M062 to overcome SAMD9 restriction in rabbit but not hare cells likely contributed to the original host range barrier that prevented MYXV from infecting hares prior to the acquisition of M159 [3, 5].
The recombinant MYXV-Tol solved this restriction by acquiring M159, a novel C7-like host range factor that provides the additional functions necessary for productive infection in hare cells [5]. Interestingly, M159 is expressed as an early/late gene and translocates to the nucleus at later time points, suggesting it may modulate host gene expression or interfere with nuclear antiviral responses in ways not required for rabbit infection [5]. The modular nature of poxvirus host range factors, where different members of the C7 family target distinct host antiviral pathways, explains how acquisition of a single gene can facilitate a species jump by complementing existing but insufficient host range functions [3, 5].
Beyond host range factors, MYXV encodes multiple immune modulators that contribute to species-specific virulence. The M029 protein, a truncated ortholog of vaccinia virus E3, functions as a double-stranded RNA binding protein that inhibits protein kinase R (PKR) and blocks the type I interferon-induced antiviral state in a highly species-specific fashion [19, 30]. In rabbit cells, MYXV completely overcomes the interferon-induced antiviral state; in human cells, this inhibition is partial; and in mouse cells, it is essentially absent [30]. This gradation of effectiveness correlates with the degree of permissivity for MYXV replication across species and underscores the importance of innate immune evasion in determining host range. Similarly, the M013 protein antagonizes both NF-κB and inflammasome signaling pathways via distinct structural motifs in its pyrin domain, further suppressing the host inflammatory response [22].
Ongoing Evolutionary Dynamics in Australia and Europe
The evolutionary trajectory of MYXV has not remained static since the early attenuation and resistance selection. Genomic analyses of Australian isolates from 2008–2017 revealed a dramatic punctuated evolutionary event, with one lineage (lineage c) exhibiting a greatly elevated evolutionary rate and breakdown of clock-like structure [26]. This rapid evolution, occurring between 1996 and 2012, coincided with significant ecological changes, including the introduction of rabbit hemorrhagic disease virus (RHDV) and prolonged drought in southeastern Australia, which drastically reduced rabbit populations [26]. The changing environment for virus transmission, fewer hosts, altered vector abundance, and shifting rabbit population structure, likely imposed new selection pressures that accelerated viral evolution. Critically, the branch leading to the rapidly evolving lineage accumulated a relatively high number of nonsynonymous substitutions, including a reversal of a mutation in M005L/R that had been fixed in all previously sequenced Australian strains [26]. This reversibility of genetic change highlights the dynamic nature of MYXV evolution in response to a fluctuating epidemiological landscape.
In the United Kingdom, genomic and phenotypic characterization of viruses isolated in 2008–2013 revealed three distinct lineages with rates of evolutionary change almost identical to those in Australia, but with remarkably little convergence at the genome level [29]. Despite similar selective pressures for transmission efficiency, UK and Australian MYXV evolved along distinct genetic pathways to achieve comparable virulence phenotypes. This observation underscores the flexibility of the MYXV genome: multiple genetic routes can produce similar phenotypic outcomes, a finding with important implications for understanding virulence evolution in large DNA viruses [29, 31]. The UK viruses exhibited a range of virulence grades (from grade 1, highly virulent to grade 5, highly attenuated) and disease phenotypes (cutaneous nodular versus amyxomatous), with some lineages displaying reading frame disruptions in genes previously considered essential for virulence yet still achieving high virulence in the field [29]. This suggests that compensatory mutations or alternative pathways can render individual genes dispensable for virulence in certain genetic backgrounds.
Ecological Consequences: Vector Ecology, Coinfections, and Population Dynamics
The transmission ecology of MYXV is inextricably linked to its arthropod vectors, primarily mosquitoes and fleas. In the UK, blood-fed Anopheles atroparvus mosquitoes collected from resting sites showed a strong feeding preference for rabbits (92% of blood meals), with 27% of these specimens testing positive for MYXV DNA [32]. This demonstrates that mosquito species within the Anopheles maculipennis complex can acquire MYXV from infected rabbits and potentially serve as competent vectors, contributing to transmission dynamics [32]. The presence of MYXV DNA in ticks (Rhipicephalus pusillus and Hyalomma lusitanicum) collected from lagomorphs in Spain suggests that ticks may also play a role in viral transmission, although their vector competence requires further elucidation [44]. The tick-derived viral sequences showed a mutation (G383A) distinguishing rabbit strains from hare strains, potentially reflecting host-specific adaptation or independent circulation [44].
Coinfections with other pathogens further complicate the ecological and evolutionary landscape. Coinfection of MYXV with rabbit hemorrhagic disease virus (RHDV) has been documented in both Finland (six of 18 MYXV-positive rabbits) and Portugal (0.52% of diseased wild rabbits) [43, 45]. The consequences of coinfection are not simply additive: previous exposure to MYXV has been shown to reduce survival of European rabbits during subsequent RHDV outbreaks, suggesting an immunosuppressive effect that compromises host resistance to other pathogens [47]. This immune-mediated trade-off has significant implications for rabbit population dynamics, particularly in regions where both viruses are endemic. Conversely, MYXV infection of Iberian hares can occur alongside polyomaviruses and anelloviruses, indicating that coinfection with multiple DNA viruses is common and may influence disease progression or viral evolution [20].
Evolutionary Implications for Conservation and Disease Management
The emergence of ha-MYXV poses urgent conservation challenges. The Iberian hare is a keystone species in Mediterranean ecosystems, serving as prey for endangered predators including the Iberian lynx (Lynx pardinus) and Spanish imperial eagle (Aquila adalberti). Population declines due to myxomatosis could trigger cascading ecological effects, including reduced prey availability and altered predator-prey dynamics [13, 33]. The failure of commercial rabbit myxomatosis vaccines to protect Iberian hares against ha-MYXV, even at standard doses, complicates conservation efforts [42]. However, higher doses of one vaccine (Mixohipra-FSA)
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