Myxoma Virus in the European Rabbit: Interactions Between the Virus and Its Susceptible Host Marianne Stanford, Steven Werden, Grant Mcfadden

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Myxoma virus in the European rabbit: interactions between the virus and its susceptible host Marianne Stanford, Steven Werden, Grant Mcfadden

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Marianne Stanford, Steven Werden, Grant Mcfadden. Myxoma virus in the European rabbit: interactions between the virus and its susceptible host. Veterinary Research, BioMed Central, 2007, 38 (2), pp.299-318. 10.1051/vetres:2006054. hal-00902835

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vet. Res. 38 (2007) 299–318 299 c INRA, EDP Sciences, 2007 DOI: 10.1051/vetres:2006054 Review article

Myxoma virus in the European rabbit: interactions between the virus and its susceptible host

Marianne M. S,StevenJ.W,GrantMF*

Biotherapeutics Research Group, Robarts Research Institute and Department of Microbiology and Immunology, University of Western Ontario, Siebens-Drake Building, Room 126, 1400 Western Road, London, Ontario, N6G 2V4, Canada

(Received 9 March 2006; accepted 20 June 2006)

Abstract – Myxoma virus (MV) is a poxvirus that evolved in Sylvilagus lagomorphs, and is the causative agent of myxomatosis in European rabbits (Oryctolagus cuniculus). This virus is not a natural pathogen of O. cuniculus, yet is able to subvert the host rabbit immune system defenses and cause a highly lethal systemic infection. The interaction of MV proteins and the rabbit immune system has been an ideal model to help elucidate host/poxvirus interactions, and has led to a greater understanding of how other poxvirus pathogens are able to cause disease in their respective hosts. This review will examine how MV causes myxomatosis, by examining a selection of the identi- ﬁed immunomodulatory proteins that this virus expresses to subvert the immune and inﬂammatory pathways of infected rabbit hosts.

myxoma virus / immunomodulation / poxvirus / Oryctolagus cuniculus

Table of contents 1. Introduction ...... 300 2. Aetiology...... 300 3. Epidemiology and clinical signs of myxomatosis...... 301 4. Pathogenesis...... 303 4.1. Virokines and viroreceptors...... 303 4.1.1. Virokine: M-T1...... 303 4.1.2. Viroreceptor: M-T2...... 305 4.1.3. Viroreceptor: M-T7...... 305 4.1.4. Virokine: Serp-1...... 306 4.1.5. Virokine: Myxoma Growth Factor (M010L) ...... 306 4.2. Apoptosis...... 306 4.2.1. Apoptosis: M-T4 ...... 307 4.2.2. Apoptosis: M-T5 ...... 307 4.2.3. Apoptosis: M11L ...... 308 4.2.4. Apoptosis: Serp-2 ...... 308 4.3. Modulation of macrophage and T cell activation ...... 309 4.3.1. Leukocyte activation: M141R ...... 309 4.3.2. Leukocyte activation: M128L...... 310 * Corresponding author: [email protected]

Article available at http://www.edpsciences.org/vetres or http://dx.doi.org/10.1051/vetres:2006054 300 M.M. Stanford et al.

4.3.3. Leukocyte activation: Myxoma Nuclear Factor (M150R) ...... 310 4.3.4. Leukocyte activation: Serp-3 ...... 310 4.3.5. Leukocyte activation: M13L ...... 311 4.3.6. Leukocyte activation: M153R...... 311 5. Prevention, control and vaccination ...... 311 6. Conclusions ...... 313

1. INTRODUCTION Europe by a landowner hoping to curb the rabbit population of his property. The sub- Myxoma virus (MV) causes the de- sequent rapid coevolution of both host and bilitating disease known as myxomatosis virus has been well documented [29]. The in domesticated European rabbits. This result has been the selection of both dra- virus is a member of the Leporipoxvirus matically attenuated field variants of MV, genus and the Chordopoxvirinae subfam- and an increasingly resistant population of ily of Poxviridae. It was first described rabbits present in Australia [8]. by Sanarelli in 1896 in South America as The entire genome of myxoma virus the causative agent of a lethal infection strain Lausanne has been sequenced, in imported European rabbits (Oryctolagus and many of both the structural pro- cuniculus). Subsequent work determined teins and immunomodulatory proteins MV to be a natural pathogen of the South have been characterized [5, 11]. There American tapeti (Sylvilagus brasilensis), are many extensive and excellent reviews and of the North American brush rabbit on the immunomodulatory proteins of (Sylvilagus bruneii) with MV strains en- poxviruses [42, 50, 64, 69, 74, 75, 87, 91, demic of these areas being designated the 106]. This review will focus primarily on Brazilian and Californian strains, respec- the interactions of MV and its suscepti- tively [31]. In its evolutionary Sylvilagus ble host, the European rabbit in vitro and hosts, this virus causes only a benign infec- in vivo. tion, characterized by a cutaneous fibroma restricted to the site of the inoculation. However, in European rabbits, virus infec- 2. AETIOLOGY tion causes the rapid systemic and lethal infection that has mortality rates at nearly MV is a member of the Poxviridae fam- 100% [30]. ily, and the prototypic member of the Lep- The introduction of the European rabbit oripoxvirus genus. Poxviruses are all large, to Australia in 1859 has had a devastating linear double stranded DNA viruses that effect on the biodiversity of that conti- have the capacity to infect both inverte- nent. In 1918, Aragão, who was involved in brates (Entomopoxvirinae) and vertebrates much of the initial characterization of MV, (Chordopoxvirinae). Poxviruses also have formally suggested using this virus to curb a classic brick shaped virion and replicate the feral rabbit population. After thorough exclusively in the cytoplasm of infected investigation, MV was used as a biological cells. Poxviruses have the ability to cause control in 1950, when the Standard Lab- both acute and persistent infections, yet no oratory Strain (SLS), also known as the poxvirus has been shown to integrate its Moses strain, isolated in Brazil in 1911, DNA into the host genome, or undergo a was released into the wild. In 1952, a sim- true latent stage infection [68]. ilar strain known as Lausanne, isolated in Poxviruses are larger than most animal Brazil in 1949, was illegally released into viruses with dimensions in the order of Myxomatosis in the European rabbit 301 several hundred nanometers, and can be scription of poxviral genes. As late viral observed by light microscopy [68]. The se- gene products accumulate, the virus un- quence of MV revealed it to have 161.8 kB dergoes morphogenesis and assembly of of DNA, encoding 171 unique genes [11]. infectious virus particles. The first infec- The central portion of the genome encodes tious forms that appear are the intracellular close to 100 genes that are among the mature virions (IMV) which travel along highly conserved set of conserved poxvi- microtubules and then become enwrapped ral genes, encoding mostly structural and in Golgi-derived membranes to form in- housekeeping functions, while the ends tracellular enveloped virus or IEV [83]. have terminal inverted repeats (TIR) and However, early tubular endosomes have closed hairpin looks at each end [32, 34]. also been shown to contribute to IEV for- These TIR and the near-terminal unique re- mation [100]. As the IEV fuses with the gions of the poxvirus genome are where cellular membrane, it loses a membrane many immunomodulatory genes involved wrapping to form the cell associated en- in subverting the host immune system map. veloped virus, or CEV, an important com- A selection of these host-interaction pro- ponent of cell to cell infection, as this virus teins of MV will be discussed in the next is propelled into the neighboring cell via section. actin tail formation, or can be released Poxviruses are enveloped viruses, and from the cell as EEV. Whereas CEV and have at least two distinct forms that can EEV are important in cell to cell spread in productively infect cells – the intracellu- vivo, IMV infection becomes important in lar mature virus (IMV) and the extracel- virus spread after cell death and membrane lular enveloped virus (EEV). These two rupture [89,104]. forms differ in their membrane compo- In addition to the numerous poxviral nents and are thought to enter cells by proteins involved in propelling the virus different mechanisms [88, 101, 102]. Al- through its replication cycle, these viruses though there have been multiple viral pro- also encode an additional plethora of proteins that have been shown to be essential teins that allow them to survive in the for virus entry/fusion into cells [70], the harsh environment of the host. These in- cellular determinants for poxvirus bind- clude expressing inhibitors of the antiviral ing are believed to be the ubiquitously pathways of the cell, preventing the cell expressed surface glycosaminoglycans or death of the infected cell and the encod- extracellular matrix proteins [15,18]. After ing anti-inflammatory proteins to dampen virus binding to cellular membranes, there the host defenses mounted against it. These is a fusion step that is generally poorly immunomodulatory proteins often mediate understood, yet results in the release of pathogenesis induced by these viruses [87] the virion core into the cytoplasm of the and some will be discussed in greater detail cell [70]. The viral transcriptosome is in in the next sections. the released core, containing the endoge- nous RNA polymerase and transcription factors. It initiates the first wave of early 3. EPIDEMIOLOGY AND CLINICAL viral gene transcription, synthesizing vi- SIGNS OF MYXOMATOSIS ral mRNA under the control of early viral promoters. This stimulates the second Today, MV is endemic on four con- uncoating stage, releasing the viral DNA tinents: North and South America, Eu- into the cytoplasm where it can serve as rope and Australia [20]. In much of South a template for viral DNA replication and America, such as Brazil and Uruguay, the subsequent intermediate and late tran- the virus is endemic in rabbits of the 302 M.M. Stanford et al.

Sylvilagus genus, particularly S. brasilien- toxemia or cytokine storm) induced by the sis. However, in countries such as Chile, virus in its host. the principal reservoir is the wild Euro- The clinical signs and high mortality pean rabbit (O. cuniculus). On the contrary, rates associated with myxomatosis are be- in North America, the Californian strain lieved to be a result of multi-organ dys- of MV is endemic to wild Sylvilagus rab- function as well as uncontrolled secondary bits in the area, particularly S. bachamani bacterial infections of the respiratory tract and this species serves as a reservoir of in- that result from the progressive impair- fection for domestic rabbits. In Australia, ment of the host immune response [29, 31, the virus has been endemic in the wild 32, 68]. The early clinical signs of typical European rabbit population since its intro- myxomatosis are swelling of the muzzle duction in 1950. In Europe, myxomatosis and anogenital region, as well as blepharo- has spread to many countries after its ini- conjuctivitis [29,32,68]. This gives the rab- tial introduction in 1952, with O. cuniculus bit a leonine-like appearance. The severity being the predominant host species. How- of clinical disease can vary between dif- ever, other lagomorphs such as the Euro- ferent strains of rabbits as well as MV pean hare (Lepus europaeus), the mountain isolates. The variety of clinical signs have hare (Lepus timidus) and other Sylvilagus been extensively reviewed elsewhere [27– species can also serve as hosts [20]. 29]. The clinical signs of myxomatosis vary In general, Californian strains of myx- widely among the evolutionary and sus- oma virus are quite pathogenic. Animals ceptible lagomorph hosts. In members of can die within a week of infection, with the Sylvilagus species, infection usually re- little clinical signs other than edema and sults in the formation of localized skin lethargy. Animals that survive 1–2 weeks pseudo-tumors at the site of virus infection. exhibit more inflammation, edema and These tumors resemble fibromas and usu- skin hemorrhages. Rabbits infected with ally appear 4–8 days after exposure, and Brazilian strains (such as the South Amer- may persist for up to 40 days [46]. Young ican Moses strain) usually survive for Sylvilagus rabbits may succumb to a gen- 10–14 days and subcutaneous gelatinous eralized disease following infection with swellings (from which the name myxo- MV [31]. matosis was derived) appear all over the Myxomatosis in European rabbits is body. Typically, MV replicates in the skin much more severe and results in a high at the site of infection and spreads from mortality rate. The severity of the clini- there into the draining lymph node. MV cal disease can differ with the strain of then replicates in the node to high titers MV used, as well as the strain of rabbit and spreads via infected leukocytes to dis- infected. To date, there are at least two tal tissues such as the spleen, testis, lung, forms of myxomatosis in European rabbits; nose, conjunctivae and skin [8, 26]. The the nodular or typical form of myxomato- formation of myxomas has not been resis and amyxomatosis or “atypical” myx- ported with all strains of virus and may omatosis. The main difference between contribute to the two classified forms of the these forms is that the latter is character- disease [20]. By the 10th day, hard convex ized by having highly reduced cutaneous lumps appear, and the primary lesion has clinical signs and is typically classified by become necrotic, whereas the secondary intense respiratory distress in the atypical lesions may also become necrotic in rab- form of the disease [60–62]. This increased bits surviving over two weeks. Infected lethality of this type of myxomatosis may rabbits usually live 8–15 days, and death be due to severe immunopathology (i.e. is often preceded by terminal convulsions. Myxomatosis in the European rabbit 303

Not all myxoma strains are lethal consequently developing a variety of so- in the European Rabbit. The laboratory- phisticated strategies to ensure successful attenuated neuroMV produces a mild dis- viral replication within the host [38,69,91]. ease with little or no mortality [20]. In One particular mechanism conceived by both Australia and Europe, attenuated MV MV to subvert the host immune response strains have emerged with lower morbidity is carried out by two related sets of and mortality [9]. These naturally attenu- immunomodulatory proteins described as ated strains, including the Urrarria strain in virokines and viroreceptors [74,87]. These Australia produce flat rather than convex virus-encoded proteins target specific lesions and generally have a much lower pathways of the host immune system used mortality rate in susceptible European rab- to coordinate antiviral and early inflam- bits [9]. matory responses directed against viral infection [64, 74]. Viroreceptors closely 4. PATHOGENESIS resemble cellular receptors and are often secreted or expressed on the surface of The pathogen-host relationship between virus infected cells [65]. They functionally MV and the European rabbit has been well bind and inhibit extracellular host ligands characterized [46, 48] and is an excellent which are intended to induce an inflamma- model to study the mechanisms by which tory or antiviral response following virus a large DNA virus can use its genetic infection [42]. On the contrary, virokines repertoire to manipulate the host immune are normally secreted and are often similar response during viral infection. Success- to host immune ligands such as cytokines, ful viral replication can be attributed to or chemokines, but usually the viral the ability of the MV to avoid recog- versions are smaller or have alternative nition and clearance by the host innate biological properties compared to the host and acquired immune systems of the in- ligands [54, 64, 90]. Examples of myxoma fected rabbit [47, 87]. A wide variety of encoded secreted immunomodulatory MV encoded immunoregulatory proteins proteins include the following: (1) M-T1, have been identified which include; vi- an inhibitor of CC-chemokines; (2) M-T2, rokines, viroreceptors, proteins that mod- / a tumor necrosis factor receptor homolog; ulate host range and or apoptotic response (3) M-T7, a homolog of the interferon- and proteins that modulate the action of gamma receptor; (4) Serp-1, a secreted macrophages and T cells (Tab. I). The pro- serine proteinase inhibitor; (5) myxoma teins discussed do not represent all MV im- growth factor, ligand for the erbB family munomodulator proteins, yet give a cross of EGF receptors. section of the scope of proteins encoded by this virus, and are the MV proteins most extensively characterized in both in vitro 4.1.1. Virokine: M-T1 and in vivo studies. These particular MV proteins have been experimentally shown The MV glycoprotein M-T1 is a mem- to function as critical virulence factors nec- ber of a large poxvirus family of se- essary for the infection and replication of creted chemokine-binding proteins [50, MV leading to the development of myxo- 55, 105]. Two copies of the M-T1 gene matosis in European rabbits. are present in the TIR of the genome and the secreted protein has a molecular mass of 43 kDa [52]. Chemical cross- 4.1. Virokines and viroreceptors linking demonstrated that M-T1 interacts Throughout evolutionary history with a broad spectrum of both CXC- poxviruses have continuously evolved, and CC-chemokine subfamilies; with a 304 M.M. Stanford et al.

Table I. Selected immunomodulatory proteins of MV.

Required for infection ORFs Gene name Protein function and/or properties Reference(s) In vitro In vivo Virokines and viroreceptors M001 L/R M-T1 Chemokine binding protein; inhibits No No [35, 51, 52] chemokine chemoattractant properties M002 L/R M-T2 TNF receptor homolog; binds rabbit Only Yes [58, 84–86, TNF-α RL-5 97] M007 L/RM-T7 IFN-γ receptor homolog; also binds No Yes [10, 49, 71, C-C and C-X-C chemokines in vitro 72, 98] M008.1L/R SERP-1 Secreted serpin No Yes [57, 96] M010L MGF EGF-like growth factor No Yes [78, 79]

Anti-apoptotic factors M004 L/R M-T4 ER-localized apoptosis regulator Only Yes [6, 39, 76, RL-5 77, 106] M005 L/R M-T5 Ankyrin-like host range; Cell RL-5, Yes [44, 73, 93] cycle/apoptosis inhibitor cancer cells M11L M11L Integral membrane protein/apoptosis Only Yes [21, 24, 78, regulator RL-5 92, 103] M151R SERP-2 Intracellular serpin No Yes [67, 80]

Immune modulators M141R vOx-2 Immunoglobulin domain/OX-2 No Yes [11, 12] /vCD200 homolog M128L vCD47 Integrin-associated protein, CD47 No Yes [11, 13] homolog M150R MNF Ankyrin-like/host range, inhibits No Yes [11, 14] NF-κB M013L M13L Pyrin-containing inhibitor of the Only Yes [43] inflammasome RL-5 M152R Serp-3 Virulence factor, viral serpin No Yes [36] M153R MV-LAP Promotes downregulation of MHC-I No No [37, 59] and CD4 on T cells preferentially higher affinity towards CC- tality rate of European rabbits infected chemokines [35]. Therefore, M-T1 func- with recombinant MV bearing the dis- tions to effectively sequester soluble host rupted M-T1 genes (vMyxT1KO) was chemokine ligands from binding their cog- identified when compared to rabbits innate cell-surface receptors in vitro, con- fected by the wild-type strain. The M-T1 sequently inhibiting chemoattractant ac- gene is thus dispensable for a productive tivities [51]. No significant difference in virus infection. However, during the first viral replication levels or the overall mor- few days post infection an increase in the Myxomatosis in the European rabbit 305 localized cellular infiltrate was observed recovery, and when the survivors were sub- in the deep dermal tissues surrounding the sequently challenged with wild type MV, site of primary infection of vMyxT1KO they were resistant to developing myxo- infected rabbits. Leukocytes such as mono- matosis [97]. Thus, both in vitro and in cytes/macrophages were present in the in- vivo studies conclude that the M-T2 gene filtrate although these phagocytes were is an essential virulence factor of MV, nec- ineffective at clearing the virus infection essary for successful disease progression in [52]. Therefore, the functional role of susceptible rabbits resulting in myxomato- M-T1 is to bind and sequester chemokines sis. responsible for the recruitment and activation of leukocytes during early stages of MV infection in susceptible rabbits. 4.1.3. Viroreceptor: M-T7

The most abundantly secreted pro- 4.1.2. Viroreceptor: M-T2 tein from MV-infected cells, M-T7, has significant sequence similarity to the The MV gene M-T2 was the first iden- human and mouse receptors for interferon- tified viroreceptor based on similar se- gamma (IFN-γ) [71]. M-T7 was ini- quence homology to the N-terminal ligand- tially identified to specifically bind to binding domain of cellular tumor necro- and inhibit the biological activity of ex- sis factor (TNF) receptors [97]. M-T2 tracellular rabbit IFN-γ [98]. Later bind- is a glycosylated protein, expressed as ing studies indicated that purified M-T7 monomeric and dimeric species that are protein also interacts with representa- secreted from virus infected cells, and tive members of the CXC-, CC-, and both have been demonstrated to bind rab- C-chemokine families by targeting the bit specific TNF with an affinity simi- conserved C-terminal heparin-binding do- lar to the rabbit TNF receptor [84, 85]. main present on many cytokines [49]. In However, the dimeric form of M-T2 was addition, M-T7 may functionally disrupt shown to be a more potent inhibitor of chemokine-glycosaminoglycan (GAG) in- TNF cytolytic activities, presumably due teractions within virus-infected cells, lead- to an increased ability to prevent TNF re- ing to the interruption of important ceptor signaling in response to the TNF chemokine gradients [10]. Virus replica- trimer [86]. Rabbit T lymphocytes (as rep- tion in cultured cells is unaffected by re- resented by the CD4+ T-cell line RL-5) combinant MV disrupted in both copies underwent extensive cellular apoptosis fol- of the M-T7 gene (vMyxT7KO) but in lowing infection by MV deficient in M-T2 vivo the M-T7 gene is required for a pro- (vMyxT2KO), therefore M-T2 also func- ductive viral infection and it is a critical tions to block apoptosis. The addition of virulence factor for virus pathogenesis in exogenous M-T2 protein to vMyxT2KO the European rabbit [72]. In comparison infected RL-5 cells was unable to in- to rabbits infected by the wild-type virus, hibit cellular apoptosis in response to viral a dramatic decrease in the clinical signs infection, suggesting that M-T2 inhibits of myxomatosis and viral dissemination apoptosis intracellularly [58]. Disease pro- into secondary sites was observed in the gression in susceptible rabbits is consid- vMyxT7KO infected rabbits. In addition, erably attenuated when rabbits have been rabbits infected by the M-T7 knockout infected with vMyxT2KO in contrast to the virus were characterized by an increase parental virus strain [97]. The majority of in the number of infiltrating leukocytes vMyxT2KO infected rabbits made a full that migrate into the virus-infected lesions. 306 M.M. Stanford et al.

An extensive number of lymphocytes in ical for virus survival in infected rabbits, the secondary immune organs, particularly leading to myxomatosis. the spleen and lymph nodes, appeared to be activated as early as four days post infection [72]. In summary, M-T7 func- 4.1.5. Virokine: Myxoma Growth Factor tions to modify the inflammatory cellular (M010L) trafficking of lymphocytes into secondary lymphoid organs by binding and neutraliz- M010L, also known as myxoma growth ing host IFN-γ, and possibly destabilizing factor (MGF), is a secreted glycoprotein, the local chemokine gradients, at the site which shares significant sequence similar- of infection. The actions of M-T7 effec- ity to members of the epithelial growth tively disrupt the connection between anti- factor (EGF) family of proteins. In vitro gen presenting cells of the infected tissue studies have demonstrated that MGF is and the effector lymphocytes of the sec- a non-essential factor for successful virus ondary lymphoid organs. replication in rabbit spleen cells [78]. In vivo, however, the MGF gene is an essential virulence factor, such that viral repli- 4.1.4. Virokine: Serp-1 cation was severely attenuated in rabbits infected with MV deficient in the MGF The MV glycoprotein serpin 1 (Serp-1) gene (vMyxMGFKO). A majority of the is a 55 kDa secreted protein, which func- rabbits infected never became seriously ill tions as an irreversible inhibitor of serine and were resistant to myxomatosis when proteases [96]. Serp-1 interacts with and re-challenged by wild-type MV [78]. Full inhibits, among others, plasmin, urokinase, disease progression was observed in rab- and tissue-type plasminogen activator [19, bits infected with vMyxMGFKO when ei- 53]. However, many of these serine pro- ther vaccinia virus growth factor, Shope teases were identified through human stud- fibroma growth factor (SFGF) or rat trans- ies and little is know how Serp-1 interacts forming growth factor alpha was used to with similar proteins in the rabbit. Rab- restore the function of MGF in the knock- bits infected with MV deficient in Serp-1 out virus [79]. MGF therefore is required (vMyxSerp1KO) suffered moderate clini- for MV replication in vivo but the mecha- cal signs of myxomatosis but the disease nism of its role in pathogenesis remains to was unable to progress to later stages. The be better clarified. majority of infected rabbits showed significant recovery within 14 days post infection and when re-challenged with the 4.2. Apoptosis parental myxoma strain, were resistant to myxomatosis [57]. Significant tissue dam- Apoptosis is a regulated process of cel- age in the vMyxSerp1KO infected rabbits lular death used to remove unwanted or was observed in histological studies which damaged cells while causing minimal dis- included infiltration of mononuclear cells ruption to surrounding tissues [45]. In and a pronounced inflammatory response. response to virus infection, cells initiate The clinical signs, however, were less se- apoptosis which is therefore a critical host- vere compared to rabbits infected with the protective mechanism for limiting virus in- wild-type virus [57]. Therefore, Serp-1 is fection and replication within the host [25]. an essential virulence factor and its func- Apoptosis is generally accompanied by the tional role in preventing the infiltration of release of cytochrome c and other pro- monocytes into the primary lesion is crit- apoptotic regulators from the mitochondria Myxomatosis in the European rabbit 307 and the activation of caspases. This leads gesting vMyxT4KO is unable to spread in to a series of downstream events ultimately vivo. Reduced virulence in infected rab- resulting in the digestion of key structural bits may be due to the low number of proteins in the cytoplasm and degradation infected T lymphocytes available to carry of chromosomal DNA. Viruses with larger virus to distal lymph nodes [6]. The mech- genomes, such as poxviruses, have evolved anism by which M-T4 protects infected immunomodulatory proteins which func- cells against apoptosis from within the tion to inhibit host-cell apoptotic responses ER remains poorly understood. Rabbits in- during early stages of viral infection [74, fected with a recombinant M-T4 RDEL− 94]. These anti-apoptotic effectors have mutant virus suffered a novel form of myx- been shown to manipulate the cell death omatosis, which was characterized by an pathways within the cell in a variety of exaggerated inflammatory response at the ways, including the inhibition of caspases secondary sites of infection, particularly and disruption of key mitochondrial check- the ears [39]. Therefore M-T4 may have points to prevent apoptosis [22, 23, 94]. a dual function in protecting infected lym- Therefore, the interaction between viral phocytes from apoptosis and in modulating proteins and host apoptotic pathway is the inflammatory response to virus infec- essential for MV to successfully repli- tion. cate in the European rabbit. Examples of MV-encoded proteins which regulate apoptosis in response to MV infection in- 4.2.2. Apoptosis: M-T5 clude the following: (1) M-T4, (2) M-T5, M-T5 is an ankyrin-repeat containing (3) M11L and (4) Serp-2. protein that possesses no significant sequence homology to non-viral proteins but 4.2.1. Apoptosis: M-T4 is a member of a larger poxviral superfamily implicated in the determination of M-T4 was identified as the first intracel- host range [73]. RL-5 cells or primary lym- lular virulence factor in MV that functions phocytes infected with M-T5 deficient MV from within the endoplasmic reticulum (vMyxT5KO) failed to result in success- (ER) of infected cells [6]. Like most pro- ful viral replication, however, the same teins specifically localized to the ER, M- mutant virus replicated with wild-type ki- T4 posses a C-terminal ER-retention motif netics in rabbit fibroblasts [73]. Therefore, (RDEL) predicted to be critical for re- M-T5 is capable of extending the tissue trieval and retention of M-T4 in the ER [6]. specificity of MV to cells of lymphoid However, removal of the RDEL motif from origin. When RL-5 T cells were infected M-T4 did not affect localization of the pro- with M-T5 knockout virus they underwent tein within the ER, but the stability of an extensive cellular apoptotic response the mutant protein was reduced [39]. M- as well as rapid inhibition of both host T4 was determined to have a functional and viral genes [73]. It has been sug- role as an anti-apoptotic protein when cul- gested that M-T5 is essential for cells tured rabbit lymphocytes infected with M- infected with myxoma to overcome cell T4 knockout mutant virus (vMyxT4KO) cycle arrest induced by viral infection, pro- underwent extensive apoptosis. Rabbits in- tecting cells from activating diverse cell fected with M-T4 knockout virus resulted death pathways resulting in apoptosis [44]. in disease attenuation, and recovered fully M-T5 was demonstrated to be a cell cy- within 10 to 21 days post infection [6]. cle regulatory protein by direct interac- The number of secondary lesions on the tion with cullin-1, a cellular E3 ubiqui- infected animals was greatly reduced sug- tin ligase which enhances the proteasomal 308 M.M. Stanford et al. degradation of cell cycle regulators such and Bax [92], both Bcl-2 family mem- as CDK-1 p27Kip [44]. Similarly to the in bers that are constitutively present on the vitro studies, susceptible laboratory rabbits mitochondria. Deletion of the M11L gene infected with vMyxT5KO lacked progres- from MV completely abrogated the abil- sion of the infection past the primary site ity of the virus to cause the characteristic of inoculation, in addition to the establish- disease clinical signs associated with myx- ment of a rapid and effective inflamma- omatosis in susceptible rabbits [78]. All tory reaction. The M-T5 knockout virus rabbits infected with the M11L knock- was unable to initiate a cellular reaction out virus recovered completely by day 40 within secondary immune organs [73]. Ac- post infection and when challenged with cording to these observations, the M-T5 wild-type MV, were found to be resis- protein permits virus spread via infected tant to developing myxomatosis. However, lymphocytes that traffic to multiple distal despite the reduced virulence of the re- sites and therefore functions as an essen- combinant virus, lesions produced by the tial virulence factor in the establishment of virus were larger and histological analysis myxomatosis in the European rabbit. revealed signs of vigorous inflammatory activity but regressed 30 days after infection [78]. In conclusion, the M11L gene 4.2.3. Apoptosis: M11L product is an important virulence deter- The MV protein M11L has no well de- minant for myxoma infection, modulating fined structural motifs except a unique 25 apoptosis, induced by virus infection, by amino acid long C-terminal sequence that multiple independent strategies that con- forms a putative transmembrane domain tribute to the blockade of apoptosis in the similar to those present in anti-apoptotic mitochondria. Bcl-2 family members [21]. M11L is specifically localized in the mitochondria 4.2.4. Apoptosis: Serp-2 where it inserts into the outer membrane and is exposed to the cytoplasmic face of MV encodes an intracellular viral ser- the organelle in MV-infected cells [21]. In pin (Serp-2) which has a molecular mass vitro studies have identified M11L as a of 34 kDa and is a member of the serpin functional anti-apoptotic protein that pre- superfamily [80]. In culture, both rabbit vents the loss of mitochondrial membrane fibroblasts and rabbit CD4+ T-cells sup- potential, which accompanies cell death, ported replication of a Serp-2 knockout by blocking staurosporine-induced apop- mutant MV (vMyxSerp2KO) with wild- tosis [24]. Based on cross-linking stud- type kinetics [67]. Serp-2 is, however, an ies, M11L forms an inhibitory complex essential MV virulence factor, and the ma- with the peripheral benzodiazepine recep- jority of rabbits infected with the Serp-2 tor (PBR), a pro-apoptotic protein located knockout virus only developed moderate on the outer mitochondrial membrane. It is clinical signs of myxomatosis and recov- through this interaction that M11L specif- ered completely within 30 days post in- ically blocks apoptosis induced by ligands fection. Histological examination revealed of PBR. Deletion of the M11L C-terminus extensive inflammation in response to abolished the functional interaction be- infection by the Serp-2 mutant virus in tween PBR and M11L, indicating that lo- contrast to wild-type MV. In addition, calization of M11L to the mitochondria rapid apoptosis was evident in the lymph is critical for its activity [24]. In addi- node lymphocytes of the vMyxSerp2KO tion, M11L has been shown to interact infected rabbits [67]. Therefore, Serp-2 is with the pro-apoptotic proteins Bak [103] a virulence factor that is critical for the Myxomatosis in the European rabbit 309 development of myxomatosis by inhibit- factors that are important for their acti- ing host inflammatory processes and by the vation, and potentially sending conflicting prevention of apoptosis in lymphocytes, signals, preventing the successful activa- thus enabling them to spread to secondary tion of these cells. These MV proteins in- sites of infection. Compared to CrmA, clude the following: (1) M141R (vCD200), the prototypic poxvirus serpin encoded by (2) M128L (vCD47), (3) M150R (myxoma cowpox virus, MV Serp-2 is a weak in- nuclear factor), (4) Serp-3, (5) M13L and hibitor of human interleukin-1β (IL-1β)- (6) M153R. converting enzyme (ICE) and granzyme B in vitro [80, 95]. Both Serp-2 and CrmA are functionally similar in vivo, however, 4.3.1. Leukocyte activation: M141R when Serp-2 was tested for its ability to M141R is a membrane-associated pro- replace CrmA in cowpox virus-infected tein which shares significant amino acid cells, Serp-2 was unable to inhibit apop- similarity to the family of cellular CD200 tosis. The results indicate that Serp-2 and (OX-2) proteins, responsible for regulating CrmA function differently in vitro [95]. the activities of myeloid lineage cells [4]. Further studies are needed to determine A number of features present in cellular if Serp-2 has a similar inhibitory function CD200/OX-2 are also found in M141R, during MV infection in the rabbit. including the C-terminal transmembrane domain, two highly conserved cysteine residues involved in the formation of the 4.3. Modulation of macrophage and V-like immunoglobin domain and several T cell activation critical residues invariably conserved in immunoglobin domains [11]. An increased The activation of both the innate and ac- level of IFN-γ was observed when pe- quired branches of the immune response is ripheral blood mononuclear cells purified critical for viral clearance. To evade such from rabbits infected with MV deficient in a response, MV has developed strategies the M141L gene (vMyx141KO) were ac- to specifically inhibit the activation of cells tivated in vitro [12]. Cultured rabbit cell important in the initiation and propagation lines infected with vMyx141KO showed of both resident and recruited leukocytes. no differences in growth or replication The resident macrophages are an impor- kinetics in comparison to the wild-type tant first line of defense against invading virus, however, M141R is essential for the pathogens, and have the ability to rapidly development of myxomatosis in suscepti- express and secrete a plethora of cytokines ble rabbits [12]. All rabbits infected with and chemokines that act as danger signals the M141R-knockout virus completely re- to surrounding tissues. T cells are educated covered with only mild signs of dis- in the lymph nodes in the proximity of in- ease progression and all were completely fection, and travel to the site of infection to protected from myxomatosis when re- produce crucial mediators and to directly challenged with the wild-type virus [12]. kill virus infected cells in what is known as In the absence of M141R, a dramatic in- the cell mediated immune response. In ad- crease in the recruitment and activation of dition to inhibiting the efficacy of secreted monocytes/macrophages and lymphocytes mediators by the production of virokines with the capacity to express high levels of and viroreceptors, MV also directly tar- the virotoxic compound nitric oxide was gets the ability of leukocytes to become observed in lymphoid organs infected with activated. It does this through the produc- MV [12]. Additionally, M141R was shown tion of viral proteins that both mimic host to reduce T-cell activation levels in lymph 310 M.M. Stanford et al. nodes and inhibit the ability of circulat- increase in the activation and/or recruit- ing T-cells to respond to T-cell antigen- ment of iNOS+ cells to the site of infection independent activation [12]. In conclusion, and in the lymphoid tissues during infec- M141R is essential for complete pathogen- tion [13]. In conclusion, M128L is a novel esis of MV and functions to inhibit tissue CD47-like immunomodulatory gene and macrophages by reducing their ability to plays a critical role in the pathogenesis antigenically prime lymphocytes and pos- of MV by inhibiting the activation of sibly provides anergic signals to T cells myeloid-lineage cells. directly.

4.3.3. Leukocyte activation: Myxoma 4.3.2. Leukocyte activation: M128L Nuclear Factor (M150R)

The membrane-associated protein MV encodes a number of ankyrin- M128L of MV is 218 amino acids in containing proteins including the gene length, predicted to have a molecular mass M150R [11], which has a total of nine of 32 kDa and is predicted to function as an ankyrin repeats (ANK), with the eighth immunoregulator [11, 13]. M128L shares having a close similarity to the nuclear significant amino acid homology to the localization signal-containing ANK of I- cellular CD47 protein (integrin-associated κBα [14]. The protein I-κB functionally protein, or IAP), a well documented reg- binds to the transcription factor NF-κB ulator of the vertebrate immune response to form an inactive I-κB/NF-κB complex which exerts multiple functions on various which sequesters NF-κB in the cytosol, in- immune cell types [99]. A number of hibiting its activity [3, 7, 17, 33]. In cells key features present in CD47 proteins treated with TNF, both M150R and NF-κB are also present in M128L, including co-localized to the nucleus and therefore an N-terminal signal sequence, a single the protein was named myxoma nuclear predicted transmembrane domain and a factor (MNF) [14]. In vivo studies have short cytoplasmic tail [13]. The classical demonstrated that MNF is a critical viru- immunoglobulin-like domain is not pre- lence factor for the development of myx- dicted to be present in M128L; however, omatosis in susceptible rabbits. In contrast there is significant conservation of cystine to rabbits infected with wild-type MV, only residues and flanking amino acids that map mild signs of virus infection were observed to the CD47 immunoglobulin boundary in rabbits infected with the MNF knockout domain [11]. M128L is fully dispensable virus [14]. These particular rabbits fully re- for virus replication and infection in vitro covered within 21 days post infection and and the vMyx128KO showed no obvious ff when infected with the wild-type virus, di erences in growth or replication ki- were resistant to developing myxomatosis. netics when compared to the wild-type In the absence of MNF, an increase in the MV [13]. Early stages of viral disease inflammatory process was observed at the progression were evident when rabbits site of virus infection suggesting that MNF were infected with vMyx128KO virus, inhibits the NF-κB-induced proinflamma- however after day seven post infection tory pathways [14]. signs of reduced virulence were observed. All rabbits made a complete recovery from viral infection and when re-challenged 4.3.4. Leukocyte activation: Serp-3 with wild type MV, they were found to be completely protected from myxomato- The MV genome encodes a third ser- sis [13]. Histological studies identified an pin protein, Serp-3, which contains a Myxomatosis in the European rabbit 311 conserved serpin motif but lacks the signif- tiviral response of the host during infection icant sequence homology present in most is critical for virus replication and there- cellular and viral serpins. Serp-3 is present fore M13L is a critical immunomodulatory inasinglecopyandthegeneislocated protein for the development of myxomato- near the right TIR and encodes a protein sis [43]. with a molecular mass of approximately 30 kDa. Serp-3 is not essential for viral replication in vitro nor in vivo [36]. MV 4.3.6. Leukocyte activation: M153R deficient in Serp-3 showed no defects in the ability to infect and replicate in either + rabbit fibroblasts or CD4 T-cell lines. His- The M153R open reading frame of tological studies of rabbits infected with MV encodes the protein, MV leukemia- the knockout virus identified an increase in associated protein (MV-LAP). M153R is the inflammatory response and the lack of an endoplasmic reticulum (ER)-resident secondary myxomas present in the lymph and is characterized by an amino-terminal nodes [36], resulting in dramatically atten- PHD/LAP motif and two centrally lo- uated virulence in vivo, reflected by the cated transmembrane domains. MV-LAP decrease in mortality rate of virus infected was demonstrated to promote the down- rabbits. Serp-3 represents a critical viru- regulation of both surface MHC-I [37] lence factor for MV and probably acts in and the T-cell co-receptor molecule CD4 synergy with other viral proteins [36]. by targeting them for degradation within the lysosomes [59]. Successful restoration of CD4 surface expression was achieved 4.3.5. Leukocyte activation: M13L through over-expression of Hrs, a ubiquitin interaction motif-containing protein The MV-encoded M13L gene is a that sorts ubiquitinated proteins into endo- putative immunomodulator containing a somes. In addition, the purified PHD/LAP PYRIN domain (PYD) which is charac- zinc finger of MV-LAP was demonstrated teristic of the PYD superfamily of apop- to be a critical factor in the formation of tosis and inflammatory regulators [43]. multi-ubiquitin adducts in vitro [59]. Rab- M13L was shown to colocalize and in- bits infected with an MV-LAP mutant virus teract with the ASC-1 component of the suffered moderate to severe respiratory in- host inflammasome, modulating the activa- fection, however experienced a higher rate tion of caspase-1 activity and processing of recovery in contrast to rabbits infected of IL-1β and IL-18. In the absence of with wild-type MV [37]. Therefore MV- M13L, virus-infected rabbits did not de- LAP is a virulence factor which functions velop myxomatosis despite enhanced acute as a membrane-bound ubiquitin ligase, is inflammatory response to infection [43]. an immune evasion mechanism that targets Disruption of the M13L gene also severely host cell immune receptors. attenuated viral replication in cultured rabbit lymphocytes, yet replication in rabbit fibroblasts was comparable to that of the 5. PREVENTION, CONTROL wild-type MV. Reduced virulence of the AND VACCINATION M13L knockout virus can be attributed to the inability of the virus to infect lymphocytes, thus inhibiting virus spread and dis- When MV was intentionally introduced semination from primary sites of infection. into the feral rabbit population in Australia The role of M13L in manipulating the an- in 1950, the biological control method 312 M.M. Stanford et al. failed because of a combination of in- ulation also results in a mild cutaneous creased host resistance and a genetic at- reaction followed by immunity that lasted tenuation of field virus strains [9, 46]. A from 4 days post infection to over 10 head-to-head comparison of the Standard months [82]. Laboratory Strain (SLS) and the attenuated In addition to these strains, deletions of Urraria (Ur) strain in susceptible and re- host range genes that have been identified sistant rabbits indicated that MV replicates to date from MV [66] would make excel- in cells that mediate the immune response; lent candidates for vaccines. Studies that i.e. dendritic cells in the skin and T cells have selectively deleted such genes have in the lymph node [9]. Infection with SLS found no reduction in their in vitro repli- resulted in far more lymphocyte depletion cation levels, yet a severe defect in their in rabbits than the Ur strain, and the less ability to cause disease in rabbits (Tab. I). virulent Ur strain had a more robust inflam- This has been shown as a proof of princi- matory response consisting primarily of ple in studies where genes such as M-T5 mononuclear cells while the more virulent have been selectively deleted [73]. Rabbits SLS strain had an infiltrate of primarily injected with the M-T5 knock out virus not polymorphonuclear cells [9]. The use of only had very little clinical signs of myx- resistant rabbit populations also had a sig- omatosis, they were also protected from nificant effect on the immune response to challenge with wild type MV [73]. Such at- both viruses, suggesting that modeling this tenuated viruses lacking host range genes infection in fully naive laboratory rabbits would make excellent vaccine candidates, provides only a starting point to understand yet comprehensive studies into their effec- the dynamics of MV/rabbit co-evolution tiveness are needed since vaccines have yet since 1950. to be completed. In areas of the world where MV is To date, MV has been shown not to be endemic, control of virus spread among able to infect any other vertebrate family the rabbit population is critical to prevent besides lagomorphs in vivo; including day outbreaks. In many cases, proper screen- old mice [1], cats [63], and humans [40]. ing to exclude arthropod vectors such as However, reports have indicated that MV mosquitoes and fleas is sufficient. In ad- can infect non-lagomorph cells in vitro; in- dition, the quarantine of new rabbits, as cluding squirrels, guinea pigs and primate well as any sick rabbits, is necessary to ef- kidney cells [16, 97]. MV also replicated fectively contain new outbreaks. The use in guinea pig sarcoma cell lines in vitro, of the closely related Shope Fibroma virus as well as in vivo [2]. A recent report has as a vaccine strain has yielded variable indicated that this virus exhibits a natural results, yet is generally effective [29]. In tropism for many human tumor cells [93], addition, a live attenuated form of MV, indicating that this virus could be used as designated MSD strain, has been used as a therapy in cancer treatment. Studies in a a vaccine strain. Immunization of rabbits mouse model of glioma has shown that this results in a mild cutaneous reaction fol- virus is quite effective in infecting tumor lowed by immunity for approximately 9 tissue in vivo and effectively ‘cured’ mice months [81]. Serial passage of this virus from these brain tumors [56]. These studies in rabbit kidney cultures has further attenu- have fostered enthusiasm in the prospect of ated the virus without sacrificing its immu- using this virus clinically to treat human nity and has been designated MSD/B [41]. cancers [66]. In this case, special consid- In addition, the live attenuated vaccine des- erations will have to be met to ensure the ignated SG33 is a vaccine that is currently safety of both wild and cultivated rabbit used in France, Belgium and Italy. Inoc- populations. Currently, oncolytic MV is Myxomatosis in the European rabbit 313 being studied with consideration to these Research Unit of the London Regional Cancer issues, and the clinical oncolytic platform Program. G. McFadden holds a Canada Re- will likely be a recombinant MV that has search Chair in Molecular Virology and is an reduced virulence in rabbits but the on- International Scholar of The Howard Hughes colytic capacity of the wild type virus. Medical Institute.

6. CONCLUSIONS REFERENCES

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