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Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB–dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short–half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

Viral diseases, whether of animals or humans, are normally considered as problems to be managed. However, in Australia, two viruses have been used as landscape-scale therapeutics to control European rabbits (Oryctolagus cuniculus), the preeminent invasive vertebrate pest species. Rabbits have caused major environmental and agricultural losses and contributed to extinction of native species. It was not until the introduction of Myxoma virus that effective control of this pest was obtained at a continental scale. Subsequent coevolution of rabbit and virus saw a gradual reduction in the effectiveness of biological control that was partially ameliorated by the introduction of the European rabbit flea to act as an additional vector for the virus. In 1995, a completely different virus, Rabbit hemorrhagic disease virus (RHDV), escaped from testing and spread through the Australian rabbit population and again significantly reduced rabbit numbers and environmental impacts. The evolutionary pressures on this virus appear to be producing quite different outcomes to those that occurred with myxoma virus and the emergence and invasion of a novel genotype of RHDV in 2014 have further augmented control. Molecular studies on myxoma virus have demonstrated multiple proteins that manipulate the host innate and adaptive immune response; however the molecular basis of virus attenuation and reversion to virulence are not yet understood.

Background: Oncolytic therapy uses live-replicating viruses to improve the immunological status of treated tumors. Critically, while these viruses are known to self-amplify in vivo, clinical oncolytic therapies still appear to display a strong dose dependence and the mechanisms mediating this dose dependence are not well understood., Methods: To explore this apparent contradiction, we investigated how the initial dose of oncolytic myxoma virus affected the subsequent ability of treatment to alter the immunological status of tumors as well as synergize with programmed cell death protein 1 (PD1) blockade., Results: Our results indicate that, due to viral self-amplification in vivo, the overall load of myxoma virus rapidly normalizes within treated tumors despite up to 3-log differences in inoculating dose. Because of this, therapeutic efficacy in the absence of checkpoint blockade is largely dose independent. Despite this rapid normalization, however, treatment with high or low doses of myxoma virus induces distinct immunological changes within treated tumors. Critically, these changes appear to be durably programmed based on the initial oncolytic dose with low-dose treatment failing to induce immunological improvements despite rapidly achieving equivalent viral burdens. Finally, due to the distinct immunological profiles induced by high and low myxoma virus doses, oncolytic efficacy resulting from combination with PD1 blockade therapy displays a strong dose dependence., Conclusions: Taken together, these data suggest that the ability of oncolytic myxoma virus to immunologically reprogram treated tumors is dependent on initial viral dose. Additionally, this work could provide a possible mechanistic explanation for clinical results observed with other oncolytic viruses., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Cancer cells seem to exploit mechanisms that evolve as part of physiological tolerance, which is a complementary and often beneficial form of defense. The study of physiological systems of tolerance can therefore provide insights into the development of a state of host tolerance of cancer, and how to break it. Analysis of these models has the potential to improve our understanding of existing immunological therapeutic targets, and help to identify future targets and rational therapeutic combinations. The treatment of cancer with immune checkpoint inhibitors aims to reverse the progression to tolerance of cancer, and achieve an immunogenic, rather than tolerogenic, homeostasis. Broadening the efficacy and durability of checkpoint inhibitors focuses on reversing tolerance and stimulating immunogenicity in the cancer, host, and environment. Two examples of important physiological states of tolerance that may inform tolerance of cancer are microbial infection and placental reproduction. These states of tolerance result from bilateral shaping of host and non-self, akin to immunoediting in cancer, and offer reliable models to study the immune tolerance paradigm., (© 2018 WILEY Periodicals, Inc.)

Poxviruses are large, DNA viruses whose protein capsid is surrounded by one or more lipid envelopes. Embedded into these lipid envelopes are three conserved viral proteins which are thought to mediate binding of virions to target cells. While the function of these proteins has been studied in vitro , their specific roles during the pathogenesis of poxviral disease remain largely unclear. Here we present data demonstrating that the putative chondroitin binding protein M083 from the leporipoxvirus myxoma virus is a significant virulence factor during infection of susceptible Oryctolagus rabbits. Removal of M083 results in a reduced capacity of virus to spread beyond the regional lymph nodes and completely eliminates infection-mediated mortality. In vitro , removal of M083 results in only minor intracellular replication defects but causes a significant reduction in the ability of myxoma virus to spread from infected epithelial cells onto primary lymphocytes. We hypothesize that the physiological role of M083 is therefore to mediate the spread of myxoma virus onto rabbit lymphocytes, allowing these cells to disseminate virus throughout infected rabbits. IMPORTANCE Poxviruses represent both a class of human pathogens and potential therapeutic agents for the treatment of human malignancy. Understanding the basic biology of these agents is therefore significant to human health in a variety of ways. While the mechanisms mediating poxviral binding have been well studied in vitro , how these mechanisms impact poxviral pathogenesis in vivo remains unclear. The current study advances our understanding of how poxviral binding impacts viral pathogenesis by demonstrating that the putative chondroitin binding protein M083 plays a critical role during the pathogenesis of myxoma virus in susceptible Oryctolagus rabbits by impacting viral dissemination through changes in the transfer of virions onto primary splenocytes., (Copyright © 2018 American Society for Microbiology.)

The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined. IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution., (Copyright © 2017 American Society for Microbiology.)

In host-pathogen arms races, increases in host resistance prompt counteradaptation by pathogens, but the nature of that counteradaptation is seldom directly observed outside of laboratory models. The best-documented field example is the coevolution of myxoma virus (MYXV) in European rabbits. To understand how MYXV in Australia has continued to evolve in wild rabbits under intense selection for genetic resistance to myxomatosis, we compared the phenotypes of the progenitor MYXV and viral isolates from the 1950s and the 1990s in laboratory rabbits with no resistance. Strikingly, and unlike their 1950s counterparts, most virus isolates from the 1990s induced a highly lethal immune collapse syndrome similar to septic shock. Thus, the next step in this canonical case of coevolution after a species jump has been further escalation by the virus in the face of widespread host resistance., Competing Interests: The authors declare no conflict of interest.

The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954-1955) and between 2008-2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

Myxoma virus (MYXV) is Leporipoxvirus that possesses a specific rabbit-restricted host tropism but exhibits a much broader  cellular host range in cultured cells. MYXV is able to efficiently  block all aspects of the type I interferon (IFN)-induced  antiviral  state  in rabbit cells, partially in  human  cells  and  very  poorly  in  mouse  cells.  The mechanism(s) of this species-specific inhibition of  type I IFN-induced antiviral state is not well understood. Here we demonstrate that MYXV encoded  protein  M029, a truncated relative of the vaccinia virus (VACV) E3 double-stranded RNA (dsRNA)  binding  protein  that  inhibits  protein  kinase  R (PKR),  can  also  antagonize the type I IFN-induced  antiviral state in a highly species-specific manner. In cells pre-treated with type I IFN prior to  infection,  MYXV  exploits  M029  to  overcome  the  induced  antiviral  state completely in rabbit cells,  partially  in  human  cells,  but  not at all in mouse cells. However, in cells pre-infected with MYXV,  IFN-induced  signaling  is fully  inhibited  even  in the  absence  of M029 in cells from all three species,  suggesting  that  other  MYXV  protein(s)  apart  from  M029  block  IFN  signaling  in  a  speciesindependent  manner.  We  also  show  that  the  antiviral  state  induced in rabbit, human or mouse cells  by  type  I IFN  can  inhibit M029-knockout MYXV even when PKR is genetically knocked-out, suggesting  that  M029  targets  other  host  proteins  for  this  antiviral state inhibition. Thus, the MYXV  dsRNA  binding  protein  M029  not  only  antagonizes  PKR  from  multiple  species  but  also blocks the  type I IFN antiviral state independently of PKR in a highly species-specific fashion.

Myxoma virus (MYXV) is a rabbit-specific poxvirus, which is highly virulent in European rabbits. The attenuation of MYXV and the increased resistance of rabbits following the release of MYXV in Australia is one of the best-documented examples of host-pathogen coevolution. To elucidate the molecular mechanisms that contribute to the restriction of MYXV infection to rabbits and MYXV attenuation in the field, we have studied the interaction of the MYXV protein M156 with the host antiviral protein kinase R (PKR). In yeast and cell-culture transfection assays, M156 only inhibited rabbit PKR but not PKR from other tested mammalian species. Infection assays with human HeLa PKR knock-down cells, which were stably transfected with human or rabbit PKR, revealed that only human but not rabbit PKR was able to restrict MYXV infection, whereas both PKRs were able to restrict replication of a vaccinia virus (VACV) strain that lacks the PKR inhibitors E3 and K3. Inactivation of M156R led to MYXV virus attenuation in rabbit cells, which was rescued by the ectopic expression of VACV E3 and K3. We further show that a mutation in the M156 encoding gene that was identified in more than 50% of MYXV field isolates from Australia resulted in an M156 variant that lost its ability to inhibit rabbit PKR and led to virus attenuation. The species-specific inhibition of rabbit PKR by M156 and the M156 loss-of-function in Australian MYXV field isolates might thus contribute to the species specificity of MYXV and to the attenuation in the field, respectively.

Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an α2,3-sialyltransferase through its M138L gene. In this study, we showed that although the absence of the enzyme was not associated with any in vitro deficit, the M138L deficient strains are highly attenuated in vivo. Indeed, while all rabbits infected with the parental and the revertant strains died within 9 days post-infection from severe myxomatosis, all but one rabbit inoculated with the M138L deficient strains survived the infection. In primary lesions, this resistance to the infection was associated with an increased ability of innate immune cells, mostly neutrophils, to migrate to the site of virus replication at 4 days post-infection. This was followed by the development of a better specific immune response against MYXV. Indeed, at day 9 post-infection, we observed an important proliferation of lymphocytes and an intense congestion of blood vessels in lymph nodes after M138L knockouts infection. Accordingly, in these rabbits, we observed an intense mononuclear cell infiltration throughout the dermis in primary lesions and higher titers of neutralizing antibodies. Finally, this adaptive immune response provided protection to these surviving rabbits against a challenge with the MYXV WT strain. Altogether, these results show that expression of the M138L gene contributes directly or indirectly to immune evasion by MYXV. In the future, these results could help us to better understand the pathogenesis of myxomatosis but also the importance of glycans in regulation of immune responses.

Although viral emergence is commonly associated with cross-species transmission, the processes and determinants of viral evolution in a novel host environment are poorly understood. We address key questions in virus emergence and evolution using data generated from two unique natural experiments: the deliberate release of myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) as biological control (biocontrol) agents against the European rabbit in Australia, and which have been of enormous benefit to Australia's ecosystem and agricultural industries. Notably, although virulence evolution in MYXV and RHDV followed different trajectories, a strongly parallel evolutionary process was observed in Australia and Europe. These biocontrol agents were also characterized by a lack of transmission to nontarget host species, suggesting that there are major barriers to successful emergence., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Gallbladder carcinoma (GBC) has a high mortality rate, requiring synergistic anti-tumor management for effective treatment. The myxoma virus (MYXV) exhibits a modest clinical value through its oncolytic potential and narrow host tropism., Methods: We performed viral replication assays, cell viability assays, migration assays, and xenograft tumor models to demonstrate that bone marrow-derived stem cells (BMSCs) may enhance efficiency of intravenous MYXV delivery., Results: We examined the permissiveness of various GBC cell lines towards MYXV infection and found two supported single and multiple rounds of MYXV replication, leading to an oncolytic effect. Furthermore, we found that BMSCs exhibited tropism for GBC cells within a Matrigel migration system. BMSCs failed to affect the growth of GBC cells, in terms of tumor volume and survival time. Finally, we demonstrated in vivo that intravenous injection of MYXV-infected BMSCs significantly improves the oncolytic effect of MYXV alone, almost to the same extent as intratumoral injection of MYXV., Conclusion: This study indicates that BMSCs are a promising novel vehicle for MYXV to clinically address gallbladder tumors.

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.

Many common neoplasms are still noncurative with current standards of cancer therapy. More therapeutic modalities need to be developed to significantly prolong the lives of patients and eventually cure a wider spectrum of cancers. Oncolytic virotherapy is one of the promising new additions to clinical cancer therapeutics. Successful oncolytic virotherapy in the clinic will be those strategies that best combine tumor cell oncolysis with enhanced immune responses against tumor antigens. The current candidate oncolytic viruses all share the common property that they are relatively nonpathogenic to humans, yet they have the ability to replicate selectively in human cancer cells and induce cancer regression by direct oncolysis and/or induction of improved anti-tumor immune responses. Many candidate oncolytic viruses are in various stages of clinical and preclinical development. One such preclinical candidate is myxoma virus (MYXV), a member of the Poxviridae family that, in its natural setting, exhibits a very restricted host range and is only pathogenic to European rabbits. Despite its narrow host range in nature, MYXV has been shown to productively infect various classes of human cancer cells. Several preclinical in vivo modeling studies have demonstrated that MYXV is an attractive and safe candidate oncolytic virus, and hence, MYXV is currently being developed as a potential therapeutic for several cancers, such as pancreatic cancer, glioblastoma, ovarian cancer, melanoma, and hematologic malignancies. This review highlights the preclinical cancer models that have shown the most promise for translation of MYXV into human clinical trials., (Published by Elsevier Ltd.)

The myxoma virus (MYXV) carries three tandem C7L-like host range genes (M062R, M063R, and M064R). However, despite the fact that the sequences of these three genes are similar, they possess very distinctive functions in vivo. The role of M064 in MYXV pathogenesis was investigated and compared to the roles of M062 and M063. We report that M064 is a virulence factor that contributes to MYXV pathogenesis but lacks the host range properties associated with M062 and M063.

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Myxoma virus (MYXV) gained importance throughout the twentieth century because of the use of the highly virulent Standard Laboratory Strain (SLS) by the Australian government in the attempt to control the feral Australian population of Oryctolagus cuniculus (European rabbit) and the subsequent illegal release of MYXV in Europe. In the European rabbit, MYXV causes a disease with an exceedingly high mortality rate, named myxomatosis, which is passively transmitted by biting arthropod vectors. MYXV still has a great impact on European rabbit populations around the world. In contrast, only a single cutaneous lesion, restricted to the point of inoculation, is seen in its natural long-term host, the South-American Sylvilagus brasiliensis and the North-American S. Bachmani. Apart from being detrimental for European rabbits, however, MYXV has also become of interest in human medicine in the last two decades for two reasons. Firstly, due to the strong immune suppressing effects of certain MYXV proteins, several secreted virus-encoded immunomodulators (e.g. Serp-1) are being developed to treat systemic inflammatory syndromes such as cardiovascular disease in humans. Secondly, due to the inherent ability of MYXV to infect a broad spectrum of human cancer cells, the live virus is also being developed as an oncolytic virotherapeutic to treat human cancer. In this review, an update will be given on the current status of MYXV in rabbits as well as its potential in human medicine in the twenty-first century.

Invitrogen's Gateway technology is a recombination-based cloning method that allows for rapid transfer of numerous open reading frames (ORFs) into multiple plasmid vectors, making it useful for diverse high-throughput applications. Gateway technology has been utilized to create an ORF library for Myxoma virus (MYXV), a member of the Poxviridae family of DNA viruses. MYXV is the prototype virus for the genus Leporipoxvirus, and is pathogenic only in European rabbits. MYXV replicates exclusively in the host cell cytoplasm, and its genome encodes 171 ORFs. A number of these ORFs encode proteins that interfere with or modulate host defense mechanisms, particularly the inflammatory responses. Furthermore, MYXV is able to productively infect a variety of human cancer cell lines and is being developed as an oncolytic virus for treating human cancers. MYXV is therefore an excellent model for studying poxvirus biology, pathogenesis, and host tropism, and a good candidate for ORFeome development.

Myxoma virus (MYXV) M062R is a functional homolog of the C7L family of host range genes from orthopoxviruses. We constructed a targeted M062R-knockout-MYXV (vMyxM062-KO) and characterized its properties in vitro and in vivo. In European rabbits, infection by vMyxM062-KO was completely asymptomatic. The surviving rabbits did not gain full protection against the subsequent lethal-dose challenge with wild-type MYXV. We also looked for cellular tropism defects in a variety of cultured cells. In all of the rabbit cells tested, vMyxM062-KO conducts an abortive infection, although it initiates viral DNA replication. In many, but not all, human cancer cells that are permissive for wild-type MYXV, vMyxM062-KO exhibited a profound replication defect. We categorized human cells tested into two groups: (i) type A, which support productive replication for wild-type MYXV but are unable to produce significant levels of progeny virus by vMyxM062-KO, and (ii) type B, which are permissive to infections by both wild-type MYXV and vMyxM062-KO. Furthermore, using proteomic strategies, we identified sterile α motif domain containing 9 (SAMD9), an interferon-regulated cellular protein implicated in human inflammatory disorders, as a unique host binding partner of M062 in human cells. Significantly, knocking down SAMD9 in type A human cancer cells led to a substantial rescue of vMyxM062-KO infection. In summary, M062 is a novel host range factor that controls productive MYXV replication in rabbit cells and in a wide variety of human cells. M062 also binds and antagonizes cellular SAMD9 in human cells, suggesting that SAMD9 is a novel innate antiviral factor against poxviruses.

Oncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.

Myxoma virus (MYXV) is a member of the Poxviridae family and prototype for the genus Leporipoxvirus. It is pathogenic only for European rabbits, in which it causes the lethal disease myxomatosis, and two North American species, in which it causes a less severe disease. MYXV replicates exclusively in the cytoplasm of the host cell. Although not infectious in humans, its genome encodes proteins that can interfere with or modulate host defense mechanisms; it is able to productively infect a number of human cancer cell lines, but not normal human cells, and has also been shown to increase survival time in mouse models of human glioma. These characteristics suggest that MYXV could be a viable therapeutic agent, e.g., in anti-inflammatory or anti-immune therapy, or as an oncolytic agent. MYXV is also an excellent model for poxvirus biology, pathogenesis, and host tropism studies. It is easily propagated in a number of cell lines, including adherent cells and suspension cultures, and minimal purification is required to provide a stock for in vivo and in vitro studies.

The European rabbit was brought to Australia as a companion animal by early settlers. It sometimes escaped, but failed to survive in the Australian bush. In 1879 wild rabbits were deliberately sent to Victoria to provide game for wealthy settlers to shoot. They soon spread all over Australia, except in the tropics, and became Australia's major animal pest. After careful testing in Australian wildlife and in humans, control by myxoma virus was introduced at various sites between 1937 and 1950, spreading all over the Murray-Darling Basin in 1950. Within one year mutations in the virus had led to slightly less virulence, and these continued for the next 50 years. In the early 21st Century testing viruses obtained from wild rabbits showed that the majority of these viruses were more virulent than the virus used to initiate the epidemic. In 1995 another virus specific for European rabbits, rabbit haemorrhagic disease virus, escaped from areas in which field trials were being carried out and spread around Australia. It was more successful than myxomatosis for rabbit control in arid regions.

The pathogenesis of South American and North American myxoma viruses was examined in two species of North American lagomorphs, Sylvilagus nuttallii (mountain cottontail) and Sylvilagus audubonii (desert cottontail) both of which have been shown to have the potential to transmit the South American type of myxoma virus. Following infection with the South American strain (Lausanne, Lu), S. nuttallii developed both a local lesion and secondary lesions on the skin. They did not develop the classical myxomatosis seen in European rabbits (Oryctolagus cuniculus). The infection at the inoculation site did not resolve during the 20-day time course of the trial and contained transmissible virus titres at all times. In contrast, S. audubonii infected with Lu had very few signs of disseminated infection and partially controlled virus replication at the inoculation site. The prototype Californian strain of myxoma virus (MSW) was able to replicate at the inoculation site of both species but did not induce clinical signs of a disseminated infection. In S. audubonii, there was a rapid response to MSW characterised by a massive T lymphocyte infiltration of the inoculation site by day 5. MSW did not reach transmissible titres at the inoculation site in either species. This might explain why the Californian myxoma virus has not expanded its host-range in North America., (Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.)

Vaccination is the best way to control myxomatosis in both pet and production rabbits. Two types of myxomatosis vaccines are commercially available, namely, a vaccine prepared from the Shope fibroma virus (SFV) and one prepared from an attenuated myxoma virus (MV) strain, e.g., SG33. The first one is weakly immunogenic and provides only short-term protection whereas atypical reactions have been described with the second one. This short review describes the vaccine strains and provides some data on the host-virus relationship, resistance, and immunity in myxomatosis. In the last section, recommended myxomatosis vaccination schemes for production and pet animals are presented.

Background: Myxoma virus (MV) has been endemic in Europe since shortly after its deliberate release in France in 1952. While the emergence of more resistant hosts and more transmissible and attenuated virus is well documented, there have been relatively few studies focused on the sequence changes incurred by the virus as it has adapted to its new host. In order to identify regions of variability within the MV genome to be used for phylogenetic studies and to try to investigate causes of MV strain attenuation we have molecularly characterised nine strains of MV isolated in Spain between the years 1992 and 1995 from wide ranging geographic locations and which had been previously graded for virulence by experimental infection of rabbits., Results: The findings reported here show the analysis of 16 genomic regions accounting for approximately 10% of the viral genomes. Of the 20 genes analysed 5 (M034L, M069L, M071L, M130R and M135R) were identical in all strains and 1 (M122R) contained only a single point mutation in an individual strain. Four genes (M002L/R, M009L, M036L and M017L) showed insertions or deletions that led to disruption of the ORFs., Conclusions: The findings presented here provide valuable tools for strain differentiation and phylogenetic studies of MV isolates and some clues as to the reasons for virus attenuation in the field.

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain ("MAV", Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863 bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain "Lausanne" and its attenuated field derivative strain "6918". The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to "Lausanne" and "6918". Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.

Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.

NF-kappaB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1beta and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-kappaB. The induction of these NF-kappaB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IkappaBalpha, which was followed by nuclear translocation of NF-kappaB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-kappaB-mediated reporter gene expression and nuclear translocation of NF-kappaB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-kappaB1, suggesting a direct physical and functional linkage between NF-kappaB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-kappaB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1beta. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-kappaB-mediated pro-inflammatory responses.

The M141 protein of myxoma virus (MYXV) is a viral CD200 homolog (also called vOX-2) that inhibits macrophage activation in infected rabbits. Here, we show that murine myeloid RAW 264.7 cells became activated when infected with MYXV in which the M141 gene was deleted (vMyx-M141KO) but not with the parental wild-type MYXV. Moreover, transcript and protein levels of tumor necrosis factor and granulocyte colony-stimulating factor were rapidly upregulated in an NF-kappaB-dependent fashion in the RAW 264.7 cells infected with vMyx-M141KO. M141 protein is present in the virion and counteracts this NF-kappaB activation pathway upon infection with the wild-type MYXV. Our data suggest that upregulation of these classic macrophage-related proinflammatory cytokine markers following infection of myeloid cells with the M141-knockout MYXV is mediated via the rapid activation of the cellular NF-kappaB pathway.

Myxoma virus (MV) is a highly lethal, rabbit-specific poxvirus that induces a disease called myxomatosis in European rabbits. In an effort to understand the function of predicted immunomodulatory genes we have deleted various viral genes from MV and tested the ability of these knockout viruses to induce lethal myxomatosis. MV encodes a unique 15 kD cytoplasmic protein (M130R) that is expressed late (12h post infection) during infection. M130R is a non-essential gene for MV replication in rabbit, monkey or human cell lines. Construction of a targeted gene knockout virus (vMyx130KO) and infection of susceptible rabbits demonstrate that the M130R knockout virus is attenuated and that loss of M130R expression allows the rabbit host immune system to effectively respond to and control the lethal effects of MV. M130R expression is a bona fide poxviral virulence factor necessary for full and lethal development of myxomatosis.

Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy.

Serp-1 is a secreted myxoma viral serine protease inhibitor (serpin) with proven, highly effective, anti-inflammatory defensive activity during host cell infection, as well as potent immunomodulatory activity in a wide range of animal disease models. Serp-1 binds urokinase-type plasminogen activator (uPA) and the tissue-type PA, plasmin, and factor Xa, requiring uPA receptor (uPAR) for anti-inflammatory activity. To define Serp-1-mediated effects on inflammatory cell activation, we examined the association of Serp-1 with monocytes and T cells, effects on cellular migration, and the role of uPAR-linked integrins and actin-binding proteins in Serp-1 cellular responses. Our results show that Serp-1 associates directly with activated monocytes and T lymphocytes, in part through interaction with uPAR (P<0.001). Serp-1, but not mammalian serpin PA inhibitor-1 (PAI-1), attenuated cellular adhesion to the extracellular matrix. Serp-1 and PAI-1 reduced human monocyte and T cell adhesion (P<0.001) and migration across endothelial monolayers in vitro (P<0.001) and into mouse ascites in vivo (P<0.001). Serp-1 and an inactive Serp-1 mutant Serp-1(SAA) bound equally to human monocytes and T cells, but a highly proinflammatory mutant, Serp-1(Ala(6)), bound less well to monocytes. Serp-1 treatment of monocytes increased expression of filamin B actin-binding protein and reduced CD18 (beta-integrin) expression (P<0.001) in a uPAR-dependent response. Filamin colocalized and co-immunoprecipitated with uPAR, and short interference RNA knock-down of filamin blocked Serp-1 inhibition of monocyte adhesion. We report here that the highly potent, anti-inflammatory activity of Serp-1 is mediated through modification of uPAR-linked beta-integrin and filamin in monocytes, identifying this interaction as a central regulatory axis for inflammation.

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). MYXV has a linear double-stranded DNA genome that encodes several factors important for evasion from the host immune system. Among them, four ankyrin (ANK) repeat proteins were identified: M148R, M149R, M150R and M-T5. To date, only M150R and M-T5 were studied and characterized as critical virulence factors. This article presents the first characterization of M148R and M149R. Green Fluorescent Protein (GFP) fusions allowed us to localize them in a viral context. Whereas M149R is only cytoplasmic, interestingly, M148R is in part located in the nucleolus, a unique feature for an ANK repeat poxviral protein. In order to evaluate their implication in viral pathogenicity, targeted M148R, M149R, or both deletions were constructed in the wild type T1 strain of myxoma virus. In vitro infection of rabbit and primate cultured cells as well as primary rabbit cells allowed us to conclude that M148R and M149R are not likely to be implicated in cell tropism or host range functions. However, in vivo experiments revealed that they are virulence factors since after infection of European rabbits with mutant viruses, a delay in the onset of clinical signs, an increase of survival time and a dramatic decrease in mortality rate were observed. Moreover, histological analysis suggests that M148R plays a role in the subversion of host inflammatory response by MYXV.

Myxoma virus and the European rabbit are one of the best-studied examples of coevolution of pathogen virulence and host resistance. Since the introduction of the virus in Spain in 1953, a decrease in its virulence has been observed; however, most strains are still considered highly virulent. To determine whether this attenuation is due to molecular differences, and to characterise the field strains in Spain and the genetic changes that have occurred since the introduction of the virus, we analysed 7,741 bp in 97 virus samples from 12 localities. We found an extremely low genetic variability and an absence of a geographic structure. We defined 35 haplotypes, none of which were identical to the original Lausanne strain. Three genetic groups were determined and were found to occur at different frequencies in different locations. Overall, virus evolution deviated from neutrality and did not conform to a strict molecular clock, probably due to the existence of a strong selective pressure that acts differently across the viral genome.

A case of a myxoma virus strain in vaccinated and non-vaccinated rabbits is described, and genetic identification of that strain was performed in this study. In two commercial farms being 150km apart, myxomatosis has been occurred after the import of animals from a common supplier. The disease was manifested firstly in the existing non-immune population of does and fatteners, and later in all vaccinated animals, being 2-3 months immune at the time of first symptoms. Morbidity was almost 100% with nasal discharge, listlessness, fever, eyelid swelling, eye and nasal purulent discharge, papules in the ears, facial oedema, and swelling of the anagenital region, with result always the death of the animals. Examination by PCR had shown the presence of a 492-bp specific product in all the symptomatic animals tested from both farms, having 100% nucleotide sequence identity with the homologous region of the myxoma virus Lausanne strain. The simultaneous occurrence of myxomatosis in the vaccinated and non-vaccinated rabbits of both farms, suggests that the supplier was possibly the source of a viral isolate with increased virulence.

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

The authors have recently demonstrated that wild-type myxoma virus (MV) tagged with gfp (vMyxgfp) can generate a tumor-specific infection that productively infects and clears human tumor-derived xenografts when injected intratumorally into human gliomas transplanted into immunodeficient mice (Lun et al, 2005). To expand the understanding of MV tropism in cancer cells from a specific tissue lineage, the authors have screened a series of human glioma cells (U87, U118, U251, U343, U373) for myxoma virus replication and oncolysis. To assess the viral tropism determinants for these infections, the authors have screened myxoma virus knockout constructs that have been deleted for specific host range genes (M-T2, M-T4, M-T5, M11L, and M063), as well as a control MV gene knockout construct with no known host range function (vMyx135KO) but is highly attenuated in rabbits. The authors report wide variation in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells as measured by early and late viral gene expression. This differential ability to support vMyx-hrKO productive viral replication is consistent with levels of endogenous activated Akt in the various gliomas. The authors have identified one vMyx-hrKO virus (vMyx63KO) and one nonhost range knockout construct (vMyx135KO) that appear to replicate in the gliomas even more efficiently than the wild-type virus and that reduce the viability of the infected gliomas. These knockout viruses also inhibit the proliferation of gliomas in a manner similar to the wild-type virus. Together these data, as well as the fact that these knockout viruses are attenuated in their natural hosts, may represent environmentally safer candidate oncolytic viruses for usage in human trials.

The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells.

The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA 103:4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.

Myxoma virus (MV) encodes a cell surface protein (M135R) that is predicted to mimic the host alpha/beta interferon receptor (IFN-alpha/beta-R) and thus prevent IFN-alpha/beta from triggering a host antiviral response. This prediction is based on sequence similarity to B18R, the viral IFN-alpha/beta-R from vaccinia virus (VV), which has been demonstrated to bind and inhibit type I interferons. However, M135R is only half the size of VV B18R. All other poxvirus-encoded IFN-alpha/beta-R homologs align only to the amino-terminal half of M135R. Peptide antibodies raised against M135R were used for immunoblotting and immunofluorescence and indicate that M135R is expressed as an early gene and that the product is a cell surface N-linked glycoprotein that is not secreted. In contrast to the predicted properties of M135R as an inhibitor of type I interferon, all binding and inhibition assays designed to demonstrate whether M135R can interact with IFN-alpha/beta have been negative. However, pathogenesis studies with a targeted M135-knockout MV construct (vMyx135KO) indicate that the deletion of M135R severely attenuates MV pathogenesis in the European rabbit. We propose that M135R is an important immunomodulatory virulence factor for myxomatosis but that the target immune ligand is not from the predicted type I interferon family and remains to be identified.

Myxoma virus (MYX) prevents apoptosis in RK-13 cells and forms thick dermal lesions with 100% mortality in rabbits. MYX encodes the virulence factor SERP2, a serine proteinase inhibitor (serpin). SERP2 was mutated to evaluate SERP2 function during MYX infection. MYXDeltaSERP2::lacZ (deleted for SERP2) did not inhibit apoptosis in RK-13 cells; infected rabbits had thin dermal lesions and <10% mortality. MYX-SERP2-D294A, a P1-site aspartate to alanine mutant, inactivated the serpin; infection was indistinguishable from MYXDeltaSERP2::lacZ. SERP2-D294E prevented inhibition of caspase-8, caspase-10 and granzyme-B; and MYX-SERP2-D294E failed to block apoptosis in RK-13 cells, but was fully virulent in rabbits. MYXDeltaSERP2::crmA expressed crmA instead of SERP2 and inhibited apoptosis in cell culture, but caused thin lesions and only 70% mortality in rabbits, hence crmA cannot fully substitute for SERP2. Control of apoptosis in culture does not correlate with virulence in rabbits. Virulence may instead depend on inhibition of proinflammatory proteinases by SERP2.

The pathogenesis of two Californian strains of myxoma virus (MSW and MSD) was examined in European rabbits (Oryctolagus cuniculus) that were either susceptible to myxomatosis (laboratory rabbits) or had undergone natural selection for genetic resistance to myxomatosis (Australian wild rabbits). MSW was highly lethal for both types of rabbits with average survival times of 7.3 and 9.4 days, respectively, and 100% mortality. Classical clinical signs of myxomatosis were not present except in one rabbit that survived for 13 days following infection. Previously described clinical signs of trembling and shaking were observed in laboratory but not wild rabbits. Despite the high resistance of wild rabbits to myxomatosis caused by South American strains of myxoma virus, the MSW strain was of such high virulence that it was able to overcome resistance. The acute nature of the infection, relatively low viral titers in the tissues and destruction of lymphoid tissues, suggested that death was probably due to an acute and overwhelming immunopathological response to the virus. No virus was found in the brain. The MSD strain was attenuated compared to previously published descriptions and therefore was only characterized in laboratory rabbits. It is concluded that Californian MSW strain of myxoma virus is at the extreme end of a continuum of myxoma virus virulence but that the basic pathophysiology of the disease induced is not broadly different to other strains of myxoma virus.

Many viruses inhibit or retard apoptosis, a strategy that subverts one of the most ancient antiviral mechanisms. M11L, a myxoma virus-encoded antiapoptotic protein, has been previously shown to localize to mitochondria and block apoptosis of virus-infected cells (H. Everett, M. Barry, S. F. Lee, X. J. Sun, K. Graham, J. Stone, R. C. Bleackley, and G. McFadden, J. Exp. Med. 191:1487-1498, 2000; H. Everett, M. Barry, X. Sun, S. F. Lee, C. Frantz, L. G. Berthiaume, G. McFadden, and R. C. Bleackley, J. Exp. Med. 196:1127-1139, 2002; and G. Wang, J. W. Barrett, S. H. Nazarian, H. Everett, X. Gao, C. Bleackley, K. Colwill, M. F. Moran, and G. McFadden, J. Virol. 78:7097-7111, 2004). This protection from apoptosis involves constitutive-forming inhibitory complexes with the peripheral benzodiazepine receptor and Bak on the outer mitochondrial membrane. Here, we extend the study to investigate the interference of M11L with Bax activation during the process of apoptosis. Myxoma virus infection triggers an early apoptotic signal that induces rapid Bax translocation from cytoplasm to mitochondria, despite the existence of various viral antiapoptotic proteins. However, in the presence of M11L, the structural activation of Bax at the mitochondrial membrane, which is characterized by the occurrence of a Bax conformational change, is blocked in both M11L-expressing myxoma-infected cells and M11L-transfected cells under apoptotic stimulation. In addition, inducible binding of M11L to the mitochondrially localized Bax is detected in myxoma virus-infected cells and in M11L/Bax-cotransfected cells as measured by immunoprecipitation and tandem affinity purification analysis, respectively. Importantly, this inducible Bax/M11L interaction is independent of Bak, demonstrated by the complete block of Bax-mediated apoptosis in myxoma-infected cells that lack Bak expression. Our findings reveal that myxoma M11L modulates apoptosis by multiple independent strategies which all contribute to the blockade of apoptosis at the mitochondrial checkpoint.

M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25(+) infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R(+) dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.