Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway.

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-κB­dependent 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 short­half-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.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated.

Viruses for Landscape-Scale Therapy: Biological Control of Rabbits in Australia.

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.

Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus).

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains' ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.

Detection of viral DNA of myxoma virus using a validated PCR method with an internal amplification control.

Recognition of myxomatosis is usually based on clinical symptoms, but amyxomatous cases of the disease require the use of laboratory methods. Nowadays PCR assays are routinely employed for detection of MYXV DNA, but none of them have had their diagnostic usefulness conclusively confirmed through validation. The aim of the study was the development and validation of a PCR with an internal amplification control (IAC) for intravital and postmortem detection of viral DNA of myxoma virus. To avoid false negative results a chimeric internal amplification control (IAC) was prepared and incorporated into the PCR and amplified by the same primer set as the target DNA (M071L). The optimal concentration of particular ingredients in the PCR mixture (including IAC concentration and volume of DNA sample) was determined. To minimize the risk of amplicon carry-over contamination, uracil N-glycosylase was added to the reaction. Before proper validation the robustness of the IAC-PCR was verified. Validation of the method encompassed the following parameters: the analytical and diagnostic specificity (ASp, DSp) and sensitivity (ASe, DSe) of the assay, repeatability, and intra-laboratory reproducibility. The assay LOD was established at 2 TCIU of the virus particles/0.2 ml tissue homogenate with a 100% capacity to detect different MYXV strains (ASp). The method was characterized by good DSp of 0.955 (0.839-0.999 CI) and DSe of 0.976 (0.914-1.00 CI). In addition, it was repeatable and reproducible and confirmed its suitability for the detection of MYXV in clinical material. The IAC-PCR developed meets OIE validation requirements for virological methods and can be used in diagnostic or epidemiological studies of rabbit myxomatosis.

Effects of myxoma virus and rabbit hemorrhagic disease virus on the physiological condition of wild European rabbits: Is blood biochemistry a useful monitoring tool?

Myxomatosis and rabbit hemorrhagic disease (RHD) are the major viral diseases that affect the wild European rabbit (Oryctolagus cuniculus). These diseases arrived in Europe within the last decades and have caused wild rabbit populations to decline dramatically. Both viruses are currently considered to be endemic in the Iberian Peninsula; periodic outbreaks that strongly impact wild populations regularly occur. Myxoma virus (MV) and rabbit hemorrhagic disease virus (RHDV) alter the physiology of infected rabbits, resulting in physical deterioration. Consequently, the persistence and viability of natural populations are affected. The main goal of our study was to determine if blood biochemistry is correlated with serostatus in wild European rabbits. We carried out seven live-trapping sessions in three wild rabbit populations over a two-year period. Blood samples were collected to measure anti-MV and anti-RHDV antibody concentrations and to measure biochemical parameters related to organ function, protein metabolism, and nutritional status. Overall, we found no significant relationships between rabbit serostatus and biochemistry. Our main result was that rabbits that were seropositive for both MV and RHDV had low gamma glutamyltransferase concentrations. Given the robustness of our analyses, the lack of significant relationships may indicate that the biochemical parameters measured are poor proxies for serostatus. Another explanation is that wild rabbits might be producing attenuated physiological responses to these viruses because the latter are now enzootic in the study area.

A case of low success of blind vaccination campaigns against myxomatosis and rabbit haemorrhagic disease on survival of adult European wild rabbits.

Vaccination campaigns against myxomatosis and rabbit haemorrhagic disease (RHD) are commonly used in translocation programs conducted for the purpose of recovering wild European rabbit populations in Iberian Mediterranean ecosystems. In most cases rabbits are vaccinated 'blind' (i.e. without assessing their prior immunological status) for economic and logistic reasons. However, there is conflicting evidence on the effectiveness of such an approach. We tested whether blind vaccination against myxomatosis and rabbit haemorrhagic disease improved rabbit survival in a rabbit translocation program where wild rabbits were kept in semi-natural conditions in three enclosures. We conducted nine capture sessions over two years (2008-2010) and used the information collected to compare the survival of vaccinated (n=511) versus unvaccinated (n=161) adult wild rabbits using capture-mark-recapture analysis. Average monthly survival was no different for vaccinated versus unvaccinated individuals, both in the period between release and first capture (short-term) and after the first capture onward (long-term). Rabbit survival was lower in the short term than in the long term regardless of whether rabbits were vaccinated or not. Lower survival in the short-term could be due to the stress induced by the translocation process itself (e.g. handling stress). However, we did not find any overall effect of vaccination on survival which could be explained by two non-exclusive reasons. First, interference of the vaccine with the natural antibodies in the donor population. Due to donor populations have high density of rabbits with, likely, high prevalence of antibodies as a result of previous natural exposure to these diseases. Second, the lack of severe outbreaks during the study period. Based on our findings we argue that blind vaccination of adult rabbits in translocation programs may be often mostly ineffective and unnecessarily costly. In particular, since outbreaks are hard to predict and vaccination of rabbits with natural antibodies is ineffective, it is crucial to assess the immunological status of the donor population before translocating adult rabbits.

Molecular species identification, host preference and detection of myxoma virus in the Anopheles maculipennis complex (Diptera: Culicidae) in southern England, UK.

BACKGROUND: Determining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An. maculipennis s.l.), identify the vertebrate hosts of field-caught blood-fed An. maculipennis s.l. , and to test for the presence of myxoma virus (Poxviridae, genus Leporipoxvirus) in specimens found to have fed on the European rabbit (Oryctolagus cuniculus). METHODS: Blood-fed An. maculipennis s.l. were collected from resting sites at Elmley Nature Reserve, Kent, between June and September 2013. Hosts that An. maculipennis s.l. had fed on were determined by a PCR-sequencing approach based on the partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes were then identified to species by sequencing a region of the internal transcribed spacer-2. DNA extracts from all mosquitoes identified as having fed on rabbits were subsequently screened using PCR for the presence of myxoma virus. RESULTS: A total of 94 blood-fed Anopheles maculipennis s.l. were collected, of which 43 (46%) provided positive blood-meal identification results. Thirty-six of these specimens were identified as Anopheles atroparvus, which had fed on rabbit (n = 33, 92%) and cattle (n = 3, 8%). Seven mosquitoes were identified as Anopheles messeae, which had fed on cattle (n = 6, 86%) and dog (n = 1, 14%). Of the 33 An. atroparvus that contained rabbit blood, nine (27%) were positive for myxoma virus. CONCLUSIONS: Results demonstrate that a single DNA extract from a blood-fed mosquito can be successfully used for molecular identification of members of the An. maculipennis complex, blood-meal identification, and for the targeted detection of a myxoma virus. This study shows that An. atroparvus has a strong feeding preference for both healthy and myxoma-infected rabbits, providing evidence that this species may play a significant role in the transmission of myxomatosis among wild rabbit populations in the United Kingdom (UK).

Myxoma virus and the Leporipoxviruses: an evolutionary paradigm.

Myxoma virus (MYXV) is the type species of the Leporipoxviruses, a genus of Chordopoxvirinae, double stranded DNA viruses, whose members infect leporids and squirrels, inducing cutaneous fibromas from which virus is mechanically transmitted by biting arthropods. However, in the European rabbit (Oryctolagus cuniculus), MYXV causes the lethal disease myxomatosis. The release of MYXV as a biological control for the wild European rabbit population in Australia, initiated one of the great experiments in evolution. The subsequent coevolution of MYXV and rabbits is a classic example of natural selection acting on virulence as a pathogen adapts to a novel host species. Slightly attenuated mutants of the progenitor virus were more readily transmitted by the mosquito vector because the infected rabbit survived longer, while highly attenuated viruses could be controlled by the rabbit immune response. As a consequence, moderately attenuated viruses came to dominate. This evolution of the virus was accompanied by selection for genetic resistance in the wild rabbit population, which may have created an ongoing co-evolutionary dynamic between resistance and virulence for efficient transmission. This natural experiment was repeated on a continental scale with the release of a separate strain of MYXV in France and its subsequent spread throughout Europe. The selection of attenuated strains of virus and resistant rabbits mirrored the experience in Australia in a very different environment, albeit with somewhat different rates. Genome sequencing of the progenitor virus and the early radiation, as well as those from the 1990s in Australia and Europe, has shown that although MYXV evolved at high rates there was no conserved route to attenuation or back to virulence. In contrast, it seems that these relatively large viral genomes have the flexibility for multiple pathways that converge on a similar phenotype.

The α2,3-sialyltransferase encoded by myxoma virus is a virulence factor that contributes to immunosuppression.

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.

Viral biocontrol: grand experiments in disease emergence and evolution.

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.

Multi-event capture-recapture modeling of host-pathogen dynamics among European rabbit populations exposed to myxoma and Rabbit Hemorrhagic Disease Viruses: common and heterogeneous patterns.

Host-pathogen epidemiological processes are often unclear due both to their complexity and over-simplistic approaches used to quantify them. We applied a multi-event capture-recapture procedure on two years of data from three rabbit populations to test hypotheses about the effects on survival of, and the dynamics of host immunity to, both myxoma virus and Rabbit Hemorrhagic Disease Virus (MV and RHDV). Although the populations shared the same climatic and management conditions, MV and RHDV dynamics varied greatly among them; MV and RHDV seroprevalences were positively related to density in one population, but RHDV seroprevalence was negatively related to density in another. In addition, (i) juvenile survival was most often negatively related to seropositivity, (ii) RHDV seropositives never had considerably higher survival, and (iii) seroconversion to seropositivity was more likely than the reverse. We suggest seropositivity affects survival depending on trade-offs among antibody protection, immunosuppression and virus lethality. Negative effects of seropositivity might be greater on juveniles due to their immature immune system. Also, while RHDV directly affects survival through the hemorrhagic syndrome, MV lack of direct lethal effects means that interactions influencing survival are likely to be more complex. Multi-event modeling allowed us to quantify patterns of host-pathogen dynamics otherwise difficult to discern. Such an approach offers a promising tool to shed light on causative mechanisms.

Early infections by myxoma virus of young rabbits (Oryctolagus cuniculus) protected by maternal antibodies activate their immune system and enhance herd immunity in wild populations.

The role of maternal antibodies is to protect newborns against acute early infection by pathogens. This can be achieved either by preventing any infection or by allowing attenuated infections associated with activation of the immune system, the two strategies being based on different cost/benefit ratios. We carried out an epidemiological survey of myxomatosis, which is a highly lethal infectious disease, in two distant wild populations of rabbits to describe the epidemiological pattern of the disease. Detection of specific IgM and IgG enabled us to describe the pattern of immunity. We show that maternal immunity attenuates early infection of juveniles and enables activation of their immune system. This mechanism associated with steady circulation of the myxoma virus in both populations, which induces frequent reinfections of immune rabbits, leads to the maintenance of high immunity levels within populations. Thus, myxomatosis has a low impact, with most infections being asymptomatic. This work shows that infection of young rabbits protected by maternal antibodies induces attenuated disease and activates their immune system. This may play a major role in reducing the impact of a highly lethal disease when ecological conditions enable permanent circulation of the pathogen.

Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations.

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.

Myxoma virus protein M029 is a dual function immunomodulator that inhibits PKR and also conscripts RHA/DHX9 to promote expanded host tropism and viral replication.

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells.

Evolutionary history and attenuation of myxoma virus on two continents.

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.

Myxoma virus M064 is a novel member of the poxvirus C7L superfamily of host range factors that controls the kinetics of myxomatosis in European rabbits.

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.

Myxomatosis in Australia and Europe: a model for emerging infectious diseases.

Myxoma virus is a poxvirus naturally found in two American leporid (rabbit) species (Sylvilagus brasiliensis and Sylvilagus bachmani) in which it causes an innocuous localised cutaneous fibroma. However, in European rabbits (Oryctolagus cuniculus) the same virus causes the lethal disseminated disease myxomatosis. The introduction of myxoma virus into the European rabbit population in Australia in 1950 initiated the best known example of what happens when a novel pathogen jumps into a completely naïve new mammalian host species. The short generation time of the rabbit and their vast numbers in Australia meant evolution could be studied in real time. The carefully documented emergence of attenuated strains of virus that were more effectively transmitted by the mosquito vector and the subsequent selection of rabbits with genetic resistance to myxomatosis is the paradigm for pathogen virulence and host-pathogen coevolution. This natural experiment was repeated with the release of a separate strain of myxoma virus in France in 1952. The subsequent spread of the virus throughout Europe and its coevolution with the rabbit essentially paralleled what occurred in Australia. Detailed molecular studies on myxoma virus have dissected the role of virulence genes in the pathogenesis of myxomatosis and when combined with genomic data and reverse genetics should in future enable the understanding of the molecular evolution of the virus as it adapted to its new host. This review describes the natural history and evolution of myxoma virus together with the molecular biology and experimental pathogenesis studies that are informing our understanding of evolution of emerging diseases.

Dying like rabbits: general determinants of spatio-temporal variability in survival.

1. Identifying general patterns of how and why survival rates vary across space and time is necessary to truly understand population dynamics of a species. However, this is not an easy task given the complexity and interactions of processes involved, and the interpopulation differences in main survival determinants. 2. Here, using European rabbits (Oryctolagus cuniculus) as a model and information from local studies, we investigated whether we could make inferences about trends and drivers of survival of a species that are generalizable to large spatio-temporal scales. To do this, we first focused on overall survival and then examined cause-specific mortalities, mainly predation and diseases, which may lead to those patterns. 3. Our results show that within the large-scale variability in rabbit survival, there exist general patterns that are explained by the integration of factors previously known to be important at the local level (i.e. age, climate, diseases, predation or density dependence). We found that both inter- and intrastudy survival rates increased in magnitude and decreased in variability as rabbits grow old, although this tendency was less pronounced in populations with epidemic diseases. Some causes leading to these higher mortalities in young rabbits could be the stronger effect of rainfall at those ages, as well as, other death sources like malnutrition or infanticide. 4. Predation is also greater for newborns and juveniles, especially in population without diseases. Apart from the effect of diseases, predation patterns also depended on factors, such as, density, season, and type and density of predators. Finally, we observed that infectious diseases also showed general relationships with climate, breeding (i.e. new susceptible rabbits) and age, although the association type varied between myxomatosis and rabbit haemorrhagic disease. 5. In conclusion, large-scale patterns of spatio-temporal variability in rabbit survival emerge from the combination of different factors that interrelate both directly and through density dependence. This highlights the importance of performing more comprehensive studies to reveal combined effects and complex relationships that help us to better understand the mechanisms underlying population dynamics.