Parallel adaptation of rabbit populations to myxoma virus.

In the 1950s the myxoma virus was released into European rabbit populations in Australia and Europe, decimating populations and resulting in the rapid evolution of resistance. We investigated the genetic basis of resistance by comparing the exomes of rabbits collected before and after the pandemic. We found a strong pattern of parallel evolution, with selection on standing genetic variation favoring the same alleles in Australia, France, and the United Kingdom. Many of these changes occurred in immunity-related genes, supporting a polygenic basis of resistance. We experimentally validated the role of several genes in viral replication and showed that selection acting on an interferon protein has increased the protein's antiviral effect.

Large-scale assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60years after first outbreak in Spain.

Myxomatosis is a viral disease that affects European rabbits (Oryctolagus cuniculus) worldwide. In Spain, populations of wild rabbits drastically decreased in the 1950s after the first outbreak of myxomatosis. Since that first appearance, it seems to be an annual epizootic in Spain with periodic outbreaks, predominantly in summer and autumn. Taking into account rabbit population structure, abundance, and genetic lineage, this paper attempts to make a large-scale characterization of myxomatosis seroprevalence based on the immune status of 29 rabbit populations distributed throughout Spain, where O. cuniculus cuniculus and O. c. algirus, the two known rabbit subspecies, naturally inhabit. A total of 654 samples were collected between 2003 and 2009, and seroprevalence of antibodies against Myxoma virus (MYXV) was determined. Overall, our results revealed that 53% of the rabbit samples were positive to antibodies against MYXV. Newborn and juvenile rabbits were the most susceptible animals to the virus, with 19% and 16% seropositivity for newborn and juveniles, respectively, while adult rabbits were the most protected, with 65% of seropositive samples. This suggests that prevalence is negatively related to the proportion of newborn and juvenile rabbits in a population. Our results also showed that seroprevalence against MYXV tended to be higher in high-abundance populations. In contrast, no differences were detected in seroprevalence between rabbit subspecies. This study confirms that >60years since first outbreak, myxomatosis is an endemic disease in Spain. Based on the results, the establishment of a myxomatosis surveillance protocol is proposed.

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.

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.

Coccidian and nematode infections influence prevalence of antibody to myxoma and rabbit hemorrhagic disease viruses in European rabbits.

The interaction among several parasites in European rabbits (Oryctolagus cuniculus) is crucial to host fitness and to the epidemiology of myxomatosis and rabbit hemorrhagic disease. These diseases have caused significant reductions in rabbit populations on the Iberian Peninsula. Most studies have focused on the epidemiology and pathogenesis of these viruses individually, and little is known about interactions between these viruses and other parasites. Taking advantage of an experimental restocking program in Spain, the effects of coccidian and nematode infections on the probability of having detectable antibody to myxoma and rabbit hemorrhagic disease viruses were tested in European wild rabbits. For 14 mo, we monitored rabbit abundance and parasite loads (coccidia and nematodes) in three reintroduced rabbit populations. While coccidian and nematode loads explained seasonal antibody prevalences to myxoma virus, the pattern was less clear for rabbit hemorrhagic disease. Contrary to expectations, prevalence of antibody to myxoma virus was inversely proportional to coccidian load, while nematode load seemed to play a minor role. These results have implications for viral disease epidemiology and for disease management intended to increase rabbit populations in areas where they are important for ecosystem conservation.

The current status and future directions of myxoma virus, a master in immune evasion.

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.

Molecular characterization of SG33 and Borghi vaccines used against myxomatosis.

Myxoma virus is a poxvirus responsible for myxomatosis in European Rabbits (Oryctolagus cuniculus). The entire genome of the myxoma virus has been sequenced, allowing a systemic survey of the functions of a large number of putative pathogenic factors that this virus expresses to subvert the immune and inflammatory pathways of infected rabbit hosts. In Italy, industrial rabbits are mostly vaccinated against myxomatosis using the attenuated myxoma virus strains Borghi or SG33. We have identified genetic markers specific for Borghi or SG33 vaccine strains and established a PCR-based assay that could be used to: (a) rapidly diagnose the presence of myxoma virus in infected organs; (b) discriminate between field strain-infected and vaccinated rabbits and (c) differentiate between Borghi or SG33 vaccine strain.

Genetic characterization of the chemokine receptor CXCR4 gene in lagomorphs: comparison between the families Ochotonidae and Leporidae.

Chemokines receptors are transmembrane proteins that bind chemokines. Chemokines and their receptors are known to play a crucial role in the immune system and in pathogen entry. There is evidence that myxoma virus, the causative agent of myxomatosis, can use the chemokine receptor CXCR4 to infect cells. This virus causes a benign disease in its natural host, Sylvilagus, but in the European rabbit (Oryctolagus cuniculus) it causes a highly fatal and infectious disease known as myxomatosis. We have characterized the chemokine receptor CXCR4 gene in five genera of the order Lagomorpha, Ochotona (Ochotonidae), and Oryctolagus, Lepus, Bunolagus and Sylvilagus (Leporidae). In lagomorphs, the CXCR4 is highly conserved, with most of the protein diversity found at surface regions. Five amino acid replacements were observed, two in the intracellular loops, one in the transmembrane domain and two in the extracellular loops. Oryctolagus features unique amino acid changes at the intracellular domains, putting this genus apart of all other lagomorphs. Furthermore, in the 37 European rabbits analysed, which included healthy rabbits and rabbits with clinical symptoms of myxomatosis, 14 nucleotide substitutions were obtained but no amino acid differences were observed.

Impact of myxomatosis in relation to local persistence in wild rabbit populations: the role of waning immunity and the reproductive period.

Many diseases are less severe when they are contracted in early life. For highly lethal diseases, such as myxomatosis in rabbits, getting infected early in life can represent the best chance for an individual to survive the disease. For myxomatosis, early infections are attenuated by maternal antibodies. This may lead to the immunisation of the host, preventing the subsequent development of the lethal form of the disease. But early infection of young individuals requires specific demographic and epidemiological contexts, such as a high transmission rate of the pathogen agent. To investigate other factors involved in the impact of such diseases, we have built a stochastic model of a rabbit metapopulation infected by myxomatosis. We show that the impact of the pathogen agent can be reduced by early infections only when the agent has a long local persistence time and/or when the host subpopulations are highly connected. The length of the reproductive period and the duration of acquired immunity are also important factors influencing the persistence of the pathogen and thus, the impact of the disease. Besides confirming the role of classical factors in the persistence of a pathogen agent, such as the size of the subpopulation or the degree of connectivity, our results highlight novel factors that can modulate the impact of diseases whose severity increase with age.

Parasite co-infection and interaction as drivers of host heterogeneity.

We examined the hypothesis that the interaction between concomitant infecting parasites modifies host susceptibility, parasite intensity and the pattern of parasite distribution within the host population. We used a 26 year time series of three common parasites in a natural population of rabbits: two gastrointestinal nematodes (Trichostrongylus retortaeformis and Graphidium strigosum) and the immunosuppressive myxoma virus. The frequency distribution of nematodes in the host population and the relationship between host age and nematode intensity were explored in rabbits with either single or dual nematode infections and rabbits infected with the nematodes and myxoma virus. The aggregation of T. retortaeformis and G. strigosum among the rabbits varied with the nature of the co-infection both in male and female hosts. The two nematodes exhibited different age-intensity profiles: G. strigosum intensity increased exponentially with host age while T. retortaeformis intensity exhibited a convex shape. The presence of a secondary infection did not change the age-intensity profile for G. strigosum but for T. retortaeformis co-infection (either both nematodes or myxoma-nematodes) resulted in significantly greater intensities in adult hosts. Results suggest that multi-species infections contributed to aggregation of parasites in the host population and to seasonal variation in intensity, but also enhanced differences in parasitism between sexes. This effect was apparent for T. retortaeformis, which appears to elicit a strong acquired immune response but not for G. strigosum which does not produce any evident immune reaction. We concluded that concomitant infections mediated by host immunity are important in modifying host susceptibility and influencing heterogeneity amongst individual hosts.

Vaccination of free-living juvenile wild rabbits (Oryctolagus cuniculus) against myxomatosis improved their survival.

For several decades, the populations of the European wild rabbit (Oryctolagus cuniculus) have declined, which is partly due to myxomatosis. Vaccination against this disease is expected to contribute to restoration of rabbit populations but the actual impact of myxomatosis is not well known and vaccination might have some negative effects. We analyzed the capture-mark-recapture data obtained in a 4-year field experiment (1991-1994) in a park near Paris, France wherein 300 out of 565 seronegative juvenile rabbits were vaccinated at first capture against myxomatosis with the nontransmissible Dervaximyxo SG33 vaccine. After accounting for weight at first capture, age-class (juvenile/adult), "trap-happiness" and season (spring/autumn) of the capture event, vaccinated rabbits had 1.8-fold greater odds of surviving than the unvaccinated rabbits. The average summer survival risk for vaccinated juveniles was 0.63 (+/-0.08 S.E.) whereas it was 0.48 (+/-0.08 S.E.) for unvaccinated juvenile rabbits.

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

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

Waning of maternal immunity and the impact of diseases: the example of myxomatosis in natural rabbit populations.

Myxomatosis is a leporipoxvirus that infects the european rabbit, inducing a high mortality rate. Observations lead us to hypothesize that a rabbit carrying maternal antibodies (or having recovered) can be infected (or re-infected) upon being exposed (or re-exposed) to the virus. Infection will lead to mild disease, boosting host immune protection. Using a modelling approach we show that this phenomenon may lead to a difference of impact of myxomatosis according to its transmission rate. Young are exposed when they still carry maternal antibodies and develop a mild disease in high transmission populations. Our results show that the impact of myxomatosis is generally higher in epidemic situations compared to populations where the virus circulates all the year. As a consequence, waning of acquired immunity and the continuous supply of newborn along the year may reduce the impact of the disease.

The poxviral scrapin MV-LAP requires a myxoma viral infection context to efficiently downregulate MHC-I molecules.

Downregulation of MHC class I molecules is a strategy developed by some viruses to escape cellular immune responses. Myxoma virus (MV), a poxvirus causing rabbit myxomatosis, encodes MV-LAP that is known to increase MHC-I endocytosis and degradation through a C(4)HC(3) motif critical for an E3 ubiquitin ligase activity. Here, we performed a functional mapping of MV-LAP and showed that not only the C(4)HC(3) motif is necessary for a marked downregulation of MHC-I but also a conserved region in the C-terminal part of the protein. We also showed that the putative transmembrane domains are responsible for a specific subcellular localization of the protein: they retain MV-LAP in the ER in transfected cells and in the endolysosomal compartments in infected cells. We observed that a specific MV infection context is necessary for a fully efficient downregulation of MHC-I. Our data suggest that the functionality of viral LAP factors, inherited by herpes- and poxviruses from mammalian cells, is more complex than anticipated.

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit.

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5.1 and 8.4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

Rabbit IL-4 was expressed in the virulent standard laboratory strain (SLS) and the attenuated Uriarra (Ur) strain of myxoma virus with the aim of creating a Th2 cytokine environment and inhibiting the development of an antiviral cell-mediated response to myxomatosis in infected rabbits. This allowed testing of a model for genetic resistance to myxomatosis in wild rabbits that have undergone 50 years of natural selection for resistance to myxomatosis. Expression of IL-4 significantly enhanced virulence of both virulent and attenuated virus strains in susceptible (laboratory) and resistant (wild) rabbits. SLS-IL-4 completely overcame genetic resistance in wild rabbits. The pathogenesis of SLS-IL-4 was compared in susceptible and resistant rabbits. The results support a model for resistance to myxomatosis of an enhanced innate immune response controlling virus replication and allowing an effective antiviral cell-mediated immune response to develop in resistant rabbits. Expression of IL-4 did not overcome immunity to myxomatosis induced by immunization.

Lessons in détente or know thy host: the immunomodulatory gene products of myxoma virus.

The poxvirus, myxoma virus, encodes within its genome at least eleven different proteins that compromise, skew, or disable the innate and adaptive responses of its hosts. In the laboratory rabbit, Oryctolagus cuniculus, these effects result in myxomatosis, a fatal condition characterized by skin lesions and systemic immunosuppression. Interestingly, while myxoma infection also causes skin lesions in its natural host and in natural populations of O. cuniculus in Australia where this novel host and the virus have co-evolved, the condition of myxomatosis does not ensue and infection is not fatal. In this review I discuss the biochemical properties of the characterized immunomodulatory proteins of myxoma virus, and their pathogenic effects in laboratory rabbits. Disruption of any one myxoma immunomodulatory gene diminishes the severity of the infection without compromising infectivity. Thus, the characterized immunomodulatory genes appear not to be required for a productive infection in vivo. The differences in the severity of their effects in laboratory-bred versus wild O. cuniculus suggest that the outcome of myxoma infection is a consequence of the interplay between the viral immunomodulatory gene products and the cells and molecules of the host immune system.

A pox on thee! Manipulation of the host immune system by myxoma virus and implications for viral-host co-adaptation.

The poxviruses have evolved a diverse array of proteins which serve to subvert innate and adaptive host responses that abort or at least limit viral infections. Myxoma virus and its rabbit host are considered to represent an ideal poxvirus-host system in which to study the effects of these immunomodulatory proteins. Studies of laboratory rabbits (Oryctolagus cuniculus) infected with gene knockout variants of myxoma virus have provided compelling evidence that several myxoma virus gene products contribute to the pathogenic condition known as myxomatosis. However, myxomatosis, which is characterized by skin lesions, systemic immunosuppression, and a high mortality rate, does not occur in the virus' natural South American host, Sylvilogus brasiliensis. Moreover, in Australia where myxoma virus was willfully introduced to control populations of O. cuniculus, myxomatosis-resistant rabbits emerged within a year of myxoma virus introduction into the field. In this review I discuss the characterized immunomodulatory proteins of myxoma virus, their biochemical properties, their pathogenic effects in laboratory rabbits, the role of the host immune system in the susceptibility or resistance to myxomatosis, and the evidence that immunomodulatory genes may have been attenuated during the co-adaptation of myxoma virus and O. cuniculus in Australia.

Immune responses to myxoma virus.

Myxoma virus causes the systemic disease myxomatosis in the European rabbit (Oryctolagus cuniculus). Originating in the South American rabbit Sylvilagus brasiliensis, where it causes a relatively localized fibroma, myxoma virus is a classic example of a virus that has jumped species to produce an exotic disease and then coevolved with its new host. Like other poxviruses, myxoma virus encodes multiple proteins capable of downregulating the host innate and acquired immune responses. Other virus-encoded proteins enable replication in host lymphocytes and monocytes, for example, by inhibiting apoptosis. Detailed studies on these proteins have demonstrated novel methods of interactions with the host immune system and added substantially to the understanding of the interaction of large DNA viruses with their hosts. Despite the increasingly detailed molecular knowledge of myxoma virus, relatively little is known about the dynamics of the interaction of the virus with the integrated host-immune system during infection and, in particular, about the evolution of resistance to the virus in wild rabbits or the species barrier. This review will focus on the detailed molecular studies that have been done with myxoma virus and discuss the more limited knowledge of the pathogenesis of myxoma virus in rabbits and the ways that the consolidated immune responses may determine genetic resistance to myxomatosis.

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins.

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.