In situ single-crystal synchrotron X-ray diffraction studies of biologically active gases in metal-organic frameworks.

Metal-organic frameworks (MOFs) are well known for their ability to adsorb various gases. The use of MOFs for the storage and release of biologically active gases, particularly nitric oxide (NO) and carbon monoxide (CO), has been a subject of interest. To elucidate the binding mechanisms and geometry of these gases, an in situ single crystal X-ray diffraction (scXRD) study using synchrotron radiation at Diamond Light Source has been performed on a set of MOFs that display promising gas adsorption properties. NO and CO, were introduced into activated Ni-CPO-27 and the related Co-4,6-dihydroxyisophthalate (Co-4,6-dhip). Both MOFs show strong binding affinity towards CO and NO, however CO suffers more from competitive co-adsorption of water. Additionally, we show that morphology can play an important role in the ease of dehydration for these two systems.

Divergent Evolutionary Pathways of Myxoma Virus in Australia: Virulence Phenotypes in Susceptible and Partially Resistant Rabbits Indicate Possible Selection for Transmissibility.

To characterize the ongoing evolution of myxoma virus in Australian rabbits, we used experimental infections of laboratory rabbits to determine the virulence and disease phenotypes of recent virus isolates. The viruses, collected between 2012 and 2015, fell into three lineages, one of which, lineage c, experienced a punctuated increase in evolutionary rate. All viruses were capable of causing acute death with aspects of neutropenic septicemia, characterized by minimal signs of myxomatosis, the occurrence of pulmonary edema and bacteria invasions throughout internal organs, but with no inflammatory response. For the viruses of highest virulence all rabbits usually died at this point. In more attenuated viruses, some rabbits died acutely, while others developed an amyxomatous phenotype. Rabbits that survived for longer periods developed greatly swollen cutaneous tissues with very high virus titers. This was particularly true of lineage c viruses. Unexpectedly, we identified a line of laboratory rabbits with some innate resistance to myxomatosis and used these in direct comparisons with the fully susceptible rabbit line. Importantly, the same disease phenotype occurred in both susceptible and resistant rabbits, although virulence was shifted toward more attenuated grades in resistant animals. We propose that selection against inflammation at cutaneous sites prolongs virus replication and enhances transmission, leading to the amyxomatous phenotype. In some virus backgrounds this creates an immunosuppressive state that predisposes to high virulence and acute death. The alterations in disease pathogenesis, particularly the overwhelming bacterial invasions that characterize the modern viruses, suggest that their virulence grades are not directly comparable with earlier studies. IMPORTANCE The evolution of the myxoma virus (MYXV) following its release as a biological control for European rabbits in Australia is the textbook example of the coevolution of virus virulence and host resistance. However, most of our knowledge of MYXV evolution only covers the first few decades of its spread in Australia and often with little direct connection between how changes in virus phenotype relate to those in the underlying virus genotype. By conducting detailed experimental infections of recent isolates of MYXV in different lines of laboratory rabbits, we examined the ongoing evolution of MYXV disease phenotypes. Our results reveal a wide range of phenotypes, including an amyxomatous type, as well as the impact of invasive bacteria, that in part depended on the level of rabbit host resistance. These results provide a unique insight into the complex virus and host factors that combine to shape disease phenotype and viral evolution.

Controlled Synthesis of Large Single Crystals of Metal-Organic Framework CPO-27-Ni Prepared by a Modulation Approach: In situ Single-Crystal X-ray Diffraction Studies.

The size of single crystals of the metal-organic framework CPO-27-Ni was incrementally increased through a series of modulated syntheses. A novel linker modulated synthesis using 2,5-dihydroxyterephthalic acid and the isomeric ligand 4,6-dihydroxyisophthalic acid yielded large single crystals of CPO-27-Ni (∼70 µm). All materials were shown to have high crystallinity and phase purity through powder X-ray diffraction, electron microscopy methods, thermogravimetry, and compositional analysis. For the first time single-crystal structure analyses were carried out on CPO-27-Ni. High BET surface areas and nitric oxide (NO) release efficiencies were recorded for all materials. Large single crystals of CPO-27-Ni showed a prolonged NO release and proved suitable for in situ single-crystal diffraction experiments to follow the NO adsorption. An efficient activation protocol was developed, leading to a dehydrated structure after just 4 h, which subsequently was NO-loaded, leading to a first NO loaded single-crystal structural model of CPO-27-Ni.

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.

Punctuated Evolution of Myxoma Virus: Rapid and Disjunct Evolution of a Recent Viral Lineage in Australia.

Myxoma virus (MYXV) has been evolving in a novel host species-European rabbits-in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017, we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic breakdown of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of nonsynonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of the M005L/R gene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in the evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in southeastern Australia, followed by the partial recovery of populations. It is therefore possible that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV, altering the course and pace of virus evolution.IMPORTANCE The coevolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural experiments in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia, we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a breakdown in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple nonsynonymous and indel mutations. We suggest that this punctuated evolutionary event may reflect a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field.

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.

Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype.

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.

An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus.

The calicivirus Rabbit haemorrhagic disease virus (RHDV) is widely used in Australia as a biocontrol agent to manage wild European rabbit (Oryctolagus cuniculus) populations. However, widespread herd immunity limits the effectiveness of the currently used strain, CAPM V-351. To overcome this, we developed an experimental platform for the selection and characterisation of novel RHDV strains. As RHDV does not replicate in cell culture, variant viruses were selected by serially passaging a highly virulent RHDV field isolate in immunologically naïve laboratory rabbits that were passively immunised 18-24 hours post-challenge with a neutralising monoclonal antibody. After seven passages, two amino acid substitutions in the P2 domain of the capsid protein became fixed within the virus population. Furthermore, a synonymous substitution within the coding sequence of the viral polymerase appeared and was also maintained in all subsequent passages. These findings demonstrate proof-of-concept that RHDV evolution can be experimentally manipulated to select for virus variants with altered phenotypes, in this case partial immune escape.

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia.

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.

Benign Rabbit Caliciviruses Exhibit Evolutionary Dynamics Similar to Those of Their Virulent Relatives.

UNLABELLED: Two closely related caliciviruses cocirculate in Australia: rabbit hemorrhagic disease virus (RHDV) and rabbit calicivirus Australia 1 (RCV-A1). RCV-A1 causes benign enteric infections in the European rabbit (Oryctolagus cuniculus) in Australia and New Zealand, while its close relative RHDV causes a highly pathogenic infection of the liver in the same host. The comparison of these viruses provides important information on the nature and trajectory of virulence evolution, particularly as highly virulent strains of RHDV may have evolved from nonpathogenic ancestors such as RCV-A1. To determine the evolution of RCV-A1 we sequenced the full-length genomes of 44 RCV-A1 samples isolated from healthy rabbits and compared key evolutionary parameters to those of its virulent relative, RHDV. Despite their marked differences in pathogenicity and tissue tropism, RCV-A1 and RHDV have evolved in a very similar manner. Both viruses have evolved at broadly similar rates, suggesting that their dynamics are largely shaped by high background mutation rates, and both exhibit occasional recombination and an evolutionary environment dominated by purifying selection. In addition, our comparative analysis revealed that there have been multiple changes in both virulence and tissue tropism in the evolutionary history of these and related viruses. Finally, these new genomic data suggest that either RCV-A1 was introduced into Australia after the introduction of myxoma virus as a biocontrol agent in 1950 or there was drastic reduction of the rabbit population, and hence of RCV-A1 genetic diversity, perhaps coincident with the emergence of myxoma virus. IMPORTANCE: The comparison of closely related viruses that differ profoundly in propensity to cause disease in their hosts offers a powerful opportunity to reveal the causes of changes in virulence and to study how such changes alter the evolutionary dynamics of these pathogens. Here we describe such a novel comparison involving two closely related RNA viruses that cocirculate in Australia, the highly virulent rabbit hemorrhagic disease virus (RHDV) and the nonpathogenic rabbit calicivirus Australia 1 (RCV-A1). Both viruses infect the European rabbit, but they differ in virulence, tissue tropism, and mechanisms of transmission. Surprisingly, and despite these fundamental differences, RCV-A1 and RHDV have evolved at very similar (high) rates and with strong purifying selection. Furthermore, candidate key mutations were identified that may play a role in virulence and/or tissue tropism and therefore warrant further investigation.

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.

Comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens.

BACKGROUND: Only one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers. METHODS: We quantified RHDV replication and shedding in 4-5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia. RESULTS: Kittens were susceptible to infection with virus doses as low as 10 ID50. Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4-5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection. CONCLUSIONS: Rabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks.

Genome scale evolution of myxoma virus reveals host-pathogen adaptation and rapid geographic spread.

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.

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.

Viral infections of rabbits.

Viral diseases of rabbits have been used historically to study oncogenesis (e.g. rabbit fibroma virus, cottontail rabbit papillomavirus) and biologically to control feral rabbit populations (e.g. myxoma virus). However, clinicians seeing pet rabbits in North America infrequently encounter viral diseases although myxomatosis may be seen occasionally. The situation is different in Europe and Australia, where myxomatosis and rabbit hemorrhagic disease are endemic. Advances in epidemiology and virology have led to detection of other lapine viruses that are now recognized as agents of emerging infectious diseases. Rabbit caliciviruses, related to rabbit hemorrhagic disease, are generally avirulent, but lethal variants are being identified in Europe and North America. Enteric viruses including lapine rotavirus, rabbit enteric coronavirus and rabbit astrovirus are being acknowledged as contributors to the multifactorial enteritis complex of juvenile rabbits. Three avirulent leporid herpesviruses are found in domestic rabbits. A fourth highly pathogenic virus designated leporid herpesvirus 4 has been described in Canada and Alaska. This review considers viruses affecting rabbits by their clinical significance. Viruses of major and minor clinical significance are described, and viruses of laboratory significance are mentioned.

In situ hybridisation assay for localisation of rabbit calicivirus Australia-1 (RCV-A1) in European rabbit (Oryctolagus cuniculus) tissues.

Recently, a new lagovirus enzootic in Australian wild rabbits was identified and described as rabbit calicivirus Australia-1 (RCV-A1). Unlike the closely related Rabbit Haemorrhagic Disease Virus (RHDV), which causes fulminant hepatitis and rabbit death, RCV-A1 does not appear to induce any clinical disease. RCV-A1 has been postulated to act as an imperfect natural vaccine to RHDV thus reducing RHDV-induced rabbit mortality, which is detrimental for bio-control of rabbits in Australia. This study was carried out to determine in which cells RCV-A1 replication occurs. An in situ hybridisation (ISH) protocol was developed using a RCV-A1 specific probe to localise the virus in rabbit tissues. The results were compared to those obtained with a quantitative RT-PCR assay that had previously been developed to measure RCV-A1 RNA in rabbit tissues. The histology of the tissues was also examined. ISH showed that virus replication, inferred by the presence of detectable RNA, was limited to a small number of epithelial cells towards the tip of the villi in the duodenum. Quantitative RT-PCR detected RCV-A1 RNA in jejunum, ileum and lymphoid tissue at day 3, 4 and 7 post-infection, but no hybridisation was detected in these tissues.

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.

A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies.

BACKGROUND: Antibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia. RESULTS: A highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity). CONCLUSIONS: This new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations.

Serological assays to discriminate rabbit haemorrhagic disease virus from Australian non-pathogenic rabbit calicivirus.

Serological cross reactivity between the virulent rabbit haemorrhagic disease virus (RHDV) and the closely related but non-pathogenic rabbit calicivirus (RCV) makes it difficult to study the epidemiology of each virus and the interaction between them when both viruses co-circulate in wild rabbit populations. ELISA methods for the diagnosis of RHDV infection are well characterized, but no specific serological tests for RCV have been developed. Following the characterization of Australian non-pathogenic RCV-A1 strains, we used virus-like-particles (VLPs) and anti-RCV-A1 specific antibodies to establish a set of isotype ELISAs for detection of IgG, IgA and IgM in rabbit sera and secretory mucosal IgA in rectal swabs, and two competition ELISAs. These assays were used to discriminate between anti-RCV-A1 and anti-RHDV antibodies in rabbits. The isotype ELISAs were highly sensitive for detection of anti-RCV-A1 antibodies, but varying levels of cross reactivity from anti-RHDV antibodies occurred in the isotype ELISAs and one competition ELISA. However, the second competition ELISA specifically detected antibodies to RCV-A1 and showed no cross reactivity to anti-RHDV sera. These ELISAs provide important tools to monitor RCV-A1 infection when it occurs alone, and to discriminate between RHDV and RCV-A1 infection when they occur in the same rabbit population. When used in parallel with RHDV serology, they could be used to monitor the dynamics of these two closely related but pathogenically distinct viruses in wild and domestic rabbit populations.

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.