In female (XX) mammals one of the two X chromosomes is inactivated to ensure an equal dose of X-linked genes with males (XY)1. X-inactivation in eutherian mammals is mediated by the non-coding RNA Xist2. Xist is not found in metatherians3 and how X-inactivation is initiated in these mammals has been the subject of speculation for decades4. Using the marsupial Monodelphis domestica we identify Rsx (RNA-on-the-silent X), an RNA that exhibits properties consistent with a role in X-inactivation. Rsx is a large, repeat-rich RNA that is expressed only in females and is transcribed from, and coats, the inactive X chromosome. In female germ cells, where both X chromosomes are active, Rsx is silenced, linking Rsx expression to X-inactivation and reactivation. Integration of an Rsx transgene on an autosome in mouse embryonic stem cells leads to gene silencing in cis. Our findings permit comparative studies of X-inactivation in mammals and pose questions about the mechanisms by which X-inactivation is achieved in eutherians.

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. IMPORTANCEThe 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. R evealing the factors that determine the evolution of virulence in viruses and identifying how changes in virulence might impact viral evolutionary dynamics are key queries in the study of infectious disease evolution. Although there is a large body of work on revealing the relationship between virulence and transmissibility, most studies of virulence evolution involve the retrospective analysis of single viruses (1-4). However, the comparison of closely related...

The introduction of rabbit hemorrhagic disease virus (RHDV) into Australia and New Zealand during the 1990s as a means of controlling feral rabbits is an important case study in viral emergence. Both epidemics are exceptional in that the founder viruses share an origin and the timing of their release is known, providing a unique opportunity to compare the evolution of a single virus in distinct naive populations. We examined the evolution and spread of RHDV in Australia and New Zealand through a genome-wide evolutionary analysis, including data from 28 newly sequenced RHDV field isolates. Following the release of the Australian inoculum strain into New Zealand, no subsequent mixing of the populations occurred, with viruses from both countries forming distinct groups. Strikingly, the rate of evolution in the capsid gene was higher in the Australian viruses than in those from New Zealand, most likely due to the presence of transient deleterious mutations in the former. However, estimates of both substitution rates and population dynamics were strongly sample dependent, such that small changes in sample composition had an important impact on evolutionary parameters. Phylogeographic analysis revealed a clear spatial structure in the Australian RHDV strains, with a major division between those viruses from western and eastern states. Importantly, RHDV sequences from the state where the virus was first released, South Australia, had the greatest diversity and were diffuse throughout both geographic lineages, such that this region was likely a source population for the subsequent spread of the virus across the country. IMPORTANCE