The spatial and temporal dynamics of RNA viruses are often reflected by their phylogenetic structure (Biek et al., 2006
; Grenfell et al., 2004
; Holmes, 2004
). As such, detailed phylogenetic analysis of viral populations provides a valuable insight into the pattern and rate of geographical dispersal, especially for viruses that are subject to little natural selection at the epidemiological scale, as is likely to be the case for lyssaviruses (Bourhy et al., 1999
; Davis et al., 2005
; Holmes et al., 2002
; Kissi et al., 1995
). The aim of the analysis presented here was to determine the phylogeographic structure of RABV on a global basis and to reconstruct the spatial and temporal dynamics of this virus.
On a broad-scale, our study places the phylogeography of RABV in a global context. Specifically, we show that the current global genetic diversity of RABV from non-flying mammals can be represented by six major and geographically distinct phylogenetic clades, thereby extending previous studies of viral biodiversity. Our analysis also suggests that these clades of terrestrial mammal RABV may have an ancestry that lies with domestic dogs from the south of the Indian subcontinent, as the latter are clearly represented by the most phylogenetically divergent clade in the N gene tree. However, this hypothesis will clearly need to be confirmed with a larger sample of sequences representing a wider range of geographical localities, and with longer sequences to achieve greater phylogenetic support. Further, using coalescent-based methods to estimate times to common ancestry, we were able to show that this evolutionary diversification most likely occurred within the last 1500 years. Consequently, any older canid RABV lineages, proposed to have circulated in the Middle-East more than 2000 years ago (Steele & Fernandez, 1991
; Theodorides, 1986
), either have not survived to be sampled in the current study, were caused by an independent spill-over from bats that later died out or were due to a different lyssavirus genotype.
Lyssaviruses are zoonotic infections that invariably spill over into non-reservoir hosts (humans, bovines, small ruminants, cats etc). Onward transmission within these dead-end hosts is not sustained, so the successful transmission of RABV in new host species is likely to represent a major adaptive challenge (Kuiken et al., 2006
). This is, in part, a reflection of the strong selective constraints that act on RABV, resulting in a high rate of deleterious mutation and hence in relatively low rates of non-synonymous substitution, including at sites that might potentially enhance fitness (Holmes et al., 2002
; Kissi et al., 1999
). At a larger level, both the N and G gene phylogenies indicate that viruses sampled from other species of the family Canidae, such as foxes and raccoon dogs, as well as hosts belonging to other families within the Carnivora –the Herpestidae in southern Africa and the Mephitidae (skunks) in America – are interspersed within the phylogenetic diversity of dog RABV. While we found no significant evidence for adaptive evolution, our observation strongly suggests that the dog has served as the main vector for inter-species RABV transmission, generating viral lineages that then spread to other taxa. Determining the genetic basis of the traits that govern cross-species transmission clearly represents a major goal for future research on RABV and for emerging viruses in general, although it is important to note that patterns of cross-species transmission may also be in part determined by the ecological factors that shape host contact rates.
Our phylogenetic analysis of migration patterns is notable in that it reveals a strong population subdivision in RABV on a global scale, in contrast to the more fluid dynamics seen when the virus spreads through a specific geographical region (Biek et al., 2007
; Real & Biek, 2007
). The geographical spread of RABV in non-flying mammals at a global level (and over a period of less than 1500 years) has therefore occurred at such a low rate that its phylogenetic structure is dominated by population subdivision rather than gene flow (Criscione & Blouin, 2005
). Hence, despite the relatively recent timescale of RABV evolution, its current biodiversity is characterized by a series of spatially distinct clusters that experience relatively little contact among them. It is likely that this lack of admixture reflects the influence of major geographical barriers to gene flow, as previously demonstrated for RABV in Europe (Bourhy et al., 1999
). Indeed, the importance of physical isolation is supported by the phylogeographical patterns observed here, which suggest that both the Himalayan mountains and the Sahara Desert have acted as barriers to gene flow; the former explaining, in part, the spatial partitioning within the Asian clade, and the latter, the different phylogenetic groups seen in Africa. Conversely, a lack of major physical barriers, thereby enabling gene flow, may explain why those viruses from the Arctic-related clade occupy such a wide geographical range. Alternatively, it may be that after initial colonization there is little viral spread to adjoining regions, perhaps because immigrating viruses have a low probability of establishment in areas where other RABV already circulate (Biek et al., 2007
; Real & Biek, 2007
). However, whether such exclusion barriers to gene flow can explain broad-scale phylogeographic patterns is uncertain.
There are now several examples illustrating how the long distance transmission of RABV is facilitated by human-mediated animal movements (Fevre et al., 2006
), including the translocation of infected raccoons from Florida to Virginia for hunting (Jenkins & Winkler, 1987
) and the importation of dog rabies in Flores Islands in Indonesia in 1997 (Windiyaningsih et al., 2004
), both of which resulted in the rapid spread of RABV. Indeed, the movement of rabid domestic dogs is clearly still a major threat for rabies-free areas (Bourhy et al., 2005
). However, the strong population subdivision observed here suggests that, other than large-scale and often inter-continental translocations, humans were not normally responsible for the dispersal of rabid animals and hence RABV. Further, the lack of admixture among clades supports the idea that, over longer timescales, the persistence of RABV in its enzootic stage does not depend upon regular immigration of infected individuals (Biek et al., 2007
). Rather, it is more likely that the dispersal of RABV reflects the gradual spatial spread of virus within animals that themselves move relatively small distances, as previously demonstrated in Europe with red foxes and raccoon dogs (Bourhy et al., 1999
) and in North American raccoons (Biek et al., 2007
). The only exceptions found in our study, which most likely reflect human intervention, are the migration of virus from Russia to Canada and Greenland and from China to the Philippines and Indonesia.
The phylogenetic pattern depicted here – of distinct, geographically based clades with few intermediate lineages – is in contrast with recent studies of raccoon RABV in North America. In this case, phylogenies of isolates sampled over a period of 30 years were characterized by high rates of branching near the root of the tree, indicative of both spatial and demographical expansion (Biek et al., 2007
). There are two explanations for the long-term phylogeographical pattern revealed here: that fitness differences among lineages have enabled some to out-compete others, resulting in a selective purging of lineages, or that intermediate lineages have died out because of stochastic processes alone. Although it is possible that the fixation of advantageous mutations that enable RABV to adapt to new host species has occurred but cannot be detected by current methods, the very low dN
values observed throughout RABV evolution, as well as their bias towards external branches, suggests that purifying, rather than positive selection, dominates evolutionary dynamics. Therefore, we propose that random processes have had a more profound effect on long-term phylogeographical patterns in RABV. Specifically, over extended time periods, many of those lineages that appear in short-term demographical expansions are lost randomly by genetic drift, leaving the spatially disjunct phylogeographical clades observed here. This stochastic picture of RABV phylogeography, analogous to an allopatric model of speciation, is also compatible with simple population genetic theory. In a haploid population, the mean time to common ancestry, 2Net
is the generation time between transmission events and Ne
the effective population size), is likely to be ~2 months in the case of canid RABV (Fekadu, 1991
). In both this study and that of Biek et al. (2007)
ranges from 102
, leading to mean TMRCAs of a few hundred years, indicating that both studies are in agreement regarding the timescale of RABV evolution, as depicted here. Hence, the dual processes of random genetic drift and geographical isolation alone are likely to be sufficient to explain the long-term phylogeographical patterns of dog-associated RABV.