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Thread: Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus.

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    Default Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus.

    Large-Scale Phylogenomic Analysis Reveals the Complex Evolutionary History of Rabies Virus in Multiple Carnivore Hosts


    Revealing how viruses jump species boundaries and establish productive infections in new hosts is key to understanding disease emergence. As most recent emerging and re-emerging viruses have RNA genomes [1], it is of central importance to understand the drivers of RNA virus evolution, diversification and cross-species transmission. Clearly, successful virus emergence has diverse causes, likely involving anthropogenic, social and environmental factors [2]. However, the capacity of the viral genome to vary and generate advantageous mutations is also an important element, enabling RNA viruses to exploit new niches, including novel host species, often more rapidly than DNA-based organisms [1, 3, 4]. One important manifestation of RNA virus evolution and diversification is the rate of evolutionary change (i.e. nucleotide substitution), with analyses of how this parameter varies by host species providing important information on the nature of virus-host interactions.

    Disease emergence results from complex mechanisms that shape the ability of a virus to be maintained within its primary host species, then be serially transmitted to a new host species and initiate a pathologic process to cause disease [5]. As such, lyssaviruses (family Rhabdoviridae), the causative agents of rabies–an acute and almost invariably fatal encephalomyelitis in humans–represent an informative case study to examine the relationship between virus genetic diversity and disease emergence. In particular, the natural history of these zoonotic viruses provides an excellent model to study how replication in different host species alters the selection pressures that act on virus genomes. Lyssaviruses are single-stranded, negative-sense RNA viruses with a genome size of approximately 12 kb that encodes five proteins: the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G) and the Large protein or polymerase (L). Currently, the lyssaviruses are classified into 14 species and one tentative species [6]. Like other RNA viruses, lyssaviruses exhibit high rates of mutation due to a lack of proofreading activity in the L protein [7]. Notably, although many mammalian species appear to be susceptible to lyssavirus infection, the virus is only able to establish sustained transmission networks in a relatively small number, indicating that there are major barriers to successful cross-species transmission [8–11].

    One species of lyssavirus, rabies virus (RABV), is present worldwide and circulates in a diverse set of reservoir hosts among the mammalian orders Chiroptera and Carnivora [12]. Its natural evolution provides an illustrative example of multiple host switches, in turn enabling comparative studies of the evolutionary patterns, processes and dynamics associated with host adaptation. Previous studies demonstrated that RABV isolates fall into two major phylogenetic groups; the bat- and the dog-related RABV groups [8, 13, 14]. The ‘bat-related’ RABV group is confined to New World viruses circulating mainly among bats, as well as in some terrestrial carnivores such as skunks and raccoons [14–17]. In contrast, the ‘dog-related’ RABV group contains viruses circulating worldwide in dogs, as well as in wildlife carnivores in specific geographic areas such as foxes and raccoon dogs in Europe, foxes in the Middle East, raccoon dogs and ferret-badgers in Asia, skunks, foxes, coyotes and mongooses in the Americas, and mongooses in Africa [14, 16, 18–22]. Importantly, dogs are responsible for more than 99% of the human rabies cases worldwide [23] and are likely the main vector for the inter-species transmission of dog-related RABV.

    Previous phylogenetic analyses have largely been performed on individual genes [13–19, 21, 24–29] with a few assessing the full-length viral genome [20, 30, 31]. In addition, most of these phylogenetic studies were performed on relatively small numbers of sequences originating from one specific geographical area and/or associated with a specific animal host [20, 22, 30, 32, 33]. Despite these limitations, these studies are consistent in showing that RABV is subject to strong purifying selection [10] coupled to geographical clustering that is occasionally disrupted by human mediated dispersion [13, 34, 35]. Recently, it was shown that nucleotide substitution rates in RABV vary markedly among those viruses infecting bats, such that rates in tropical and subtropical species were markedly higher than those from temporal bat species, perhaps reflecting a combination of host and environmental factors [36]. However, equivalent data for dog-related RABV are lacking. In addition, whether evolutionary rates in RABV vary among wild carnivores and domestic dogs is unknown, although studies in other systems have revealed that rates of RNA virus evolution may differ between wild and domestic animals [37]. Clearly, the large-scale analysis of RABV, particularly comprising full-length genome sequences, is needed to reveal the nature of the selection pressures associated with host switching. That the RABV genome encodes a limited number of proteins that necessarily have multifunctional roles [38], and hence potentially large-scale epistasis, also means that these selection pressures may be complex.

    Herein we present the first phylogenetic study of RABV on a genome-wide and global scale, utilizing a data set of 321 whole-genome sequences sampled from 66 countries over a time period of 65 years, with the aim of inferring those evolutionary patterns and processes associated with host-switching. In particular, we compared RABV from wild carnivores and in domestic dogs with respect to selection pressures, evolutionary rates, and the time-scale of their evolutionary history. Importantly, the size of the data set allowed us to reveal any heterogeneity in evolutionary rates among RABV adapted to different primary hosts, and determine the complex evolutionary dynamics of RABV as it adapts to new hosts.

    Host and geographical clustering of RABV

    A phylogenetic analysis was performed on the (99%) full-length genome sequences of 321 RABV sequences sampled from 66 countries (S1 Fig, S1 Table). Of these viruses, 170 were newly sequenced as part of this study. As expected given the low levels of recombination in RABV, the topology of the maximum likelihood (ML) tree performed on the five concatenated RABV genes (Fig 1) was similar to that obtained for each individual gene (N, P, M, G and L genes) and for the concatenated non-coding sequence (S2 Fig). In particular, two major phylogenetic groups were apparent, corresponding to bat- and dog-related RABVs, each of which can be further subdivided into several major clades. This is consistent with previous analyses of smaller data sets and on individual RABV genes [13, 14, 16, 29].

    Fig 1. Maximum likelihood phylogeny of 321 RABV sequences from five concatenated genes.
    The major clades of RABV are indicated in boxes. The names of subclades and lineages defined for the Arctic-related, Asian and Cosmopolitan clades are detailed in S1 Table, with corresponding bootstrap values shown for major nodes. The tree is mid-point rooted for clarity only, and shows the division into bat-related RABV including the RAC-SK and bat clades, and dog-related RABV including the Africa-2, Africa-3, Arctic-related, Asian, Indian subcontinent and Cosmopolitan clades.

    Variation in evolutionary rates among hosts and selection pressures along the RABV genome

    The root-to-tip regression analysis also revealed that different groups of dog-related RABV have seemingly evolved at different rates (S4C Fig), with a number appearing as distinct outliers. Interestingly, these outliers were confined to RABV circulating in mongooses in Africa (Africa-3 clade) and in ferret-badgers in Asia (the SEA5 subclade and SEA2b lineage), suggesting that they might represent species-specific variation. To address this, we compared the evolutionary rates of these clusters to both the entire dog-related RABV group and to subsets of this group representing dog-related viruses circulating in Africa and Asia, and to mongoose viruses circulating in the Caribbean. For this analysis we focused on the N and G genes as they comprise the largest data sets. This analysis revealed that the N gene of those viruses circulating in ferret-badgers in Asia (n = 81) and in mongooses in Africa (n = 47) evolved between 2–4 times more rapidly than those of the whole dog-related group (n = 248), at rates of 7.82 x 10−4 subs/site/year (95% HPD 3.14–12.83 x 10−4 subs/site/year) and 5.88 x 10−4 subs/site/year (95% HPD 3.67–8.11 x 10−4 subs/site/year), respectively (Fig 2B). Importantly, these estimates and their associated uncertainty do not overlap with those for the dog-related group as a whole. This finding is confirmed using smaller subsets of dog-related RABV from more closely related geographically settings in Asia and in Africa (Fig 2B). Interestingly, the rate of RABV evolution in mongooses in Africa is two times higher than that of RABVs from mongooses in the Caribbean (i.e. Puerto Rico, Cuba and Grenada) that belong to the Cosmopolitan clade (Fig 2B). Although less rate variation was observed in the G gene, RABV associated with ferret-badgers in Asia still evolved considerably more rapidly than those obtained with the different subsets of dog-related RABV (Fig 2B). These results were confirmed by using different nucleotide substitution models and a hierarchical phylogenetic model approach (S4 Table) [45, 46].

    To determine if the variation in rates of evolutionary change might result from differing selection pressures, we first compared the ratio of nonsynonymous (dN) to synonymous (dS) substitutions per site. This analysis was performed on each of the five RABV genes of the two major RABV groups. For each gene, the dN/dS ratios of the bat- and dog-related groups are very similar (and very low) and followed the same ascending order between genes: N, L, M, G and P genes (Table 1). Furthermore, we explored the number of positively selected sites using several different approaches (SLAC, FUBAR and FEL) [47, 48]. In each of the two major RABV groups, one position was identified as positively selected by at least two of these methods: positions 496 and 484 in the G protein for the bat- and dog-related groups, respectively (Table 1). Interestingly, the dN/dS of the N and G genes for the branches leading to sequences found to be outliers in the analysis of evolutionary rates (Africa-3 clade and ferret-badgers in Asia) were 1.4 to 4.7 times higher than those of dog-related RABV data sets used as controls (S4C Fig and S5 Table), but still relatively low. Together, these results are generally indicative of strong purifying selection among all sites and branches of the RABV phylogeny.

    To investigate selection pressures in greater detail we utilized a modified MEME analysis that considered internal branches of the tree only (as external branches often contain transient deleterious nonsynonymous substitutions yet to be removed by purifying selection) [49]. Using this MEME-internal analysis, we identified nine positions to be under positive selection (N436, P55, P154, P265, G198, G476, L430, L681, L2091). In addition, position G484 that was identified as positively selected using SLAC, FUBAR and FEL was not significant (at the p<0.05 level) in the MEME-internal analysis (p-value = 0.084).

    Finally, it was also clear that specific amino acid substitutions characterized RABV circulating in mongooses in Africa (Africa-3 clade) and in ferret-badgers in Asia (SEA5 subclade and SEA2b lineage) (S6 Table). Four substitutions were specific (i.e. not present in any other dog-related RABV sequences) to mongoose RABV: two in the nucleoprotein, from Asp to Asn at codon position 88 (Asp-N88-Asn) and Leu-N108-Ile, and two in the glycoprotein–Ser-G223-Asn and Pro-G386-Ser. The case of the ferret-badger was more interesting as the host jump to this species from dogs has occurred independently in the SEA5 and SEA2b clades, allowing us to determine whether cross-species transmission in this case is associated with parallel viral evolution. This analysis revealed that two amino acid substitutions were common to all ferret-badger viruses across both clades: Leu-N374-Ser and Lys-L200-Arg. The Leu-N374-Ser substitution is particularly noteworthy as it only occurs in the ferret-badger, this residue is normally highly conserved in RABV, and Leu-to-Ser is a non-conservative amino acid change. Hence, we suspect that Leu-N374-Ser, and perhaps Lys-L200-Arg, facilitate RABV adaptation to ferret-badgers. Notably, neither of these sites was found to be subject to positive selection using the methods employed here (Table 1).


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