Cross-species transmission (CST) has led to many devastating epidemics, but is still a poorly understood phenomenon. HIV-1 and HIV-2 (human immunodeficiency virus 1 and 2), which have collectively caused over 35 million deaths, are the result of multiple CSTs from chimpanzees, gorillas, and sooty mangabeys. While the immediate history of HIV is known, there are over 45 lentiviruses that infect specific species of primates, and patterns of host switching are not well characterized. We thus took a phylogenetic approach to better understand the natural history of SIV recombination and CST. We modeled host species as a discrete character trait on the viral phylogeny and inferred historical host switches and the pairwise transmission rates between each pair of 24 primate hosts. We identify 14 novel, well-supported, ancient cross-species transmission events. We also find that lentiviral lineages vary widely in their ability to infect new host species: SIVcol (from colobus monkeys) is evolutionarily isolated, while SIVagms (from African green monkeys) frequently move between host subspecies. We also examine the origins of SIVcpz (the predecessor of HIV-1) in greater detail than previous studies, and find that there are still large portions of the genome with unknown origins. Observed patterns of CST are likely driven by a combination of ecological circumstance and innate immune factors.
As demonstrated by the recent epidemics of EBOV and MERS, and by the global HIV pandemic, viral cross-species transmissions (CST) can be devastating [1,2]. As such, understanding the propensity and ability of viral pathogens to cross the species barrier is of vital public health importance. Of particular interest are transmissions that not only “spillover” into a single individual of a new host species, but that result in a virus actually establishing a sustained chain of transmission and becoming endemic in the new host population (“host switching”) [3]. HIV is the product of not just one successful host switch, but a long chain of host switch events [4,5]. There are two human immunodeficiency viruses, HIV-2 and HIV-1. HIV-2 arose from multiple cross-species transmissions of SIVsmm (simian immunodeficiency virus, sooty mangabey) from sooty mangabeys to humans [6–8]. HIV-1 is the result of four independent cross-species transmissions from chimpanzees and gorillas. Specifically, SIVcpz was transmitted directly from chimpanzees to humans twice; one of these transmissions generated HIV-1 group M, which is the primary cause of the human pandemic [9]. SIVcpz was also transmitted once to gorillas, generating SIVgor [10], which was in turn transmitted twice to humans [11]. Looking further back, SIVcpz itself was also generated by lentiviral host switching and recombination. Based on the SIV sequences available at the time, early studies identified SIVmon/-mus/-gsn (which infect mona, mustached, and greater spot-nosed monkeys, respectively) and SIVrcm (which infects red-capped mangabeys) as probable donors [12]. Functional analysis of accessory genes from these putative parental lineages indicate that the specific donors and genomic locations of these recombination event (s) were crucial for enabling what became SIVcpz to cross the high species barrier and establish an endemic lineage in hominids [13]. The complex evolutionary history resulting in HIV illustrates the importance of natural history to modern day viral diversity, and although the history leading to HIV is well detailed, broader questions regarding cross-species transmission of primate lentiviruses remain [14]. With over 45 known extant primate lentiviruses, each of which is endemic to a specific host species [4,5, 15], we sought to characterize the history of viral transmission between these species to the degree possible given the precision allowed from a limited sample of modern-day viruses. Here, we utilized phylogenetic inference to reconstruct the evolutionary history of primate lentiviral recombination and cross-species transmission. We assembled datasets from publicly available lentiviral genome sequences and conducted discrete trait analyses to infer rates of transmission between primate hosts. We find evidence for extensive interlineage recombination and identify many novel host switches that occurred during the evolutionary history of lentiviruses. We also find that specific lentiviral lineages exhibit a broad range of abilities to cross the species barrier. Finally, we also examined the origins of each region of the SIVcpz genome in greater detail than previous studies to yield a more nuanced understanding of its origins.
In order to reconstruct the lentiviral phylogeny, we first had to address the issue of recombination, which is frequent among lentiviruses [16]. In the context of studying cross-species transmission, this is both a challenge and a valuable tool. Evidence of recombination between viral lineages endemic to different hosts is also evidence that at one point in time, viruses from those two lineages were in the same animal (i. e. , a cross-species transmission event must have occurred in order to generate the observed recombinant virus). However, this process also results in portions of the viral genome having independent evolutionary—and phylogenetic—histories. To address the reticulate evolutionary history of SIVs, we set out to identify the extent and nature of recombination between lentiviral lineages. Extensive sequence divergence between lineages masks site-based methods for linkage estimation (S1 Fig). However, topology-based measures of recombination allow for “borrowing” of information across nearby sites, and are effective for this dataset. We thus utilized a phylogenetic model to group segments of shared ancestry separated by recombination breakpoints instantiated in the HyPhy package GARD [17]. For this analysis, we used a version of the SIV compendium alignment from the Los Alamos National Lab (LANL), modified slightly to reduce the overrepresentation of HIV sequences (N = 64, see Methods) [18]. Importantly, because each virus lineage has only a few sequences present in this alignment, these inferences refer to inter-lineage recombination, and not the rampant intra-lineage recombination common among lentiviruses. GARD identified 13 locations along the genome that had strong evidence of inter-lineage recombination (Fig 1). Here, evidence for a particular model is assessed via Akaike Information Criterion (AIC) and differences in AIC between models indicate log probabilities, so that a delta-AIC of 10 between two models would indicate that one model is e10/2 = ~148 as likely as the other [19]. In our case, delta-AIC values ranged from 154 to 436 for each included breakpoint, indicating that these breakpoints are strongly supported by the underlying tree likelihoods. The 14 resulting segments ranged in length from 351 to 2316 bases; in order to build reliable phylogenies, we omitted two of the less supported breakpoints from downstream analyses, yielding 12 segments ranging in length from 606–2316 bases. We found no evidence to suggest linkage between non-neighboring segments (S2 Fig). While it has been previously appreciated that several lineages of SIV are recombinant products (e. g. , [12,20]), the 13 breakpoints identified here provide evidence that there have been at least 13 inter-lineage recombination events during the evolution of SIVs. Identifying these recombination breakpoints allowed us to construct a putatively valid phylogeny for each segment of the genome that shares an internally cohesive evolutionary history. We then looked for phylogenetic evidence of cross-species transmission in the tree topologies of each of the 12 genomic segments. For this and all further analyses, we constructed a dataset from all publicly available primate lentivirus sequences, which we curated and subsampled by host and virus lineage to ensure an equitable distribution of data (see Methods). This primary dataset consists of virus sequences from the 24 primate hosts with sufficient data available (5–25 sequences per viral lineage, N = 423, S3 Fig). Alignments used the fixed compendium alignment as a template (see Methods). In phylogenetic trees of viral sequences, cross-species transmission appears as a mismatch between the host of a virus and the host of that virus’s ancestor. To identify this pattern and estimate how frequently each pair of hosts has exchanged lentiviruses, we used the established methods for modeling evolution of discrete traits, as implemented in BEAST [21,22]. In our case, the host of each viral sample was modeled as a discrete trait. This is analogous to treating the “host state” of each viral sample as an extra column in an alignment, and inferring the rate of transition between all pairs of host states along with inferring ancestral host states across the phylogeny. This approach is similar to common phylogeographic approaches that model movement of viruses across discrete spatial regions [23] and has previously been applied to modeling discrete host state in the case of rabies virus [21]. Here, we took a fully Bayesian approach and sought the posterior distribution across phylogenetic trees, host transition rates and ancestral host states. We integrate across model parameters using Markov chain Monte Carlo (MCMC). The resulting model provides phylogenetic trees for each segment annotated with ancestral host states alongside inferred transition rates. Fig 2 shows reconstructed phylogenies for 3 segments along with inferred ancestral host states. Trees are color coded by known host state at the tips, and inferred host state at internal nodes/branches; color saturation indicates the level of certainty for each ancestral host assignment. A visual example of how the model identifies cross-species transmissions can be seen in the SIVmon/SIVtal clade, which infect mona- and talapoin monkeys, respectively (starred in Fig 2A–2C). Due to the phylogenetic placement of the SIVmon tips, the internal node at the base of this clade is red, indicating that the host of the ancestral virus was most likely a mona monkey. This contrasts with the host state of the samples isolated from talapoin monkeys (tips in green). These changes in the host state across the tree are what inform our estimates of the rate of transmission between host pairs. In total, the support for each possible transmission is derived from both A) whether the transmission is supported across the posterior distribution of phylogenies for a particular segment, and B) whether this is true for multiple genomic segments. Notably, the tree topologies are substantially different between segments, which emphasizes both the extent of recombination and the different evolutionary forces that have shaped the phylogenies of individual portions of the genome. In all segments’ trees, we also see frequent changes in the host state between internal nodes (illustrated as changes in color going up the tree), suggestive of frequent ancient cross-species transmissions. On average, primate lentiviruses switch hosts once every 6. 25 substitutions per site per lineage across the SIV phylogeny. The cross-species transmission events inferred by the model are illustrated in Fig 3, with raw rates and Bayes factors (BF) in S4 Fig. As shown, the model correctly infers nearly all pairs of hosts with previously identified (to our knowledge) CST events [7,9, 11,12,24,25], with the exception of the putative CST from sooty mangabeys to sabaeus monkeys reported by [20] (see Discussion). Importantly, we also identify 14 novel cross-species transmission events with strong statistical support (cutoff of BF ≥ 10. 0). Each of these transmissions is clearly and robustly supported by the tree topologies (all 12 trees are illustrated in S5 Fig). To control for sampling effects, we repeated the analysis with a supplemental dataset built with fewer hosts, and more sequences per host (15 host species, subsampled to 16–40 sequences per viral lineage, N = 510), and see consistent results. As illustrated in S6–S8 Figs, we find qualitatively similar results. When directly comparing the average indicator values, host state transition rates, and BF values between analyses, the results from the main and supplemental datasets are strongly correlated, indicating robust quantitative agreement between the two analyses (S9 Fig). We also see similar agreement between replicates when we regenerate the main dataset via independent sampling draws (see online repository). Taken together, these results represent a far more extensive pattern of CST among primate lentiviruses than previously described [4,5]; nearly every primate clade has at least one inbound, robustly supported viral transmission from another clade. We thus conclude that the majority of lentiviruses have arisen from a process of host switching, followed by a combination of intraclade host switches and host-virus coevolution. While most SIVs are the product of ancient recombination and host switching, the distribution of these host switches is not uniform; when we assess the network centrality of each virus we find a broad range (Figs 3, S7 and S11, as node size), indicating some hosts act as sources in the SIV transmission network and other hosts act as sinks. From this, we infer that some viruses have either had greater opportunity or have a greater ability to cross the species barrier than others. In particular, the SIVs from the four closely related species of African green monkeys (SIVsab, SIVtan, SIVver, SIVgrv; collectively, SIVagm) appear to exchange viruses with other host species frequently (Fig 3,12’ o-clock). An example of SIVagm CST events can be seen in the tree topologies from gag, prot, reverse transcriptase (RT), and vif (Figs 2 and S5, segments 2,3, 4, & 6). Here, SIVtan isolates reported by Ayouba et al. [26] (denoted with ✝ in Fig 2) clearly cluster with SIVsab, in a distant part of the phylogeny from the rest of the SIVagm viruses (including the majority of SIVtan isolates). For all other segments, however, the SIVagms cluster together. We thus concur with the conclusion of Ayouba et al. that these samples represent a recent spillover of SIVsab from sabaeus monkeys to tantalus monkeys, and the model appropriately identifies this transmission. Contrastingly, previous studies of the lentiviral phylogeny have noted that SIVcol is typically the outgroup to other viral lineages, and have hypothesized that this may implicate SIVcol as the “original” primate lentivirus [27]. We find this hypothesis plausible, but the evidence remains inconclusive. For the majority of genomic segments, we also observe SIVcol as the clear outgroup (Figs 2 and S5). In contrast, for portions of gag/pol (segments 3 and 4) and some of the accessory genes (segment 7), we find that there is not a clear outgroup. For these segments, many other lineages of SIV are just as closely related to SIVcol as they are to each other. However, with the occasional exception of single heterologous taxa with poorly supported placement, SIVcol remains a monophyletic clade (N = 16), and does not intercalate within the genetic diversity of any other lineage in our dataset. Based on these collective tree topologies, our model does not identify strong evidence for any specific transmissions out of colobus monkeys, and identifies only a single, weakly supported inbound transmission (likely noise in the model caused by the fact that red-capped mangabeys are the marginally supported root host state; see below). This is consistent with previous findings that the colobinae—in a different genus than most of the Cercopithecus primates in our dataset—have a unique variant of the APOBEC3G gene, which is known to restrict lentiviral infection and speculated to be a barrier to cross-species transmission [27]. These observations generally support the idea of SIVcol as having maintained a specific relationship with its host over evolutionary time. Additionally, while most host species carry only one lineage of SIV, mandrils and mustached monkeys carry 2 and 3 lineages of SIV, respectively [28–31]. In agreement with these previous studies, SIVmnd-1 and SIVmnd-2 do not always cluster together in the phylogeny; the same is true for SIVmus-1, SIVmus-2, and SIVmus-3, indicating that each of these viral lineages likely has a unique origin. This stands in stark contrast to baboons, which have only been infected by an SIV via a single documented spillover event [24]. Collectively, these examples demonstrate that the nature of the host-virus relationship is highly variable for primate lentiviruses, with some viruses switching hosts often while others putatively maintain strict host specificity. Likewise, while some hosts have acquired multiple SIV lineages, most are infected by only one SIV, or do not have an endemic SIV. Unlike SIVcol, SIVcpz appears to be the product of multiple CSTs and recombination events. SIVcpz actually encompasses two viral lineages: SIVcpzPtt infects chimpanzees of the subspecies Pan troglodytes troglodytes, and SIVcpzPts infects chimpanzees of the subspecies Pan troglodytes schweinfurthii [32]. There are two additional subspecies of chimpanzees that have not been found harbor an SIV despite extensive surveys, suggesting that SIVcpzPtt was acquired after chimpanzee subspeciation [25]. Both this previous work and our own results support the hypothesis that SIVcpz was later transmitted from one chimpanzee subspecies to the other, and SIVcpzPtt is the only SIVcpz lineage that has crossed into humans. Given the shared ancestry of the two lineages of SIVcpz, we use “SIVcpz” to refer specifically to SIVcpzPtt. Based on the lentiviral sequences available in 2003, Bailes et al [12] suggested that the SIVcpz genome is a recombinant of just two parental lineages. SIVrcm (which infects red-capped mangabeys) was identified as the 5’ donor, and an SIV from the SIVmon/-mus/-gsn clade (which infect primates in the Cercopithecus genus) was identified as the 3’ donor. Since the time of this previous investigation many new lentiviruses have been discovered and sequenced. In incorporating these new data, we find clear evidence that the previous two-donor hypothesis may be incomplete. The tree topologies from env in the 3’ end of the genome (segments 8–11) support the previous hypothesis [12] that this region came from a virus in the SIVmon/-mus/-gsn clade. These viruses form a clear sister clade to SIVcpz with high posterior support (Fig 2C and 2D). We find strong evidence for transmissions from mona monkeys (SIVmon) to mustached monkeys (SIVmus), and from mustached monkeys to greater spot-nosed monkeys (SIVgsn) (see Discussion of potential coevolution below). We also find more evidence in support of a transmission from mona monkeys to chimpanzees than from the other two potential donors, but more sampling is required to firmly resolve which of these viruses was the original donor of the 3’ end of SIVcpz. We find phylogenetic evidence to support the previous hypothesis [12,13] that the integrase and vif genes of SIVcpz (segments 4–6) originated from SIVrcm; however, we find equally strong evidence to support the competing hypothesis that pol came from SIVmnd-2, which infects mandrils (Fig 2B and 2D). In these portions of the genome, SIVmnd-2 and SIVrcm together form a clear sister clade to SIVcpz. The vpr gene, in segment 7, is also closely related to both SIVrcm and SIVmnd-2, but this sister clade also contains SIVsmm from sooty mangabeys. Notably, we infer a transmission from red-capped mangabeys to mandrils, but we cannot determine whether this portion of the SIVcpz genome was acquired directly from SIVrcm or from SIVmnd-2. Interestingly, we do not find evidence to support either SIVrcm/mnd-2 or SIVmon/mus/gsn as the donor for the 5’ most end of the genome (segments 1–5), including the 5’ LTR, gag, and RT genes. This is also true for the 3’ LTR (segment 12). SIVcpz lacks a clear sister clade or ancestor in this region, and SIVrcm groups in a distant clade; we therefore find no evidence to suggest that an ancestor of an extant SIVrcm was the parental lineage of SIVcpz in the 5’ most end of the viral genome as previously believed (Fig 2A and 2D). This may support the possibility of a third parental lineage, or a number of other plausible scenarios (discussed below).
Paleovirology, biogeography, and statistical models of lentiviral evolution estimate that primate lentiviruses share a common ancestor approximately 5–10 million years ago [27,34–36]. This, along with the putative viral coevolution during primate speciation, suggests that many of these transmissions were ancient, and have been acted on by selection for millions of years. Thus, given that the observed transmissions almost exclusively represent evolutionarily successful host switches, it is remarkable that lentiviruses have been able to repeatedly adapt to so many new host species. In the context of this vast evolutionary timescale, however, we conclude that while lentiviruses have a far more extensive history of host switching than previously understood, these events remain relatively rare overall. As noted above, our results illustrate that some SIVs cross the species barrier more readily than others, and some primate host species become infected with new viral lineages more commonly than others. This is likely governed by both ecological and biological factors. Ecologically, frequency and form of exposure are likely key determinants of transmission [3], but these relationships can be difficult to describe statistically. For example, many primates are chronically exposed to many exogenous lentiviruses through predation [5,15,37]. Using log body mass ratios [38] as a proxy for predation, we do not see a statistically significant association between body mass ratio and non-zero transmission rate (Fig 4A, blue; p = 0. 678, coef. 95% CI [-0. 311,0. 202]). We believe the lack of signal is likely due to the imperfect proxy, although it is also possible that predator-prey relationships do not strongly structure the CST network. It is also likely that geographic overlap and habitat similarity are ecological determinants of SIV CST, but modern primate habitats are likely very different since the time that these transmissions actually occurred. Biologically, increasing host genetic distance has a clear negative association with the probability of cross-species transmission (Fig 4B, blue, p<0. 001, coef. 95% CI [-7. 633, -2. 213]). Importantly, as already discussed, the strength of this association may be inflated by instances of lineage tracking (virus/host cospeciation). However, it is well established that increasing host genetic distance is associated with a higher species barrier [33]. As previously documented in the literature we expect this is due to several factors, such as the divergence of host restriction factor genes, which are key components of the innate immune system (reviewed in [39]) and differences in host cell receptor phenotypes (e. g. , [40–42]). Functional assays of these host phenotypes against panels of SIVs, while outside the scope of this study, will be important for further identifying the molecular bases of the species barriers that have led to the transmission patterns identified here. Here, we have shown that lentiviruses have a far more extensive history of host switching than previously described. Our findings also demonstrate that the propensity of each lentiviral lineage to switch between distant hosts, or to spillover among related hosts, is highly variable. In examining specific lineages, our findings are consistent with the prevalent hypothesis that SIVcol has evolved in isolation from other SIVs. Contrastingly, we have also demonstrated that the mosaic origins of SIVcpz are far more complex than previously recognized; the currently available sequence data is unable to resolve the ancestry of nearly half of the SIVcpz genome. Together, our analyses move closer to a full understanding of the pattern of cross-species transmission among primate lentiviruses, but additional efforts to obtain high quality viral genome sequences from under sampled lineages will be necessary to fully resolve the natural history of these viruses.
Lentiviral genomes are translated in multiple reading frames; we therefore utilized nucleotide sequence data for all analyses.
Virus cross-species transmissions (CSTs) can cause devastating epidemics, including EBOV, MERS, and HIV. HIV is actually one of over 45 similar viruses, called SIVs, which infect primates; the emergence of HIV as a human pathogen was the result of multiple CSTs, and yet it is not understood how commonly these viruses switch hosts. We used publicly available genetic sequence data to reconstruct the evolutionary history of these viruses. We found evidence for at least 13 instances of recombination between lineages of SIV, and identified at least 14 ancient cross-species transmission events where an SIV switched hosts. These results provide important ecological context to the HIV pandemic, suggesting that while SIV CST is relatively rare over evolutionary time, it is an important factor that has shaped SIV biology and evolution.
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Modern-day SIV viral diversity generated by extensive recombination and cross-species transmission