key: cord-1021492-5dvc1dpt
authors: Wertheim, Joel O.; Hostager, Reilly; Ryu, Diane; Merkel, Kevin; Angedakin, Samuel; Arandjelovic, Mimi; Ayimisin, Emmanuel Ayuk; Babweteera, Fred; Bessone, Mattia; Brun-Jeffery, Kathryn J.; Dieguez, Paula; Eckardt, Winnie; Fruth, Barbara; Herbinger, Ilka; Jones, Sorrel; Kuehl, Hjalmar; Langergraber, Kevin E.; Lee, Kevin; Madinda, Nadege F.; Metzger, Sonja; Ormsby, Lucy Jayne; Robbins, Martha M.; Sommer, Volker; Stoinski, Tara; Wessling, Erin G.; Wittig, Roman M.; Yuh, Yisa Ginath; Leendertz, Fabian H.; Calvignac-Spencer, Sébastien
title: Discovery of Novel Herpes Simplexviruses in Wild Gorillas, Bonobos, and Chimpanzees Supports Zoonotic Origin of HSV-2
date: 2021-03-15
journal: Mol Biol Evol
DOI: 10.1093/molbev/msab072
sha: 6f92f5281b0a11a2831a5829663457668cd0e593
doc_id: 1021492
cord_uid: 5dvc1dpt

Viruses closely related to human pathogens can reveal the origins of human infectious diseases. Human herpes simplexvirus type 1 (HSV-1) and type 2 (HSV-2) are hypothesized to have arisen via host-virus codivergence and cross-species transmission. We report the discovery of novel herpes simplexviruses during a large-scale screening of fecal samples from wild gorillas, bonobos, and chimpanzees. Phylogenetic analysis indicates that, contrary to expectation, simplexviruses from these African apes are all more closely related to HSV-2 than to HSV-1. Molecular clock-based hypothesis testing suggests the divergence between HSV-1 and the African great ape simplexviruses likely represents a codivergence event between humans and gorillas. The simplexviruses infecting African great apes subsequently experienced multiple cross-species transmission events over the past 3 My, the most recent of which occurred between humans and bonobos around 1 Ma. These findings revise our understanding of the origins of human herpes simplexviruses and suggest that HSV-2 is one of the earliest zoonotic pathogens.

The origins of pathogens that infect humans can be revealed by the characterization of closely related microorganisms in wild and domesticated animals. Phylogenetic analysis has revealed the source of zoonoses both recent (e.g., HIV/ AIDS, Ebola, influenza, MERS, SARS, COVID-19; Leroy et al. 2005; Hon et al. 2008; Smith et al. 2009; Sharp and Hahn 2011; Dudas et al. 2018; Nelson and Worobey 2018; Lu et al. 2020) and ancient (e.g., measles, smallpox, bubonic plague; Wertheim and Kosakovsky Pond 2011; Wagner et al. 2014; Duggan et al. 2016; Dux et al. 2020) alike. In contrast to these zoonoses, some pathogens may have been infecting our primate ancestors and have since evolved into human-specific forms via codivergence (e.g., varicella zoster virus and human mastadenovirus species C; Weiss 2007; Grose 2012; Hoppe et al. 2015) . The human herpes simplexviruses (HSVs) appear to have arisen from both of these mechanisms: codivergence and zoonosis (Luebcke et al. 2006; Weiss 2007; Severini et al. 2013; Wertheim et al. 2014) .

HSVs belong to a diverse family of viruses with doublestranded DNA genomes (Herpesviridae), whose members infect a broad range of vertebrates and invertebrates, reflecting a remarkably ancient association of these viruses with their hosts (McGeoch et al. 1995; Davison et al. 2009 ). Within this family, HSVs are part of a subfamily (Alphaherpesvirinae) and genus (Simplexvirus) within which codivergence events are very frequent. This pattern is exemplified by primate-infecting simplexviruses whose phylogeny recapitulates that of Platyrrhini (New World monkeys), Cercopithecidae (Old World monkeys), and Homininae (African great apes, including humans) (Eberle and Black 1993) . Accordingly, most primate species have only been associated with a single simplexvirus. Humans are a striking exception in that they are infected with two: HSV-1 (Human alphaherpesvirus 1) and HSV-2 (Human alphaherpesvirus 2). Cross-sectional estimates indicate that HSV-1 infects over two-thirds of adults worldwide, whereas HSV-2 has a lower prevalence around 11% (Smith and Robinson 2002) . HSV-1 is predominantly associated with oral transmission early in life, and HSV-2 is more commonly associated with sexual transmission; however, the disease presentation by these viruses is indistinguishable and both viruses can transmit through oral and genital routes. Both viruses can experience long periods of latency between infection and transmission, which when coupled with robust DNA repair mechanisms, may explain their exceptionally slow rate of evolution: around 10 À8 substitutions/site/year (Norberg et al. 2011) .

HSV-1 and HSV-2 are evolutionarily very closely related. They are not, however, each other's closest relatives. HSV-2 is more closely related to the chimpanzee herpes virus (ChHV; Panine alphaherpesvirus 3), which was discovered in a captive chimpanzee (Pan troglodytes), than either of these viruses is to HSV-1 (Luebcke et al. 2006; Severini et al. 2013) . Two alternate scenarios have been articulated to account for this pattern, which both assume that ChHV is a codiverged simplexvirus infecting (wild) chimpanzees and suggest a potential zoonotic origin for one of the HSVs. Pairwise genetic distance analysis suggested that HSV-1 was of zoonotic origin from a hypothetical orangutan simplexvirus ancestor and that HSV-2 had codiverged with ChHV (Luebcke et al. 2006; Severini et al. 2013) . In contrast, we previously suggested that HSV-2 was of recent zoonotic origin from the ancestor of modern P. troglodytes and that HSV-1 has its origins in codivergence (Wertheim et al. 2014 ). Both of these scenarios assume there exist, or once existed (Johnson et al. 2003) , simplexviruses infecting great apes (Hominidae) other than chimpanzees: bonobo (Pan paniscus), gorilla (Gorilla spp.), and orangutan (Pongo spp.) . Great apes can be infected with HSV-1 and HSV-2 (Eberle and Hilliard 1989) ; however, the presence of extant nonhuman Homininae simplexviruses is supported by serology in wild Gorilla beringei beringei populations that demonstrates intermediate ELISA reactivity to both HSV-1 and HSV-2 antigens, with preferential reactivity toward HSV-2 (Eberle 1992) .

Both hypothesized scenarios about the origins of HSV-1 and HSV-2 make specific predictions about the phylogenetic placement of uncharacterized viruses. The HSV-1 zoonotic origin hypothesis assumes the split between HSV-2 and ChHV represents a codivergence event and predicts that a gorilla simplexvirus would lie basal to HSV-2 and ChHV, but that HSV-1 would lie basal to these three viruses. In contrast, the HSV-2 zoonotic origin hypothesis assumes the split between HSV-1 and ChHV represents a codivergence event and predicts that a gorilla simplexvirus would lie basal to HSV-1, HSV-2, and ChHV. Here, we report the results of a large-scale molecular screening of fecal samples from wild animals representing all African great ape species, including seven of nine African great ape subspecies. This screening allowed us to confirm ChHV infection in wild West African chimpanzees (P. troglodytes verus) as well as detect simplexviruses infecting wild mountain gorillas (G. beringei beringei) and bonobos (P. paniscus). We determined partial gene sequences from virus infecting these three host species, and we assembled a partial genome sequence for the simplexvirus infecting mountain gorillas. Phylogenetic and molecular clock analyses suggest a complicated history of cross-species transmission of simplexviruses among Homininae species.

We screened a large set of African great ape feces, identifying eight positive samples with a nested PCR system targeting a short fragment of the glycoprotein B (gB) coding sequence. We found a single positive sample in the set of feces from a pregnant Western chimpanzee (P. troglodytes verus), two positive samples from one site and one from another site, representing at least two bonobos (P. paniscus), and four positive samples from a pregnant (n ¼ 3) and another lactating (n ¼ 1) mountain gorilla (G. beringei beringei); for all these samples, we sequenced this 124-bp gB fragment (UL27). We attempted to obtain more sequence information by using additional semi-nested and nested PCR systems targeting gB and UL53 but were only successful with samples from Novel Great Ape Herpes Simplexviruses . doi:10.1093/molbev/msab072 MBE pregnant mountain gorillas. Sequencing these amplicons allowed us to sequence 410 and 272 bp of the gB and UL53 coding sequences, respectively.

To collect genome-wide sequence information, we also attempted to enrich high-throughput sequencing libraries generated from all positive samples in simplexvirus sequences using hybridization capture. We generated a total of 60,741,470 reads (2,163,068-22,702 ,256 reads per sample, after filtering). Only the libraries generated from the pregnant mountain gorilla contained enough simplexvirus reads to allow for the reconstruction of a significant fraction of the genome. We were able to unambiguously characterize 103,446 positions of the according viral genome, representing 74.8% of its unique and internal repeat regions, from which we identified 58 coding sequences homologous (and syntenic) to those found in HSV-2.

The herpes simplexvirus maximum likelihood phylogeny including previously described catarrhine simplexviruses and the newly described viruses supports a general pattern of viral codivergence with their host species, with strong node support between major lineages ( fig. 1 and see Supplementary Material online). All viruses from Homininae are reciprocally monophyletic with respect to viruses from Cercopithecidae (ultrafast bootstrap support [UF] ¼ 100). Each Homininae species appears to have a distinct simplexvirus, with the known exception of HSV-1 and HSV-2 in Homo sapiens. However, these Homininae simplexviruses do not recapitulate their host phylogeny ( fig. 2A) . We caution against overinterpretation of the tanglegrams (de Vienne 2019) and note the differences in structure between the host and virus trees. Specifically, the nonhuman Homininae simplexviruses are more closely related to HSV-2 than they are to HSV-1 (UF ¼ 100). The closest relative of the novel gorilla simplexvirus is ChHV (UF ¼ 93), and the closet relative of HSV-2 is the novel bonobo simplexvirus (UF ¼ 79). In contrast, P. troglodytes and P. paniscus are most closely related and both more closely related to H. sapiens than they are to G. gorilla. The identical topology is observed across the phylogeny if we exclude the shorter sequences from G. gorilla, P. troglodytes, P. paniscus, and Macaca fuscata from the alignment prior to phylogenetic inference. A phylogeny inferred from all taxa restricted to The tree includes 74 taxa and was inferred using an alignment of 29 coding genes totaling 43,797 bp in length. Node support from ultrafast bootstrap analysis is denoted on major branches separating virus infecting different primate species. HSV-1 and HSV-2, human herpes simplexviruses 1 and 2; ChHV, chimpanzee herpes virus; McHV-1, macacine herpes virus 1; PaHV-2, Baboon herpes virus 2; CeHV-2, Cercopithecus herpes virus 2. Tree with strain labels and support values is available on Data Dryad. Wertheim et al. . doi:10.1093/molbev/msab072 MBE the glycoprotein B (UL27) gene in which many of these gene fragments reside is highly similar to the partial genome inference, albeit with lower UF (supplementary fig. 1 , Supplementary Material online). We did not detect evidence of positive, diversifying selection in UL27 (supplementary fig.  1 , Supplementary Material online), suggesting that this lower topological support is due to its shorter length, rather than biased inference due to positive selection.

The close phylogenetic relatedness of HSV-2 to the novel bonobo simplexvirus is supported by only a single nonsynonymous nucleotide substitution across the 124 nucleotide novel bonobo simplexvirus sequence: cytosine (isoleucine in all basal lineages) to guanine (methionine in HSV-2 and the P. paniscus simplexvirus) (supplementary fig. 2 , Supplementary Material online). Regardless of the specific placement of the novel bonobo simplexvirus, the remaining phylogenetic relationships are consistent with multiple crossspecies transmission of simplexvirus among Homininae. If the ancestor of this clade infected the immediate ancestor of gorillas, then the primate precursors to both humans and chimpanzees would have acquired this virus via crossspecies transmission; however, the order and direction of these events remain ambiguous.

Two major genera within the Cercopithecidae family, Papio and Macaca, are each represented by a distinct viral clade: PaHV-2 (Papiine alphaherpesvirus 2, formerly known as HVP2) and McHV-1 (Macacine alphaherpesvirus 1, formerly known as B virus). The placement of CeHV-2 from Chlorocebus pygerythrus is consistent with a cross-species transmission event. The lack of host-phylogeny recapitulation by the Papio viruses (supplementary fig. 3 , Supplementary Material online) may reflect the ongoing transmission among captive baboons, which are often housed in multispecies facilities (Rogers et al. 2003 ) (see Supplementary Material online for a detailed discussion). In contrast, the Macaca viruses from each of the five represented species (M. fascicularis, fuscata, mulatta, nemestrina, and silenus) are reciprocally monophyletic and the relationships among them accurately recapitulate the host phylogeny ( fig. 2B ).

Calibrating viral diversification events using a molecular clock in primate simplexviruses is complicated by the saturation of clock-like signal as one moves deeper into the viral phylogeny (Wertheim et al. 2014 ). Therefore, we calibrated a strict molecular clock using only shallow diversification events Methods for details). We consistently inferred a substitution rate on the order of 10 À8 substitutions/site/year (table 1) , congruent with previous rate estimates from primate simplexviruses (Norberg et al. 2011; Wertheim et al. 2014 ). Accordingly, we inferred TMRCAs around 0.5 Ma for HSV-1 and 0.1 Ma for HSV-2 (table 2) , which are broadly in agreement with previous estimates (Norberg et al. 2011; Wertheim et al. 2014; Burrel et al. 2017 ). The inferred TMRCA for HSV-1 and all other Homininae simplexviruses ranged between around 7 and 12 Ma, which is consistent with the split between Homo and another extant Hominidae genera: Pan, Gorilla, or Pongo (Raaum et al. 2005) (table 2 and fig. 3 ). This timing suggests that HSV-1 arose via codivergence. The inferred TMRCA for HSV-2 and all other Homininae simplexviruses (excluding HSV-1) was around 2-3 Ma, far more recent than the speciation between Homo and Pan genera. This recency suggests that HSV-2 arose via crossspecies transmission. We note that the ages inferred under narrow calibrations priors tended to be slightly older than ages inferred under wide priors.

The TMRCA for HSV-2 and the novel bonobo simplexvirus was bimodal due to the ambiguous phylogenetic relationship between these viruses. If these two viruses are monophyletic, as they are in around 89% of posterior trees, then we infer a median TMRCA less than 1 Ma (table 3) . However, if the novel bonobo simplexvirus is more closely related to ChHV and the novel gorilla simplexvirus, as they are in around 11% of posterior samples, then HSV-2 is equally divergent from all three Homininae herpes simplexviruses.

To determine whether the assumption of a strict molecular clock is appropriate for a hypothesis testing framework, we repeated the molecular clock phylogenetic inference assuming a relaxed clock using an uncorrelated lognormal distribution (Drummond et al. 2006) . We found no evidence for significant deviations from a strict molecular clock. Across the three calibration scenarios, the 95% highest posterior density (HPD) for the standard deviation of the lognormal distribution (a proxy for deviation from a strict clock) included or abutted against zero.

Our molecular clock calibrations were based on four calibrated nodes from McHV-1 infecting members of Macaca. We explored the internal consistency of these calibrations on the simplexvirus phylogeny using a leave-one-out cross-validation approach. The inferred TMRCAs of the great ape simplexviruses and HSV-1 or HSV-2 were robust when wide calibration priors were applied (supplementary fig. 5 , Supplementary Material online). As expected, these TMRCAs were more sensitive when narrow calibration priors were applied. Excluding the calibration representing the common ancestors of 1) M. mulatta and fuscata or 2) M. mulata, fuscata, and fascicularis tended to produce younger TMRCAs among the Homininae simplexviruses. Excluding the narrow calibration on M. silenus and nemestrina resulted in older Homininae simplexviruses TMRCAs using the Fabre and 

In order to formalize our understanding of primate simplexvirus evolution, we performed a series of hypothesis tests using Bayes Factors (BF), based on marginal likelihood estimates (MLEs) obtained via a Generalized Steeping Stone (GSS) approach. The first of these tests was a direct comparison of strict versus relaxed molecular clocks. In all three calibration scenarios, we observed a BF <20 in favor of relaxing the molecular clock: Fabre (BF ¼ 4.6), Perelman (BF ¼ 5.0), and Springer (BF ¼ 15). Therefore, we can confidently test hypotheses concerning the history of these herpes simplexviruses under the assumption of a strict molecular clock.

We performed a series of tests to determine whether we could reject scenarios in which the divergence of HSV-1 or HSV-2 from other simplexviruses represents a host divergence event. We added an additional constraint to the TMRCA of either HSV-1 or HSV-2 and the other Homininae simplexviruses corresponding to the estimated divergence events between H. sapiens and either P. troglodytes, G. beringei, or P. pygmaeus using a narrow calibration prior, according to the published dated primate phylogenies (Fabre et al. 2009; Perelman et al. 2011; Springer et al. 2012) . As in the molecular clock comparison, we assessed the support for models using BF based on MLEs obtained via a GSS approach. These BF values represent the comparisons of models with Macacaonly calibrations against models with Macaca and an additional Hominidae calibration. In order to prevent the poor phylogenetic signal for the placement of novel bonobo simplex virus and other viral sequence fragments, these calibrations were only placed on the ancestors of the partial-genome MBE sequences. We included P. pygmaeus divergence dates in this analysis, because even though an orangutan simplexvirus has never been characterized, the lineage leading to the Homininae simplexviruses could represent Pongo divergence.

Using the GSS framework, we can strongly reject nearly every scenario in which the divergence of HSV-2 and a Homininae simplexvirus corresponds to a host divergence event (BF >100; fig. 4 ; supplementary table 1, Supplementary Material online). The evidence for rejection was only moderately strong using the Fabre dates with wide calibration constraints for a scenario in which the most recent common ancestor of HSV-2 and the Homininae simplexviruses corresponded with divergence between H. sapiens and P. troglodytes (BF ¼ 91). Hence, it is unlikely that HSV-1 is of zoonotic origin.

The most consistently supported scenario is one in which the split between HSV-1 and the Homininae simplexviruses represents the divergence between H. sapiens and G. beringei ( fig. 4 ; BF <20). This scenario was never rejected using wide calibration priors ( fig. 4B ; BF <20). Although it was weakly rejected (BF ¼ 51) using the Perelman dates with narrow calibration priors, this scenario still had the highest MLE of any hypothesis tested using the Perelman dates with narrow calibration priors ( fig. 4A ).

When applying narrow calibration priors ( fig. 4A ), we can reject our previously hypothesized scenario (Wertheim et al. 2014) in which the most recent common ancestor of HSV-1 and Homininae simplexviruses corresponds with the divergence between H. sapiens and P. troglodytes (BF >100), except when using the Springer dates (BF ¼ 71). Similarly, when applying the narrow calibration priors, we can strongly reject this ancestor representing the split between H. sapiens and Pongo spp. (BF >100), except when using the Fabre dates (BF ¼ 74).

When applying wide calibration priors ( fig. 4B ), we cannot reject our previously hypothesized scenario in which the most recent common ancestor of HSV-1 and Homininae simplexviruses corresponds with the divergence between H. sapiens and P. troglodytes (BF <20). Similarly, we cannot reject that this ancestor corresponds with the divergence between H. sapiens and Pongo spp. using the Fabre calibrations (BF <20). There is less support for the Pongo codivergence scenario using the Perelman (BF ¼ 40) and Springer (BF ¼ 71) calibrations.

We report the discovery of herpes simplexviruses in wild members of three extant African great ape (Homininae) species: bonobo (P. paniscus), mountain gorilla (G. beringei beringei), and western chimpanzee (P. troglodytes verus). Phylogenetic analysis is consistent with each Homininae species being infected with its own specific herpes simplexvirus. Although the herpes simplexviruses in Homininae are all closely related, their phylogeny is not compatible with a pattern of strict codivergence. Rather the Homininae herpes simplexviruses appear to have been repeatedly transmitted between African great ape species. Molecular clock analysis supports a scenario in which HSV-1 arose via codivergence, with the preponderance of evidence favoring its divergence from other Homininae simplexvirus corresponding with the evolutionary divergence between H. sapiens and G. beringei lineages. HSV-2, in contrast, is likely of zoonotic origin and shares a common ancestor with a novel bonobo herpes simplexvirus in the last million years.

In our previous study into the origins of HSV-1 and HSV-2 (Wertheim et al. 2014), we constructed a hypothesis testing framework that implicitly assumed that the divergence between ChHV and either HSV-1 or HSV-2 corresponded with the divergence between P. troglodytes and H. sapiens. This framework led us to predict the existence of a novel gorilla herpes simplexvirus and that this virus would lie basal to both HSV-2 and ChHV on a phylogenetic tree. The latter half of this prediction proved to be incorrect. Rather, the more inclusive hypothesis testing framework implemented here suggests that the divergence between HSV-1 and other Homininae simplexviruses (including ChHV) more likely corresponds with the divergence between the genera Gorilla and Homo. Nonetheless, we cannot definitively reject scenarios in which this viral ancestor corresponds with the divergence between Homo and Pongo or Homo and Pan lineages.

The novel bonobo simplexvirus is most likely the closest relative to HSV-2, and these two viruses likely diverged within the past 1 My. The maximum likelihood phylogeny and the bulk of the posterior trees (!89%) from our Bayesian inference favor a scenario in which the viruses transmitted between H. sapiens and P. paniscus sometime after P. paniscus diverged from P. troglodytes around 2.1 Ma (Stone et al. 2010; Bjork et al. 2011 ). However, we cannot determine the directionality of transmission based on the phylogeny alone, and it is possible that the bonobo simplexvirus arose via reversezoonosis from a human ancestor. Further, this phylogenetic relationship is based on a single nonsynonymous synapomorphy, so we cannot discard a scenario in which Gorilla and Pan simplexviruses are monophyletic. Therefore, the specific precursor to modern humans that was involved in the transmission event or events remains ambiguous (Wertheim et al. 2014; Houldcroft et al. 2017) .

We accounted for uncertainty in divergence time estimates by separately exploring three different sets of published calibrations and by incorporating various levels of precision into these calibrations. This level of precision in our calibration priors (wide vs. narrow, see Materials and Methods) is then magnified by uncertainty in branch length and substitution rate inference inherent in our phylogenetic analysis. Nonetheless, the zoonotic origin of HSV-2, rather than HSV-1, is supported across all three sets of calibrations and both levels of precision.

We do not find evidence for the alternative scenario for the origins of the HSV-1 and HSV-2 in which the virus duplicated within an ancestral great ape species and each descendent species had two simplexviruses (as outlined and argued against in Wertheim et al. [2014] ). The duplication scenario predicts that the divergence between HSV-2 and another simplexvirus corresponds with a host divergence event, which our hypothesis testing does not support. The duplication Wertheim et al. . doi:10.1093/molbev/msab072 MBE scenario also predicts two divergent simplexviruses within all Homininae species. Although our sampling of simplexvirus diversity in Homininae is sparse, we found no indication of more than one simplexvirus infecting any African great species. If this sampling is robust, the phylogeny is consistent with a scenario in which Homininae simplexviruses have competitively excluded their predecessor after cross-species transmission. The serological similarity of these HSV-2 and the Homininae simplexviruses (Eberle 1992; Luebcke et al. 2006) may have prevented the persistence of closely related simplexviruses. Notably, we cannot exclude the possibility that some Homininae simplexviruses failed to diverge with their host species, leaving a vacant niche for a related simplexvirus to exploit (Johnson et al. 2003) . Nonetheless, the evolutionary distance between HSV-1 and HSV-2 may facilitate their ability to simultaneously infect different niches in the same human host.

Estimates of deep divergence times in viruses using phylogenetics can be biased by strong purifying selection, resulting in the underestimation of branch lengths in a phylogenetic tree (Wertheim and Kosakovsky Pond 2011; Aiewsakun and Katzourakis 2016). Our current approach avoided a reliance on molecular clock calibrations deeper in the primate phylogenetic tree. Rather, we calibrated the molecular clock using recent evidence of codivergence between five Macaca species and their simplexviruses. By calibrating evolutionary rates for Homininae viruses using the pattern of codivergence within the Macaca viruses, we were able to infer the evolutionary rate using the recent portion of the phylogeny unlikely to be biased by purifying selection. In this context, the estimated age of internal nodes deep in the phylogeny will not affect our rate estimates inferred under a strict molecular clock. If, however, the biases related to purifying selecting are present deeper in the Homininae viruses, the true TMRCA of HSV-2 and the nonhuman Homininae simplexviruses would shift even younger, favoring the scenario in which HSV-2 is the result of zoonotic transmission.

Our hypothesis testing framework used Hominidae divergence dates estimated from these same publications, rather than using speciation dates estimated from other sources. Alternate methods of dating the divergence between Hominidae species (e.g., slower pedigree-derived rates) would result in different absolute ages for the divergence events between primates (Langergraber et MBE and Moorjani 2020). However, our approach is robust to alternative dating schemes, because it relied on the internal consistency within a phylogeny, rather than absolute divergence times. We acknowledge that the deep divergence dates among primates provided by Perelman tend to be older than typically reported (dos Reis et al. 2018) ; however, the Macaca and Homininae calibrations employed here are medial compared with other published estimates. Given this potential for uncertainty, we note that a slight increase in the substitution rate in Homininae simplexviruses would tend to favor a Pan origin of HSV-2, whereas a somewhat slower substitution rate would favor a Pongo origin of HSV-2. Fecal samples have been a noninvasive, productive source of knowledge about many infectious agents of primate species, more or less irrespective of the agent's suspected cellular tropism (Liu et al. 2010; Sharp and Hahn 2011; Calvignac-Spencer et al. 2012) . Although the results of this first largescale fecal screening clearly suggest noninvasive detection of viruses capable of establishing latency in neural tissues (such as simplexviruses) is very challenging, it is indeed possible. Another potential source of simplexvirus nucleic acids might be brain material from necropsies performed on naturally deceased great apes in the wild, which is however a very rare resource (Hoffmann et al. 2017) . We anticipate that additional fecal and necropsy samples from gorillas, bonobos, chimpanzees, and potentially orangutans will further clarify the origin of human herpes simplexviruses.

In total, 1,273 fecal samples were collected at 19 sites in ten sub-Saharan countries from seven great ape species/subspecies (supplementary table 2, Supplementary Material online): Pan troglodytes ellioti (n ¼ 159), Pan troglodytes troglodytes (n ¼ 116), Pan troglodytes schweinfurthii (n ¼ 79), Pan troglodytes verus (n ¼ 420), Pan paniscus (n ¼ 135), Gorilla gorilla gorilla (n ¼ 148), Gorilla beringei beringei (n ¼ 216). Authorizations of sampling were obtained from responsible local and national authorities. Except for P. troglodytes verus from Taï National Park, P. paniscus from Salonga National Park and G. beringei beringei, feces were obtained opportunistically from unhabituated individuals. All unhabituated P. troglodytes fecal samples except those from Loango National Park were collected under the umbrella of the Pan African Programme: The Cultured Chimpanzee (Kuhl et al. 2019 ). We did not attempt to determine the number of unhabituated individuals that were sampled. All P. troglodytes verus samples from Taï National Park and a fraction of G. beringei beringei fecal samples were obtained from pregnant chimpanzees (n ¼ 222, representing 40 pregnancies by 27 individuals) and gorillas (n ¼ 98, representing ten pregnancies and gorillas), respectively, under the assumption that pregnancy may favor active herpetic disease and, following, virus shedding (De Nys et al. 2014 ). Finally, 24 G. beringei beringei samples were collected from eight individuals with skin lesions suggestive of herpetic disease. DNA was extracted using the Stool DNA Kit (Roboklon, Berlin, Germany). As part of our routine quality control, about 5% of the extracts were assessed for the presence of PCR inhibitors using an inhouse qPCR assay (Calvignac-Spencer et al. 2013) ; no inhibition was detected.

To screen DNA extracts for simplexvirus genetic material, we used a seminested PCR approach targeting short fragments of the glycoprotein B (gB; UL27) gene: the first round oligonucleotides-HS-1f 5 0 -gCRggAggTggACgAgATg-3 0 and HS-1r 5 0 -gCCAggTAgTACTgCRSCTg-3 0 -targeted a 225-bp fragment, within which a 160-bp fragment was targeted by the second round oligonucleotides-HS-2f-B9 5 0 -CSSCTCSTTCCgMTTCTC-3 0 and HS-2r 5 0 -SAYgTgCgTSSCgTTgTA-3 0 . PCRs were carried out in a total volume of 25 ml and seeded with 3 ml DNA extract (first round) or 1 ml of the first round PCR product diluted 40 times (second round). Reactions contained 0.2 mM dNTP (with dUTP replacing dTTP), 4 mM MgCl 2 , 0.2 lM of each primer, 1.25 U Platinum Taq Polymerase (Invitrogen), and 2.5 ll 10Â PCR Buffer (Invitrogen, Carlsbad, CA). Cycling condition were the same for the two rounds: 95 C 5 min, 40 cycles (95 C 30 s, 58 C 30 s, 72 C 30 s), and 72 C 10 min. In case a sample was positive with this assay, we tried to increase the gB sequence length by using two additional seminested PCR systems under the same conditions as aforementioned: 1) using first round oligonucleotides B3 (5 0 -TTCACCGTGGSCTGGGACTGG-3 0 ) and HS-1r (280 bp) and second round oligonucleotides B3 and HS-2r (260 bp), 2) using first round oligonucleotides HS-1f and B10m (5 0 -GAGGASGTGGTCTTGATGCGYTCCACG-3 0 ) (413bp) and HS-2f and B10m (380 bp); and we also tried to generate amplicons for another coding sequence (UL53) using a seminested PCR system which was also run as aforementioned, using the first round oligonucleotides UL53-f1 (5 0 -CCSGTSACCTTCYTGTACC-3 0 ) and UL53-r1 (5 0 -GCCKCTGRATCTCCTGYTCGTA-3 0 ) (390 bp) and the second round oligonucleotides UL53-f1 and UL53-r2 (5 0 -ATNCCSGASAGGATGATGGA-3 0 ) (310 bp). PCR products were cleaned up with ExoSAP-IT (Affymetrix, Santa Clara, CA) and sequenced in both directions according to Sanger's method using the BigDye Terminator kit v3.1 (Thermofischer, Waltham, MA). All chromatograms were evaluated using the software Geneious Prime (Biomatters Ltd., Auckland, New Zealand) (Kearse et al. 2012) .

PCR positive extracts were fragmented using a Covaris S220 Focused-Ultrasonicator in a total volume of 130 ml (filled with low EDTA TE buffer), using settings aiming to generate approximately 400-bp fragments (intensity ¼ 4, duty cycle ¼ 10%, cycles per burst ¼ 200, treatment time ¼ 55 s, temperature ¼ 7 C). Fragmented DNAs were then concentrated using a MinElute PCR purification kit and eluted into 2 Â 10 ml low EDTA TE buffer. DNA concentration was measured using a Qubit dsDNA High Sensitivity kit. About 1 lg DNA or all available remaining DNA extract were used for subsequent library preparation using the Accel-NGS 2S DNA library kit following the standard protocol and a sample specific unique index (Swift Biosciences, Ann Arbor, MI). Quantification was conducted using a KAPA HiFi Library Wertheim et al. . doi:10.1093/molbev/msab072 MBE Quantification Kit (Roche, Basel, Switzerland) and libraries were then amplified using a KAPA Hot Start Library Amplification Kit (Roche) and Illumina adapter specific primers (5 0 -AATGATACGGCGACCACCGA-3 0 and 5 0 -CAAGCAGAAGACGGCATACGA-3 0 ), 45 s at 98 C, variable number of cycles (15 s at 98 C, 30 s at 65 C, 45 s at 72 C), 1 min at 72 C. Following amplification libraries were requantified to ensure the desired amount of starting material for capture was available (>100 ng). Libraries were pooled per individual and separate hybridization capture reactions were set up. We designed 80mer RNA baits to span the RefSeq genomes of the three human alphaherpesviruses (HSV-1: NC_001806, HSV-2: NC_001798, human herpesvirus 3: NC_001348) with a 2Â tiling density, which we used following the Mybaits Sequence Enrichment for Targeted Sequencing protocol (Version 2.3.1; Arbor Biosciences, Ann Arbor, MI). Following an initial round of capture, capture products were quantified and reamplified to generate 100-500 ng starting material for a second round of capture. After reamplification, the final capture products were diluted to 4 nM and sequenced on an Illumina MiSeq (v3 2x300 Chemistry; Illumina, San Diego, CA).

Raw reads were filtered (adapter removal and quality filtering) using Trimmomatic (Bolger et al. 2014) with the following settings: LEADING: 30 TRAILING: 30 SLIDINGWINDOW: 4:30 MINLEN: 30. Filtered reads were imported in Geneious, where overlapping paired-end reads were first merged using BBmerge (Bushnell et al. 2017) . Merged and unmerged reads were then mapped onto the HSV-2 RefSeq genome, from which we had removed terminal inverted repeats, using the built-in mapper with default settings. The resulting map was exported and deduplicated using the SAMtools command rmdup (Li et al. 2009 ). The final map was imported in Geneious and used to call a consensus genome for which an unambiguous base call required that at least ten unique reads covered a position, of which 90% had to agree on the identified base. Coding sequences were identified in the consensus by similarity to coding sequences in the HSV-2 RefSeq genome, using a permissive 60% similarity threshold.

Herpes simplexvirus sequence data from Catarrhine species (including Homininae and Cercopithecidae) were downloaded from GenBank. Full-length viral genomes were available from ten host species: humans, Homo sapiens; vervet monkeys, Chlorocebus pygerythrus; chimpanzees, Pan troglodytes; crab-eating macaques, Macaca fascicularis; rhesus macaques, Macaca mulatta; southern pig-tailed macaques, Macaca nemestrina; lion-tailed macaques, Macaca silenus; chacma baboons, Papio ursinus; yellow baboons, Papio cynocephalus; and olive baboon, Papio anubis. We limited the number of HSV-1 and HSV-2 sequences to representatives of major clades (Burrel et al. 2017 ). In addition, we included three glycoprotein B (UL27) gene fragments from McHV-1 infecting Japanese macaques, Macaca fuscata.

Many of the McHV-1 and PaHV-2 sequences were obtained from captive animals that had been housed with related Macaca and Papio species. Therefore, we investigated the provenance of each virus to determine if 1) the host species could not be reliably determined due to cohousing of multiple species belonging to the same genus or 2) the virus detected in one species in captivity likely infected a different wild species. See Supplementary Material online  for a detailed description and supplementary table 3 , Supplementary Material online, for accession numbers and references.

We identified genes in the novel gorilla simplexvirus that contained at least 500 nonambiguous nucleotides and had homologous genes in both Homininae and Cercopithecidae simplexviruses. Homologous coding regions in these related viruses were defined according to their GenBank annotations. Sequences for each gene were aligned separately as translated amino acids in Muscle v3.8 in Aliview v1.23 (Edgar 2004; Larsson 2014) . We then added the partial glycoprotein B (UL27) viral sequences from Macaca fuscata (267 bp for two sequences and 1,944 bp for the other), P. troglodytes verus (124 bp), P. paniscus (124 bp), and G. beringei beringei (124 bp for two sequences and 365 bp for the other); glycoprotein K (UL53) sequence from two G. beringei beringei were also included (272 bp). Genes with known recombinant regions within alphaherpes simplexvirus (i.e., UL15, UL29, UL30, and UL39; Burrel et al. 2017) were excluded and the remaining 29 genes were concatenated into a single partial-genome alignment.

Recombination is pervasive in human herpes simplexviruses, including between HSV-1 and HSV-2 (Lamers et al. 2015; Burrel et al. 2017 ) and among PaHV-2 (Tyler and Severini 2006) . The genomic regions analyzed here were restricted to regions with high-sequencing coverage in the novel gorilla simplexvirus genome and known recombinant regions in HSV-2 (UL15, UL29, UL30, and UL39) were excised from the alignment (Burrel et al. 2017) . We screened the 74 taxon alignment for recombination using RDP4 and found no robust evidence for recombination between these viral lineages in the alignment. However, we cannot exclude the possibility of recombination between the novel gorilla simplexvirus and other Homininae simplexviruses in other genomic regions.

The final alignment included 74 taxa and, after removing recombinant regions and genes with <500-bp coverage in the novel gorilla simplexvirus, was 43,797 bp in length.

We inferred a simplexvirus maximum likelihood phylogenetic tree using IQTree v1.6.3 under a GTRþC 4 substitution model (Nguyen et al. 2015) . Node support was determined using 1,000 ultrafast bootstrap replicates (Hoang et al. 2018 

A test for diversifying selection was performed using BUSTED with synonymous site variation on the Datamonkey server Weaver et al. 2018; Wisotsky et al. 2020 ).

Bayesian phylogenetic inference was conducted using BEAST v1.10.4 . The bias in TMRCA estimation deep in viral phylogenies due to purifying selection has been previously demonstrated to strongly affect branch length estimation separating the major catarrhine simplexvirus lineages (Wertheim et al. 2014) ; however, the shallower divergences within this phylogeny were not affected. Therefore, we calibrated the molecular clock using divergence events in the McHV-1 clade. This calibration is based on the assumption of codivergence within the McHV-1 viruses and their corresponding five Macaca host species, because the McHV-1 phylogenetic relationships perfectly recapitulated those of their hosts. We identified three published studies on primate divergence times that included estimates for all four Macaca divergence events represented in the simplexvirus phylogeny: Fabre, Perelman, and Springer (supplementary fig. 4 , Supplementary Material online). These studies inferred Macaca TMRCAs using different molecular clock approaches based on different sources of external calibrations. Further, each of the studies also provided estimates for TMRCAs within Hominidae, which are necessary for subsequent hypothesis testing.

We explored two different sets of priors based on the estimated node ages from the literature: narrow (normal distribution with standard deviation of 1 Â 10 À4 ) and wide (normal distribution encompassing reported confidence intervals from the literature). We opted to use the reported node ages as prior distributions, rather than incorporate the fossil data itself, because of the sparsity of known primate simplexviruses relative to the number of corresponding primate fossils. Each of these six scenarios was run in duplicate for a Markov chain Monte Carlo (MCMC) length of 10 million generations, sampling ever 10,000 generations. We used a GTRþC 4 substitution model assuming empirical base frequencies in a birthdeath framework employing strict or relaxed (uncorrelated lognormal distribution) molecular clock. Convergence (effective sample sizes !200) and mixing were assessed in Tracer v1.7 , after removing the first 10% generations as burn-in. A maximum clade credibility (MCC) tree displaying median node heights was generated using TreeAnnotator. Subsampling of the posterior TMRCA distributions of HSV-2 and the novel bonobo simplexvirus based on monophyly and paraphyly was achieved using the ETE3 package (Huerta-Cepas et al. 2016 ).

A leave-one-out cross-validation sensitivity analysis was performed in order to assess the robustness of the four Macaca calibrations. Sensitivity was assessed on the TMRCAs between HSV-1/HSV-2 and the Homininae herpes viruses. As in the primary MCMC analysis, cross-validation was performed using both narrow and wide calibration priors.

We performed a series of hypothesis tests to determine whether the TMRCAs of Homininae simplexviruses and HSV-1 or HSV-2 corresponded to the TMRCA of H. sapiens and Pan, Gorilla, or Pongo. Even though we do not have evidence for the existence of an orangutan (Pongo) herpes simplexvirus, it has been hypothesized that the split between HSV and Homininae simplexviruses may actually represent this deeper codivergence event. To incorporate this testing into the MCMC framework, for each of the three published calibration schemes (Fabre, Perleman, and Springer) , we included additional calibration priors at the ancestor of either 1) HSV-1, HSV-2, ChHV, and the novel gorilla simplexvirus or 2) HSV-2, ChHV, and the novel gorilla simplexvirus. These priors were applied as normal distributions with narrow standard deviations (10 À4 ), as wider calibrations were too accommodating. Taxa with only partial-gene sequences were excluded from the calibration prior specifications but maintained in the phylogenetic analysis.

We also compared the marginal likelihood estimates between the strict and relaxed molecular clock analyses, as described above.

MLEs are intuitive estimations of model fit that are founded on probability (Oaks et al. 2019) . To compare the likelihood of each calibration set, we selected a GSS approach Baele et al. 2016 ), a streamlined development of path- (Lartillot and Philippe 2006) and stepping stone- (Xie et al. 2011 ) sampling which accommodates phylogenetic uncertainty (Baele et al. 2012) . We applied a burn-in of one million from the original MCMC, an MLE chain length of 250,000, and 75 path steps. Optimal chain length was determined through exploratory analysis on a single set of calibrations varying chain length (10,000-1,000,000) and path steps (50-150). Working priors from the MCMC were applied under the destination of the MLE. GSS analyses with joint, prior, or likelihood estimated sample size <200 or duplicate runs whose MLEs differed >15 BF, suggesting poor convergence, were repeated. Due to the variability of MLEs from duplicate runs (Baele et al. 2016 ), we used a significance threshold of 100 BF for establishing strong evidence for rejecting a model.

Supplementary data are available at Molecular Biology and Evolution online.

Time-dependent rate phenomenon in viruses

Improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty

Genealogical working distributions for Bayesian model testing with phylogenetic uncertainty

Evolutionary history of chimpanzees inferred from complete mitochondrial genomes

Trimmomatic: a flexible trimmer for Illumina sequence data

Ancient recombination events between human herpes simplex viruses

BBMerge -accurate paired shotgun read merging via overlap

Wild great apes as sentinels and sources of infectious disease

Carrion fly-derived DNA as a tool for comprehensive and cost-effective assessment of mammalian biodiversity

Evolution of the mutation rate across primates

The order Herpesvirales

Malaria parasite detection increases during pregnancy in wild chimpanzees

Tanglegrams are misleading for visual evaluation of tree congruence

Using phylogenomic data to explore the effects of relaxed clocks and calibration strategies on divergence time estimation: primates as a test case

Relaxed phylogenetics and dating with confidence

MERS-CoV spillover at the camel-human interface

17(th) century variola virus reveals the recent history of smallpox

Measles virus and rinderpest virus divergence dated to the sixth century BCE

Evidence for an alpha-herpesvirus indigenous to mountain gorillas

Sequence analysis of herpes simplex virus gB gene homologs of two platyrrhine monkey alpha-herpesviruses

Serological evidence for variation in the incidence of herpesvirus infections in different species of apes

MUSCLE: multiple sequence alignment with high accuracy and high throughput

Patterns of macroevolution among Primates inferred from a supermatrix of mitochondrial and nuclear DNA

Choosing among partition models in Bayesian phylogenetics

Pangaea and the out-of-Africa model of Varicella-Zoster virus evolution and phylogeography

UFBoot2: improving the ultrafast bootstrap approximation

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Evidence of the recombinant origin of a bat severe acute respiratory syndrome (SARS)-like coronavirus and its implications on the direct ancestor of SARS coronavirus

Multiple cross-species transmission events of human adenoviruses (HAdV) during hominine evolution

Migrating microbes: what pathogens can tell us about population movements and human evolution

ETE 3: reconstruction, analysis, and visualization of phylogenomic data

When do parasites fail to speciate in response to host speciation?

Novel Great Ape Herpes Simplexviruses

Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data

Human impact erodes chimpanzee behavioral diversity

Global diversity within and between human herpesvirus 1 and 2 glycoproteins

Generation times in wild chimpanzees and gorillas suggest earlier divergence times in great ape and human evolution

AliView: a fast and lightweight alignment viewer and editor for large datasets

Computing Bayes factors using thermodynamic integration

Fruit bats as reservoirs of Ebola virus

Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and SAMtools

Origin of the human malaria parasite Plasmodium falciparum in gorillas

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Isolation and characterization of a chimpanzee alphaherpesvirus

RDP4: detection and analysis of recombination patterns in virus genomes

Molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses

Gene-wide identification of episodic selection

Origins of the 1918 pandemic: revisiting the swine "Mixing Vessel" hypothesis

IQ-TREE: a fast and effective stochastic algorithm for estimating maximumlikelihood phylogenies

A genome-wide comparative evolutionary analysis of herpes simplex virus type 1 and varicella zoster virus

Marginal likelihoods in phylogenetics: a review of methods and applications

A molecular phylogeny of living primates

Catarrhine primate divergence dates estimated from complete mitochondrial genomes: concordance with fossil and nuclear DNA evidence

Posterior summarisation in Bayesian phylogenetics using Tracer 1.7

Pathogenicity of different baboon herpesvirus papio 2 isolates is characterized by either extreme neurovirulence or complete apathogenicity

Genome sequence of a chimpanzee herpesvirus and its relation to other primate alphaherpesviruses

Origins of HIV and the AIDS pandemic

Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic

Age-specific prevalence of infection with herpes simplex virus types 2 and 1: a global review

Macroevolutionary dynamics and historical biogeography of primate diversification inferred from a species supermatrix

More reliable estimates of divergence times in Pan using complete mtDNA sequences and accounting for population structure

Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10

The complete genome sequence of herpesvirus papio 2 (Cercopithecine herpesvirus 16) shows evidence of recombination events among various progenitor herpesviruses

Yersinia pestis and the plague of Justinian 541-543 AD: a genomic analysis

Datamonkey 2.0: a modern web application for characterizing selective and other evolutionary processes

Lessons from naked apes and their infections

Purifying selection can obscure the ancient age of viral lineages

Evolutionary origins of human herpes simplex viruses 1 and 2

Synonymous site-to-site substitution rate variation dramatically inflates false positive rates of selection analyses: ignore at your own peril

Improving marginal likelihood estimation for Bayesian phylogenetic model selection

Wittiger, and Klaus Zuberbuehler for their help with organizing the collection/collecting great ape fecal samples. We also thank Guy Baele and Philippe Lemey for their guidance on performing and optimizing the generalized stepping stone analysis. This work was supported by the United States National Institutes of Health (NIAID R21AI115701 and R01AI135992) and the German Research Council projects LE1813/14-1 (Great Ape Health in Tropical Africa).

Raw reads and assembled sequences are available at the European Nucleotide Archive under study accession PRJEB39611. Alignments, trees, and XML input files can be accessed on Data Dryad at https://datadryad.org/stash/dataset/doi:10.6076/D13S3X.