Novel Polyomaviruses in Mammals from Multiple Orders and Reassessment of Polyomavirus Evolution and Taxonomy.

As the phylogenetic organization of mammalian polyomaviruses is complex and currently incompletely resolved, we aimed at a deeper insight into their evolution by identifying polyomaviruses in host orders and families that have either rarely or not been studied. Sixteen unknown and two known polyomaviruses were identified in animals that belong to 5 orders, 16 genera, and 16 species. From 11 novel polyomaviruses, full genomes could be determined. Splice sites were predicted for large and small T antigen (LTAg, STAg) coding sequences (CDS) and examined experimentally in transfected cell culture. In addition, splice sites of seven published polyomaviruses were analyzed. Based on these data, LTAg and STAg annotations were corrected for 10/86 and 74/86 published polyomaviruses, respectively. For 25 polyomaviruses, a spliced middle T CDS was observed or predicted. Splice sites that likely indicate expression of additional, alternative T antigens, were experimentally detected for six polyomaviruses. In contrast to all other mammalian polyomaviruses, three closely related cetartiodactyl polyomaviruses display two introns within their LTAg CDS. In addition, the VP2 of Glis glis (edible dormouse) polyomavirus 1 was observed to be encoded by a spliced transcript, a unique experimental finding within the Polyomaviridae family. Co-phylogenetic analyses based on LTAg CDS revealed a measurable signal of codivergence when considering all mammalian polyomaviruses, most likely driven by relatively recent codivergence events. Lineage duplication was the only other process whose influence on polyomavirus evolution was unambiguous. Finally, our analyses suggest that an update of the taxonomy of the family is required, including the creation of novel genera of mammalian and non-mammalian polyomaviruses.

Primates, Lice and Bacteria: Speciation and Genome Evolution in the Symbionts of Hominid Lice.

Insects with restricted diets rely on symbiotic bacteria to provide essential metabolites missing in their diet. The blood-sucking lice are obligate, host-specific parasites of mammals and are themselves host to symbiotic bacteria. In human lice, these bacterial symbionts supply the lice with B-vitamins. Here, we sequenced the genomes of symbiotic and heritable bacterial of human, chimpanzee, gorilla, and monkey lice and used phylogenomics to investigate their evolutionary relationships. We find that these symbionts have a phylogenetic history reflecting the louse phylogeny, a finding contrary to previous reports of symbiont replacement. Examination of the highly reduced symbiont genomes (0.53-0.57 Mb) reveals much of the genomes are dedicated to vitamin synthesis. This is unchanged in the smallest symbiont genome and one that appears to have been reorganized. Specifically, symbionts from human lice, chimpanzee lice, and gorilla lice carry a small plasmid that encodes synthesis of vitamin B5, a vitamin critical to the bacteria-louse symbiosis. This plasmid is absent in an old world monkey louse symbiont, where this pathway is on its primary chromosome. This suggests the unique genomic configuration brought about by the plasmid is not essential for symbiosis, but once obtained, it has persisted for up to 25 My. We also find evidence that human, chimpanzee, and gorilla louse endosymbionts have lost a pathway for synthesis of vitamin B1, whereas the monkey louse symbiont has retained this pathway. It is unclear whether these changes are adaptive, but they may point to evolutionary responses of louse symbionts to shifts in primate biology.

Lineage-Specific Changes in Biomarkers in Great Apes and Humans.

Although human biomedical and physiological information is readily available, such information for great apes is limited. We analyzed clinical chemical biomarkers in serum samples from 277 wild- and captive-born great apes and from 312 healthy human volunteers as well as from 20 rhesus macaques. For each individual, we determined a maximum of 33 markers of heart, liver, kidney, thyroid and pancreas function, hemoglobin and lipid metabolism and one marker of inflammation. We identified biomarkers that show differences between humans and the great apes in their average level or activity. Using the rhesus macaques as an outgroup, we identified human-specific differences in the levels of bilirubin, cholinesterase and lactate dehydrogenase, and bonobo-specific differences in the level of apolipoprotein A-I. For the remaining twenty-nine biomarkers there was no evidence for lineage-specific differences. In fact, we find that many biomarkers show differences between individuals of the same species in different environments. Of the four lineage-specific biomarkers, only bilirubin showed no differences between wild- and captive-born great apes. We show that the major factor explaining the human-specific difference in bilirubin levels may be genetic. There are human-specific changes in the sequence of the promoter and the protein-coding sequence of uridine diphosphoglucuronosyltransferase 1 (UGT1A1), the enzyme that transforms bilirubin and toxic plant compounds into water-soluble, excretable metabolites. Experimental evidence that UGT1A1 is down-regulated in the human liver suggests that changes in the promoter may be responsible for the human-specific increase in bilirubin. We speculate that since cooking reduces toxic plant compounds, consumption of cooked foods, which is specific to humans, may have resulted in relaxed constraint on UGT1A1 which has in turn led to higher serum levels of bilirubin in humans.

Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: Evidence of Trichomonadidae infections.

Sexually transmitted diseases (STDs) can persist endemically, are known to cause sterility and infant mortality in humans, and could have similar impacts in wildlife populations. African apes (i.e., chimpanzees, bonobos, and to a lesser extent gorillas) show multi-male mating behavior that could offer opportunities for STD transmission, yet little is known about the prevalence and impact of STDs in this endangered primate group. We used serology and PCR-based detection methods to screen biological samples from wild and orphaned eastern chimpanzees and gorillas (N = 172 individuals, including adults, and juveniles) for four classes of pathogens that either commonly cause human STDs or were previously detected in captive apes: trichomonads, Chlamydia spp., Treponema pallidum (syphilis and yaws), and papillomaviruses. Based on results from prior modeling and comparative research, we expected STD prevalence to be highest in females versus males and in sexually mature versus immature individuals. All samples were negative for Chlamydia, Treponema pallidum, and papillomaviruses; however, a high percentage of wild chimpanzee urine and fecal samples showed evidence of trichomonads (protozoa). Analysis revealed that females were more likely than males to have positive urine-but not fecal-samples; however, there was no evidence of age (sexual maturity) differences in infection status. Sequence analysis of chimpanzee trichomonad samples revealed a close relationship to previously described trichomonads within the genus Tetratrichomonas. Phylogenetic comparisons to archived sequences from multiple vertebrate hosts suggests that many of the chimpanzee parasites from our study are likely transmitted via fecal-oral contact, but the transmission of some Tetratrichomonas sequence-types remains unknown and could include sexual contact. Our work emphasizes that only a fraction of infectious agents affecting wild apes are presently known to science, and that further work on great ape STDs could offer insights for the management of endangered great apes and for understanding human STD origins.

Multiple Cross-Species Transmission Events of Human Adenoviruses (HAdV) during Hominine Evolution.

Human adenoviruses (HAdV; species HAdV-A to -G) are highly prevalent in the human population, and represent an important cause of morbidity and, to a lesser extent, mortality. Recent studies have identified close relatives of these viruses in African great apes, suggesting that some HAdV may be of zoonotic origin. We analyzed more than 800 fecal samples from wild African great apes and humans to further investigate the evolutionary history and zoonotic potential of hominine HAdV. HAdV-B and -E were frequently detected in wild gorillas (55%) and chimpanzees (25%), respectively. Bayesian ancestral host reconstruction under discrete diffusion models supported a gorilla and chimpanzee origin for these viral species. Host switches were relatively rare along HAdV evolution, with about ten events recorded in 4.5 My. Despite presumably rare direct contact between sympatric populations of the two species, transmission events from gorillas to chimpanzees were observed, suggesting that habitat and dietary overlap may lead to fecal-oral cross-hominine transmission of HAdV. Finally, we determined that two independent HAdV-B transmission events to humans occurred more than 100,000 years ago. We conclude that HAdV-B circulating in humans are of zoonotic origin and have probably affected global human health for most of our species lifetime.

High prevalence and diversity of species D adenoviruses (HAdV-D) in human populations of four Sub-Saharan countries.

BACKGROUND: Human adenoviruses of species D (HAdV-D) can be associated with acute respiratory illness, epidemic keratoconjunctivitis, and gastroenteritis, but subclinical HAdV-D infections with prolonged shedding have also been observed, particularly in immunocompromised hosts. To expand knowledge on HAdV-D in Sub-Saharan Africa, we investigated the prevalence, epidemiology and pathogenic potential of HAdV-D in humans from rural areas of 4 Sub-Saharan countries, Côte d'Ivoire (CI), Democratic Republic of the Congo (DRC), Central African Republic (CAR) and Uganda (UG). METHODS: Stool samples were collected from 287 people living in rural regions in CI, DRC, CAR and UG. HAdV-D prevalence and diversity were determined by PCR and sequencing. A gene block, spanning the genes pV to hexon, was used for analysis of genetic distance. Correlation between adenovirus infection and disease symptoms, prevalence differences, and the effect of age and gender on infection status were analyzed with cross tables and logistic regression models. RESULTS: The prevalence of HAdV-D in the investigated sites was estimated to be 66% in CI, 48% in DRC, 28% in CAR (adults only) and 65% in UG (adults only). Younger individuals were more frequently infected than adults; there was no difference in HAdV-D occurrence between genders. No correlation could be found between HAdV-D infection and clinical symptoms. Highly diverse HAdV-D sequences were identified, among which a number are likely to stand for novel types. CONCLUSIONS: HAdV-D was detected with a high prevalence in study populations of 4 Sub-Saharan countries. The genetic diversity of the virus was high and further investigations are needed to pinpoint pathological potential of each of the viruses. High diversity may also favor the emergence of recombinants with altered tropism and pathogenic properties.

Novel polyomaviruses of nonhuman primates: genetic and serological predictors for the existence of multiple unknown polyomaviruses within the human population.

Polyomaviruses are a family of small non-enveloped DNA viruses that encode oncogenes and have been associated, to greater or lesser extent, with human disease and cancer. Currently, twelve polyomaviruses are known to circulate within the human population. To further examine the diversity of human polyomaviruses, we have utilized a combinatorial approach comprised of initial degenerate primer-based PCR identification and phylogenetic analysis of nonhuman primate (NHP) polyomavirus species, followed by polyomavirus-specific serological analysis of human sera. Using this approach we identified twenty novel NHP polyomaviruses: nine in great apes (six in chimpanzees, two in gorillas and one in orangutan), five in Old World monkeys and six in New World monkeys. Phylogenetic analysis indicated that only four of the nine chimpanzee polyomaviruses (six novel and three previously identified) had known close human counterparts. To determine whether the remaining chimpanzee polyomaviruses had potential human counterparts, the major viral capsid proteins (VP1) of four chimpanzee polyomaviruses were expressed in E. coli for use as antigens in enzyme-linked immunoassay (ELISA). Human serum/plasma samples from both Côte d'Ivoire and Germany showed frequent seropositivity for the four viruses. Antibody pre-adsorption-based ELISA excluded the possibility that reactivities resulted from binding to known human polyomaviruses. Together, these results support the existence of additional polyomaviruses circulating within the human population that are genetically and serologically related to existing chimpanzee polyomaviruses.

Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses.

The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC-MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC-MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC-MS is a valuable tool for the validation of immunoassays.

Novel adenoviruses in wild primates: a high level of genetic diversity and evidence of zoonotic transmissions.

Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events.