Oesophagostomum stephanostomum causing parasitic granulomas in wild chimpanzees (Pan troglodytes verus) of Taï National Park, Côte d'Ivoire.

Nematodes belonging to the genus Oesophagostomum frequently infect wild chimpanzees (Pan troglodytes) across widely separated field sites. Nodular lesions (granulomas) containing Oesophagostomum are commonly seen in the abdomen of infected chimpanzees post-mortem. At Taï National Park, Côte d'Ivoire, previous studies have identified larvae of a variety of Oesophagostomum spp. in wild chimpanzee stool, based on sequencing of larval DNA, and nodular lesions associated with Oesophagostomum, identified morphologically to the genus level but not sequenced. Here we present three recent cases of parasitic granulomas found post-mortem in chimpanzees at Taï. We complement descriptions of gross pathology, histopathology and parasitology with PCR and sequencing of DNA isolated from the parasitic nodules and from adult worms found inside the nodules. In all three cases, we identify Oesophagostomum stephanostomum as the causative agent. The sequences from this study were identical to the only other published sequences from nodules in nonhuman primates-those from the wild chimpanzees of Gombe, Tanzania.

Evolutionary Insight into the Association between New Jersey Polyomavirus and Humans.

Advances in viral discovery techniques have led to the identification of numerous novel viruses in human samples. However, the low prevalence of certain viruses in humans raises doubts about their association with our species. To ascertain the authenticity of a virus as a genuine human-infecting agent, it can be useful to investigate the diversification of its lineage within hominines, the group encompassing humans and African great apes. Building upon this rationale, we examined the case of the New Jersey polyomavirus (NJPyV; Alphapolyomavirus terdecihominis), which has only been detected in a single patient thus far. In this study, we obtained and analyzed sequences from closely related viruses infecting all African great ape species. We show that NJPyV nests within the diversity of these viruses and that its lineage placement is compatible with an ancient origin in humans, despite its apparent rarity in human populations.

The African Network for Improved Diagnostics, Epidemiology and Management of common infectious Agents.

BACKGROUND: In sub-Saharan Africa, acute respiratory infections (ARI), acute gastrointestinal infections (GI) and acute febrile disease of unknown cause (AFDUC) have a large disease burden, especially among children, while respective aetiologies often remain unresolved. The need for robust infectious disease surveillance to detect emerging pathogens along with common human pathogens has been highlighted by the ongoing novel coronavirus disease 2019 (COVID-19) pandemic. The African Network for Improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA) is a sentinel surveillance study on the aetiology and clinical characteristics of ARI, GI and AFDUC in sub-Saharan Africa. METHODS: ANDEMIA includes 12 urban and rural health care facilities in four African countries (Côte d'Ivoire, Burkina Faso, Democratic Republic of the Congo and Republic of South Africa). It was piloted in 2018 in Côte d'Ivoire and the initial phase will run from 2019 to 2021. Case definitions for ARI, GI and AFDUC were established, as well as syndrome-specific sampling algorithms including the collection of blood, naso- and oropharyngeal swabs and stool. Samples are tested using comprehensive diagnostic protocols, ranging from classic bacteriology and antimicrobial resistance screening to multiplex real-time polymerase chain reaction (PCR) systems and High Throughput Sequencing. In March 2020, PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and analysis of full genomic information was included in the study. Standardised questionnaires collect relevant clinical, demographic, socio-economic and behavioural data for epidemiologic analyses. Controls are enrolled over a 12-month period for a nested case-control study. Data will be assessed descriptively and aetiologies will be evaluated using a latent class analysis among cases. Among cases and controls, an integrated analytic approach using logistic regression and Bayesian estimation will be employed to improve the assessment of aetiology and associated risk factors. DISCUSSION: ANDEMIA aims to expand our understanding of ARI, GI and AFDUC aetiologies in sub-Saharan Africa using a comprehensive laboratory diagnostics strategy. It will foster early detection of emerging threats and continued monitoring of important common pathogens. The network collaboration will be strengthened and site diagnostic capacities will be reinforced to improve quality management and patient care.

Genome-wide analysis of 944 133 individuals provides insights into the etiology of haemorrhoidal disease.

OBJECTIVE: Haemorrhoidal disease (HEM) affects a large and silently suffering fraction of the population but its aetiology, including suspected genetic predisposition, is poorly understood. We report the first genome-wide association study (GWAS) meta-analysis to identify genetic risk factors for HEM to date. DESIGN: We conducted a GWAS meta-analysis of 218 920 patients with HEM and 725 213 controls of European ancestry. Using GWAS summary statistics, we performed multiple genetic correlation analyses between HEM and other traits as well as calculated HEM polygenic risk scores (PRS) and evaluated their translational potential in independent datasets. Using functional annotation of GWAS results, we identified HEM candidate genes, which differential expression and coexpression in HEM tissues were evaluated employing RNA-seq analyses. The localisation of expressed proteins at selected loci was investigated by immunohistochemistry. RESULTS: We demonstrate modest heritability and genetic correlation of HEM with several other diseases from the GI, neuroaffective and cardiovascular domains. HEM PRS validated in 180 435 individuals from independent datasets allowed the identification of those at risk and correlated with younger age of onset and recurrent surgery. We identified 102 independent HEM risk loci harbouring genes whose expression is enriched in blood vessels and GI tissues, and in pathways associated with smooth muscles, epithelial and endothelial development and morphogenesis. Network transcriptomic analyses highlighted HEM gene coexpression modules that are relevant to the development and integrity of the musculoskeletal and epidermal systems, and the organisation of the extracellular matrix. CONCLUSION: HEM has a genetic component that predisposes to smooth muscle, epithelial and connective tissue dysfunction.

Monkeypox virus emergence in wild chimpanzees reveals distinct clinical outcomes and viral diversity.

Monkeypox is a viral zoonotic disease on the rise across endemic habitats. Despite the growing importance of monkeypox virus, our knowledge on its host spectrum and sylvatic maintenance is limited. Here, we describe the recent repeated emergence of monkeypox virus in a wild, human-habituated western chimpanzee (Pan troglodytes verus, hereafter chimpanzee) population from Taï National Park, Ivory Coast. Through daily monitoring, we show that further to causing its typical exanthematous syndrome, monkeypox can present itself as a severe respiratory disease without a diffuse rash. By analysing 949 non-invasively collected samples, we identify the circulation of at least two distinct monkeypox virus lineages and document the shedding of infectious particles in faeces and flies, suggesting that they could mediate indirect transmission. We also show that the carnivorous component of the Taï chimpanzees' diet, mainly consisting of the sympatric monkeys they regularly hunt, did not change nor shift towards rodent consumption (the presumed reservoir) before the outbreaks, suggesting that the sudden emergence of monkeypox virus in this population is probably due to changes in the ecology of the virus itself. Using long-term mortality surveillance data from Taï National Park, we provide evidence of little to no prior viral activity over at least two decades. We conclude that great ape sentinel systems devoted to the longitudinal collection of behavioural and health data can help clarify the epidemiology and clinical presentation of zoonotic pathogens.

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.

Cytomegalovirus distribution and evolution in hominines.

Herpesviruses are thought to have evolved in very close association with their hosts. This is notably the case for cytomegaloviruses (CMVs; genus Cytomegalovirus) infecting primates, which exhibit a strong signal of co-divergence with their hosts. Some herpesviruses are however known to have crossed species barriers. Based on a limited sampling of CMV diversity in the hominine (African great ape and human) lineage, we hypothesized that chimpanzees and gorillas might have mutually exchanged CMVs in the past. Here, we performed a comprehensive molecular screening of all 9 African great ape species/subspecies, using 675 fecal samples collected from wild animals. We identified CMVs in eight species/subspecies, notably generating the first CMV sequences from bonobos. We used this extended dataset to test competing hypotheses with various degrees of co-divergence/number of host switches while simultaneously estimating the dates of these events in a Bayesian framework. The model best supported by the data involved the transmission of a gorilla CMV to the panine (chimpanzee and bonobo) lineage and the transmission of a panine CMV to the gorilla lineage prior to the divergence of chimpanzees and bonobos, more than 800,000 years ago. Panine CMVs then co-diverged with their hosts. These results add to a growing body of evidence suggesting that viruses with a double-stranded DNA genome (including other herpesviruses, adenoviruses, and papillomaviruses) often jumped between hominine lineages over the last few million years.

CD4 receptor diversity in chimpanzees protects against SIV infection.

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

Bushmeat Hunting and Zoonotic Transmission of Simian T-Lymphotropic Virus 1 in Tropical West and Central Africa.

Simian T-lymphotropic virus 1 (STLV-1) enters human populations through contact with nonhuman primate (NHP) bushmeat. We tested whether differences in the extent of contact with STLV-1-infected NHP bushmeat foster regional differences in prevalence of human T-lymphotropic virus 1 (HTLV-1). Using serological and PCR assays, we screened humans and NHPs at two Sub-Saharan African sites where subsistence hunting was expected to be less (Taï region, Côte d'Ivoire [CIV]) or more (Bandundu region, Democratic Republic of the Congo [DRC]) developed. Only 0.7% of human participants were infected with HTLV-1 in CIV (n = 574), and 1.3% of humans were infected in DRC (n = 302). Two of the Ivorian human virus sequences were closely related to simian counterparts, indicating ongoing zoonotic transmission. Multivariate analysis of human demographic parameters and behavior confirmed that participants from CIV were less often exposed to NHPs than participants from DRC through direct contact, e.g., butchering. At the same time, numbers of STLV-1-infected NHPs were higher in CIV (39%; n = 111) than in DRC (23%; n = 39). We conclude that similar ultimate risks of zoonotic STLV-1 transmission-defined as the product of prevalence in local NHP and human rates of contact to fresh NHP carcasses-contribute to the observed comparable rates of HTLV-1 infection in humans in CIV and DRC. We found that young adult men and mature women are most likely exposed to NHPs at both sites. In view of the continued difficulties in controlling zoonotic disease outbreaks, the identification of such groups at high risk of NHP exposure may guide future prevention efforts.IMPORTANCE Multiple studies report a high risk for zoonotic transmission of blood-borne pathogens like retroviruses through contact with NHPs, and this risk seems to be particularly high in tropical Africa. Here, we reveal high levels of exposure to NHP bushmeat in two regions of Western and Central tropical Africa. We provide evidence for continued zoonotic origin of HTLV-1 in humans at CIV, and we found that young men and mature women represent risk groups for zoonotic transmission of pathogens from NHPs. Identifying such risk groups can contribute to mitigation of not only zoonotic STLV-1 transmission but also transmission of any blood-borne pathogen onto humans in Sub-Saharan Africa.

Assessing the feasibility of fly based surveillance of wildlife infectious diseases.

Monitoring wildlife infectious agents requires acquiring samples suitable for analyses, which is often logistically demanding. A possible alternative to invasive or non-invasive sampling of wild-living vertebrates is the use of vertebrate material contained in invertebrates feeding on them, their feces, or their remains. Carrion flies have been shown to contain vertebrate DNA; here we investigate whether they might also be suitable for wildlife pathogen detection. We collected 498 flies in Taï National Park, Côte d'Ivoire, a tropical rainforest and examined them for adenoviruses (family Adenoviridae), whose DNA is frequently shed in feces of local mammals. Adenoviral DNA was detected in 6/142 mammal-positive flies. Phylogenetic analyses revealed that five of these sequences were closely related to sequences obtained from local non-human primates, while the sixth sequence was closely related to a murine adenovirus. Next-generation sequencing-based DNA-profiling of the meals of the respective flies identified putative hosts that were a good fit to those suggested by adenoviral sequence affinities. We conclude that, while characterizing the genetic diversity of wildlife infectious agents through fly-based monitoring may not be cost-efficient, this method could probably be used to detect the genetic material of wildlife infectious agents causing wildlife mass mortality in pristine areas.

Genome Sequences of Polyomaviruses from the Wild-Living Red Colobus (Piliocolobus badius) and Western Chimpanzee (Pan troglodytes verus).

We identified with PCR and sequencing the full genomes of the recently discovered Pan troglodytes verus polyomavirus 8 and Piliocolobus badius polyomavirus 2 in a western chimpanzee and a western red colobus free-ranging in Taï National Park of Côte d'Ivoire.

Assessing Host-Virus Codivergence for Close Relatives of Merkel Cell Polyomavirus Infecting African Great Apes.

UNLABELLED: It has long been hypothesized that polyomaviruses (PyV; family Polyomaviridae) codiverged with their animal hosts. In contrast, recent analyses suggested that codivergence may only marginally influence the evolution of PyV. We reassess this question by focusing on a single lineage of PyV infecting hominine hosts, the Merkel cell polyomavirus (MCPyV) lineage. By characterizing the genetic diversity of these viruses in seven African great ape taxa, we show that they exhibit very strong host specificity. Reconciliation analyses identify more codivergence than noncodivergence events. In addition, we find that a number of host and PyV divergence events are synchronous. Collectively, our results support codivergence as the dominant process at play during the evolution of the MCPyV lineage. More generally, our results add to the growing body of evidence suggesting an ancient and stable association of PyV and their animal hosts. IMPORTANCE: The processes involved in viral evolution and the interaction of viruses with their hosts are of great scientific interest and public health relevance. It has long been thought that the genetic diversity of double-stranded DNA viruses was generated over long periods of time, similar to typical host evolutionary timescales. This was also hypothesized for polyomaviruses (family Polyomaviridae), a group comprising several human pathogens, but this remains a point of controversy. Here, we investigate this question by focusing on a single lineage of polyomaviruses that infect both humans and their closest relatives, the African great apes. We show that these viruses exhibit considerable host specificity and that their evolution largely mirrors that of their hosts, suggesting that codivergence with their hosts played a major role in their diversification. Our results provide statistical evidence in favor of an association of polyomaviruses and their hosts over millions of years.

Genetic identification of cytomegaloviruses in a rural population of Côte d'Ivoire.

BACKGROUND: Cytomegaloviruses (CMVs) are herpesviruses that infect many mammalian species, including humans. Infection generally passes undetected, but the virus can cause serious disease in individuals with impaired immune function. Human CMV (HCMV) is circulating with high seroprevalence (60-100 %) on all continents. However, little information is available on HCMV genoprevalence and genetic diversity in subsaharan Africa, especially in rural areas of West Africa that are at high risk of human-to-human HCMV transmission. In addition, there is a potential for zoonotic spillover of pathogens through bushmeat hunting and handling in these areas as shown for various retroviruses. Although HCMV and nonhuman CMVs are regarded as species-specific, potential human infection with CMVs of non-human primate (NHP) origin, shown to circulate in the local NHP population, has not been studied. FINDINGS: Analysis of 657 human oral swabs and fecal samples collected from 518 individuals living in 8 villages of Côte d'Ivoire with generic PCR for identification of human and NHP CMVs revealed shedding of HCMV in 2.5 % of the individuals. Determination of glycoprotein B sequences showed identity with strains Towne, AD169 and Toledo, respectively. NHP CMV sequences were not detected. CONCLUSIONS: HCMV is actively circulating in a proportion of the rural Côte d'Ivoire human population with circulating strains being closely related to those previously identified in non-African countries. The lack of NHP CMVs in human populations in an environment conducive to cross-species infection supports zoonotic transmission of CMVs to humans being at most a rare event.

Genome Sequence of a Central Chimpanzee-Associated Polyomavirus Related to BK and JC Polyomaviruses, Pan troglodytes troglodytes Polyomavirus 1.

We amplified and sequenced the genome of a polyomavirus infecting a central chimpanzee (Pan troglodytes troglodytes). This virus, which is closely related to BK and JC polyomaviruses, may help shed a new light on these human pathogens' evolutionary history.

Phylogenomic evidence for recombination of adenoviruses in wild gorillas.

Human adenoviruses (HAdVs) of species Human mastadenovirus B (HAdV-B) are genetically highly diverse and comprise several pathogenic types. AdVs closely related to members of HAdV-B infect African great apes and the evolutionary origin of HAdV-B has recently been determined in ancient gorillas. Genetic evidence for intra- and inter-species recombination has been obtained for AdVs of humans and captive great apes, but evidence from wild great apes is lacking. In this study, potential HAdV-B members of wild Eastern gorillas were analysed for evidence of recombination. One near-complete genome was amplified from primary sample material and sequenced, and from another six individuals genome fragments were obtained. In phylogenomic analysis, their penton base, pVII-pVI, hexon and fiber genes were compared with those of all publicly available HAdV-B full-genome sequences of humans and captive great apes. Evidence for intra-species recombination between different HAdV-B members of wild gorillas as well as between HAdV-B members of chimpanzees and gorillas was obtained. Since zoonotic AdVs have been reported to cause respiratory outbreaks in both humans and monkeys, and humans in West and Central Africa frequently hunt and butcher primates thereby increasing the chance of zoonotic transmission, such HAdV-B recombinants might widen the pool of potential human pathogens.

Adenovirus in Rural Côte D'Ivoire: High Diversity and Cross-Species Detection.

The Taï region in Western Côte d'Ivoire is characterized by extensive overlap of human and animal habitats. This could influence patterns of adenovirus transmission between humans and domestic animals. Fecal samples from humans and various domestic animals were tested for the presence of adenoviruses by PCR. Phylogenetic and species delineation analyses were performed to further characterize the adenoviruses circulating in the region and to identify potential cross-species transmission events. Among domestic animals, adenovirus shedding was frequent (21.6% of domestic mammals and 41.5% of chickens) and the detected strains were highly diverse, several of them representing novel types. Although no evidence for zoonotic transmission of animal adenovirus was obtained, the present study provides concordant evidence in favor of common cross-species transmission of adenoviruses between different animal species and first indications for adenovirus transmission from humans to animals. These findings underline the thus far underestimated importance of reverse zoonotic transmission of viruses and of the role of domestic animals as pathogen reservoirs, "bridge species," or intermediate hosts.

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.

The ecology of primate retroviruses - an assessment of 12 years of retroviral studies in the Taï national park area, Côte d׳Ivoire.

The existence and genetic make-up of most primate retroviruses was revealed by studies of bushmeat and fecal samples from unhabituated primate communities. For these, detailed data on intra- and within-species contact rates are generally missing, which makes identification of factors influencing transmission a challenging task. Here we present an assessment of 12 years of research on primate retroviruses in the Taï National Park area, Côte d'Ivoire. We discuss insights gained into the prevalence, within- and cross-species transmission of primate retroviruses (including towards local human populations) and the importance of virus-host interactions in determining cross-species transmission risk. Finally we discuss how retroviruses ecology and evolution may change in a shifting environment and identify avenues for future research.

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.