Performing monkeys of Bangladesh: characterizing their source and genetic variation.

The acquisition and training of monkeys to perform is a centuries-old tradition in South Asia, resulting in a large number of rhesus macaques kept in captivity for this purpose. The performing monkeys are reportedly collected from free-ranging populations, and may escape from their owners or may be released into other populations. In order to determine whether this tradition involving the acquisition and movement of animals has influenced the population structure of free-ranging rhesus macaques in Bangladesh, we first characterized the source of these monkeys. Biological samples from 65 performing macaques collected between January 2010 and August 2013 were analyzed for genetic variation using 716 base pairs of mitochondrial DNA. Performing monkey sequences were compared with those of free-ranging rhesus macaque populations in Bangladesh, India and Myanmar. Forty-five haplotypes with 116 (16 %) polymorphic nucleotide sites were detected among the performing monkeys. As for the free-ranging rhesus population, most of the substitutions (89 %) were transitions, and no indels (insertion/deletion) were observed. The estimate of the mean number of pair-wise differences for the performing monkey population was 10.1264 ± 4.686, compared to 14.076 ± 6.363 for the free-ranging population. Fifteen free-ranging rhesus macaque populations were identified as the source of performing monkeys in Bangladesh; several of these populations were from areas where active provisioning has resulted in a large number of macaques. The collection of performing monkeys from India was also evident.

A Seminomadic Population in Bangladesh with Extensive Exposure to Macaques Does Not Exhibit High Levels of Zoonotic Simian Foamy Virus Infection.

Simian foamy viruses (SVF) are ubiquitous in nonhuman primates (NHP). SFV can be zoonotically transmitted to humans who either work with or live commensally with NHP. We analyzed the blood of 45 Bangladeshi performing monkey owners (an ethnic group called the Bedey) for SFV infection. Surprisingly, a PCR assay failed to detect SFV infection in any of these participants. This is in contrast to our previously reported infection rate of about 5% among Bangladeshi villagers.

Diversity and molecular phylogeny of mitochondrial DNA of rhesus macaques (Macaca mulatta) in Bangladesh.

While studies of rhesus macaques (Macaca mulatta) in the eastern (e.g., China) and western (e.g., India) parts of their geographic range have revealed major genetic differences that warrant the recognition of two different subspecies, little is known about genetic characteristics of rhesus macaques in the transitional zone extending from eastern India and Bangladesh through the northern part of Indo-China, the probable original homeland of the species. We analyzed genetic variation of 762 base pairs of mitochondrial DNA from 86 fecal swab samples and 19 blood samples from 25 local populations of rhesus macaque in Bangladesh collected from January 2010 to August 2012. These sequences were compared with those of rhesus macaques from India, China, and Myanmar. Forty-six haplotypes defined by 200 (26%) polymorphic nucleotide sites were detected. Estimates of gene diversity, expected heterozygosity, and nucleotide diversity for the total population were 0.9599 ± 0.0097, 0.0193 ± 0.0582, and 0.0196 ± 0.0098, respectively. A mismatch distribution of paired nucleotide differences yielded a statistically significantly negative value of Tajima's D, reflecting a population that rapidly expanded after the terminal Pleistocene. Most haplotypes throughout regions of Bangladesh, including an isolated region in the southwestern area (Sundarbans), clustered with haplotypes assigned to the minor haplogroup Ind-2 from India reflecting an east to west dispersal of rhesus macaques to India. Haplotypes from the southeast region of Bangladesh formed a cluster with those from Myanmar, and represent the oldest rhesus macaque haplotypes of Bangladesh. These results are consistent with the hypothesis that rhesus macaques first entered Bangladesh from the southeast, probably from Indo-China, then dispersed westward throughout eastern and central India.

A novel Bayesian method for detection of APOBEC3-mediated hypermutation and its application to zoonotic transmission of simian foamy viruses.

Simian Foamy Virus (SFV) can be transmitted from non-human primates (NHP) to humans. However, there are no documented cases of human to human transmission, and significant differences exist between infection in NHP and human hosts. The mechanism for these between-host differences is not completely understood. In this paper we develop a new Bayesian approach to the detection of APOBEC3-mediated hypermutation, and use it to compare SFV sequences from human and NHP hosts living in close proximity in Bangladesh. We find that human APOBEC3G can induce genetic changes that may prevent SFV replication in infected humans in vivo.

Simian foamy virus infection of rhesus macaques in Bangladesh: relationship of latent proviruses and transcriptionally active viruses.

Simian foamy viruses (SFV) are complex retroviruses that are ubiquitous in nonhuman primates (NHP) and are zoonotically transmitted to humans, presumably through NHP saliva, by licking, biting, and other behaviors. We have studied SFV in free-ranging rhesus macaques in Bangladesh. It has been previously shown that SFV in immunocompetent animals replicates to detectable levels only in superficial epithelial cells of the oral mucosa, although latent proviruses are found in most, if not all, tissues. In this study, we compare DNA sequences from latent SFV proviruses found in blood cells of 30 Bangladesh rhesus macaques to RNA sequences of transcriptionally active SFV from buccal swabs obtained from the same animals. Viral strains, defined by differences in SFV gag sequences, from buccal mucosal specimens overlapped with those from blood samples in 90% of animals. Thus, latent proviruses in peripheral blood mononuclear cells (PBMC) are, to a great extent, representative of viruses likely to be transmitted to other hosts. The level of SFV RNA in buccal swabs varied greatly between macaques, with increasing amounts of viral RNA in older animals. Evidence of APOBEC3-induced mutations was found in gag sequences derived from the blood and oral mucosa.

Naturally acquired picornavirus infections in primates at the Dhaka zoo.

The conditions in densely populated Bangladesh favor picornavirus transmission, resulting in a high rate of infection in the human population. Data suggest that nonhuman primates (NHP) may play a role in the maintenance and transmission of diverse picornaviruses in Bangladesh. At the Dhaka Zoo, multiple NHP species are caged in close proximity. Their proximity to other species and to humans, both zoo workers and visitors, provides the potential for cross-species transmission. To investigate possible interspecies and intraspecies transmission of picornaviruses among NHP, we collected fecal specimens from nine NHP taxa at the Dhaka Zoo at three time points, August 2007, January 2008, and June 2008. Specimens were screened using real-time PCR for the genera Enterovirus, Parechovirus, and Sapelovirus, and positive samples were typed by VP1 sequencing. Fifty-two picornaviruses comprising 10 distinct serotypes were detected in 83 fecal samples. Four of these serotypes, simian virus 19 (SV19), baboon enterovirus (BaEV), enterovirus 112 (EV112), and EV115, have been solely associated with infection in NHP. EV112, EV115, and SV19 accounted for 88% of all picornaviruses detected. Over 80% of samples from cages housing rhesus macaques, olive baboons, or hamadryas baboons were positive for a picornavirus, while no picornaviruses were detected in samples from capped langurs or vervet monkeys. In contrast to our findings among synanthropic NHP in Bangladesh where 100% of the picornaviruses detected were of human serotypes, in the zoo population, only 15% of picornaviruses detected in NHP were of human origin. Specific serotypes tended to persist over time, suggesting either persistent infection of individuals or cycles of reinfection.

Characterizing the picornavirus landscape among synanthropic nonhuman primates in Bangladesh, 2007 to 2008.

The term synanthropic describes organisms that thrive in human-altered habitats. Where synanthropic nonhuman primates (NHP) share an ecological niche with humans, cross-species transmission of infectious agents can occur. In Bangladesh, synanthropic NHP are found in villages, densely populated cities, religious sites, and protected forest areas. NHP are also kept as performing monkeys and pets. To investigate possible transmission of enteric picornaviruses between humans and NHP, we collected fecal specimens from five NHP taxa at16 locations in Bangladesh during five field sessions, from January 2007 to June 2008. Specimens were screened using real-time PCR assays for the genera Enterovirus, Parechovirus, and Sapelovirus; PCR-positive samples were typed by VP1 sequencing. To compare picornavirus diversity between humans and NHP, the same assays were applied to 211 human stool specimens collected in Bangladesh in 2007 to 2008 for acute flaccid paralysis surveillance. Picornaviruses were detected in 78 of 677 (11.5%) NHP fecal samples. Twenty distinct human enterovirus (EV) serotypes, two bovine EV types, six human parechovirus serotypes, and one virus related to Ljungan virus were identified. Twenty-five additional enteroviruses and eight parechoviruses could not be typed. Comparison of the picornavirus serotypes detected in NHP specimens with those detected in human specimens revealed considerable overlap. Strikingly, no known simian enteroviruses were detected among these NHP populations. In conclusion, enteroviruses and parechoviruses may be transmitted between humans and synanthropic NHP in Bangladesh, but the directionality of transmission is unknown. These findings may have important implications for the health of both human and NHP populations.

Population dynamics of rhesus macaques and associated foamy virus in Bangladesh.

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses.

Zoonotic simian foamy virus in Bangladesh reflects diverse patterns of transmission and co-infection.

Simian foamy viruses (SFVs) are ubiquitous in non-human primates (NHPs). As in all retroviruses, reverse transcription of SFV leads to recombination and mutation. Because more humans have been shown to be infected with SFV than with any other simian borne virus, SFV is a potentially powerful model for studying the virology and epidemiology of viruses at the human/NHP interface. In Asia, SFV is likely transmitted to humans through macaque bites and scratches that occur in the context of everyday life. We analyzed multiple proviral sequences from the SFV gag gene from both humans and macaques in order to characterize retroviral transmission at the human/NHP interface in Bangladesh. Here we report evidence that humans can be concurrently infected with multiple SFV strains, with some individuals infected by both an autochthonous SFV strain as well as a strain similar to SFV found in macaques from another geographic area. These data, combined with previous results, suggest that both human-facilitated movement of macaques leading to the introduction of non-resident strains of SFV and retroviral recombination in macaques contribute to SFV diversity among humans in Bangladesh.

Influenza virus infection in nonhuman primates.

To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza.

From the mouths of monkeys: detection of Mycobacterium tuberculosis complex DNA from buccal swabs of synanthropic macaques.

Although the Mycobacterium tuberculosis complex (MTBC) infects a third of all humans, little is known regarding the prevalence of mycobacterial infection in nonhuman primates (NHP). For more than a century, tuberculosis has been regarded as a serious infectious threat to NHP species. Advances in the detection of MTBC open new possibilities for investigating the effects of this poorly understood pathogen in diverse populations of NHP. Here, we report results of a cross-sectional study using well-described molecular methods to detect a nucleic acid sequence (IS6110) unique to the MTBC. Sample collection was focused on the oral cavity, the presumed route of transmission of MTBC. Buccal swabs were collected from 263 macaques representing 11 species in four Asian countries and Gibraltar. Contexts of contact with humans included free ranging, pets, performing monkeys, zoos, and monkey temples. Following DNA isolation from buccal swabs, the polymerase chain reaction (PCR) amplified IS6110 from 84 (31.9%) of the macaques. In general, prevalence of MTBC DNA was higher among NHP in countries where the World Health Organization reports higher prevalence of humans infected with MTBC. This is the first demonstration of MTBC DNA in the mouths of macaques. Further research is needed to establish the significance of this finding at both the individual and population levels. PCR of buccal samples holds promise as a method to elucidate the mycobacterial landscape among NHP, particularly macaques that thrive in areas of high human MTBC prevalence.

Naturally acquired Mycobacterium tuberculosis complex in laboratory pig-tailed macaques.

Here we present a case series from a primate research facility. The index case, a 4-year-old pig-tailed macaque (Macaca nemestrina) experimentally infected with chimeric simian-human immunodeficiency virus (SHIVSF162 P4), developed weight loss and was euthanized. Based on necropsy results the animal was diagnosed with opportunistic atypical mycobacteriosis associated with simian AIDS (SAIDS). Subsequently, tissues from the index animal, as well as tissues and oral mucosal swabs from six SHIV-infected contacts, were analyzed using molecular methods and found to contain nucleic acid sequences characteristic of Mycobacterium tuberculosis complex (MTBC). These data suggest that existing protocols fail to reliably detect MTBC infection in laboratory primates used as experimental models.

Diverse contexts of zoonotic transmission of simian foamy viruses in Asia.

In Asia, contact between persons and nonhuman primates is widespread in multiple occupational and nonoccupational contexts. Simian foamy viruses (SFVs) are retroviruses that are prevalent in all species of nonhuman primates. To determine SFV prevalence in humans, we tested 305 persons who lived or worked around nonhuman primates in several South and Southeast Asian countries; 8 (2.6%) were confirmed SFV positive by Western blot and, for some, by PCR. The interspecies interactions that likely resulted in virus transmission were diverse; 5 macaque taxa were implicated as a potential source of infection. Phylogenetic analysis showed that SFV from 3 infected persons was similar to that from the nonhuman primate populations with which the infected persons reported contact. Thus, SFV infections are likely to be prevalent among persons who live or work near nonhuman primates in Asia.

Unique pattern of enzootic primate viruses in Gibraltar macaques.

Because Gibraltar's macaques (Macaca sylvanus) have frequent contact with humans, we assayed 79 macaques for antibodies to enzootic primate viruses. All macaques were seronegative for herpesvirus B, simian T-cell lymphotropic virus, simian retrovirus, simian immunodeficiency virus, and rhesus cytomegalovirus. Seroprevalence of simian foamy virus reached 88% among adult animals.

Sensitive assays for simian foamy viruses reveal a high prevalence of infection in commensal, free-ranging Asian monkeys.

Foamy viruses (FV) are retroviruses that naturally infect many hosts, including most nonhuman primates (NHPs). Zoonotic infection by primate FV has been documented in people in Asia who reported contact with free-ranging macaques. FV transmission in Asia is a concern, given abundant human-NHP contact, particularly at monkey temples and in urban settings. We have developed three assays capable of detecting the presence of FV in Asian NHP species that are commensal with humans: enzyme-linked immunosorbent assay (ELISA), Western blot assays using recombinant viral Gag protein, and an indicator cell line that can detect macaque FV. The recombinant ELISA correlates very well with the presence of FV sequences detected by PCR. We have used these assays to demonstrate both that FV is highly prevalent among free-ranging NHPs and that seroconversion occurs at a young age in these animals. These assays should also prove useful for large-scale analysis of the prevalence of FV infections in human populations in Asia that are commensal with free-ranging NHPs.

Exposure to human respiratory viruses among urban performing monkeys in Indonesia.

Performing monkeys, a common phenomena in Asia, occupy a unique urban niche that comprises a number of factors influencing the likelihood of cross-species transmission of pathogens. Here we present the first documented evidence of exposure to measles, rubella, and parainfluenza in a population of performing monkeys. Evidence of exposure to these endemic human respiratory viruses in the performing monkeys confirms human-to-primate transmission and suggests the possibility of primate-to-human transmission. Urban animal markets, the likely source of these performing monkeys, may represent an environment conducive to the mixing of animals and pathogens, making these monkeys a potential conduit for infectious agents passing from a variety of animals found in animal markets to humans. The potential significance of these results to human public health and the unique contexts of disease transmission associated with the urban ecology of the performance monkeys are discussed. Given the level of overseas travel, this potential threat is not confined solely to Asia.

Considering human-primate transmission of measles virus through the prism of risk analysis.

Measles is a respiratory virus that is endemic to humans. Human-nonhuman primate (NHP) transmission of the measles virus has been shown to cause significant morbidity and mortality in NHP populations. We investigated serological evidence of exposure to measles virus in two free-ranging populations of macaques at the Bukit Timah (BTNR) and Central Catchment Nature (CCNR) reserves in Singapore and the Swoyambhu Temple in Katmandu, Nepal. At BTNR/CCNR none of the 38 macaques (Macaca fascicularis) sampled were seropositive for antibodies to measles virus. In contrast, at Swoyambhu 100% (n = 39) of the macaques (M. mulatta) sampled were seropositive for antibodies to the measles virus. Here the contrasting seroprevalences of the two sites are analyzed using risk analysis. These case studies show how risk analysis can be used to approach the phenomenon of cross-species pathogen transmission.

Disease risk analysis: A paradigm for using health-based data to inform primate conservation and public health.

Risk analysis is a multidisciplinary process used to evaluate existing knowledge in order to prioritize risks associated with the spread of disease. A principle aim of risk analysis is to facilitate the development of cost-effective management strategies. Risk analysis calls for a multidisciplinary approach to piece together and integrate the numerous factors that influence disease transmission. The seven papers included in this volume of AJP present current primatological research as viewed through the prism of risk analysis. Issues such as interspecies disease transmission, public health, and conservation of endangered species are addressed, and risk analysis is put forward as a possible paradigm to promote understanding of infectious disease and its impact on nonhuman primate and human populations.

Temple monkeys and health implications of commensalism, Kathmandu, Nepal.

The threat of zoonotic transmission of infectious agents at monkey temples highlights the necessity of investigating the prevalence of enzootic infectious agents in these primate populations. Biological samples were collected from 39 rhesus macaques at the Swoyambhu Temple and tested by enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction, or combination of these tests for evidence of infection with rhesus cytomegalovirus (RhCMV), Cercopithecine herpesvirus 1 (CHV-1), simian virus 40 (SV40), simian retrovirus (SRV), simian T-cell lymphotropic virus (STLV), simian immunodeficiency virus (SIV), and simian foamy virus (SFV). Antibody seroprevalence was 94.9% to RhCMV (37/39), 89.7% to SV40 (35/39), 64.1% to CHV-1 (25/39), and 97.4% to SFV (38/39). Humans who come into contact with macaques at Swoyambhu risk exposure to enzootic primateborne viruses. We discuss implications for public health and primate management strategies that would reduce contact between humans and primates.