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

Full Article Figures & data References Citations Metrics Licensing Reprints & Permissions PDF AbstractSimian 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.Keywords: Bangladeshemerging infectious diseasesretroviral recombinationsimian foamy viruszoonotic transmission IntroductionFoamy viruses (FVs) are a subfamily of retroviruses that are widely distributed in vertebrate hosts, including non-human primates, cats, cows and horses.1Meiering CD, Linial ML.Historical perspective of foamy virus epidemiology and infection. Clin Microbiol Rev2001;14:165–176. [Crossref], [Web of Science ®], [Google Scholar] FVs are ancient exogenous viruses that are efficiently transmitted between hosts but are not known to cause disease.2Switzer WM, Salemi M, Shanmugam Vet al.Ancient co-speciation of simian foamy viruses and primates. Nature2005;434:376–380. [Crossref], [Web of Science ®], [Google Scholar] Although nearly ubiquitous in non-human primates (NHPs), simian foamy virus (SFV) is not endemic in Homo sapiens. Zoonotic infection of humans with SFV has been previously detected in several countries and in various contexts of interspecies contact including NHP laboratory and zoo workers in North America, ‘monkey temple’ workers, pet owners and people living around free-ranging macaques in South and Southeast Asia and bushmeat hunters in Central Africa.3Gessain A, Rua R, Betsem E, Turpin J, Mahieux R.HTLV-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, cross-species transmission and molecular virology. Virology2013;435:187–199. [Crossref], [Google Scholar]To date, NHP-to-human transmission of SFV has been documented in >100 people, far more than the number of cross-species transmission events for the other known NHP-borne retroviruses: simian immunodeficiency virus, simian retrovirus D or simian T-cell lymphotrophic virus.3Gessain A, Rua R, Betsem E, Turpin J, Mahieux R.HTLV-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, cross-species transmission and molecular virology. Virology2013;435:187–199. [Crossref], [Google Scholar],4Khabbaz RF, Heneine W, George JRet al.Infection of a laboratory worker with simian immunodeficiency virus. N Engl J Med1994;330:172–177. [Crossref], [Google Scholar],5Lerche NW, Switzer WM, Yee JLet al.Evidence of infection with simian type D retrovirus in persons occupationally exposed to nonhuman primates. J Virol2001;75:1783–1789. [Crossref], [Google Scholar],6Switzer WM, Bhullar V, Shanmugam Vet al.Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates. J Virol2004;78:2780–2789. [Crossref], [Web of Science ®], [Google Scholar],7Kalish ML, Wolfe ND, Ndongmo CBet al.Central African hunters exposed to simian immunodeficiency virus. Emerg Infect Dis2005;11:1928–1930. [Crossref], [Web of Science ®], [Google Scholar] Given the high prevalence of SFV in NHP and the diversity and extent of the interface between humans and NHP, it is estimated that globally the number of infected individuals may reach the tens of thousands.8Engel G, Hungerford LL, Jones-Engel Let al.Risk assessment: a model for predicting cross-species transmission of simian foamy virus from macaques (M. fascicularis) to humans at a monkey temple in Bali, Indonesia. Am J Primatol2006;68:934–948. [Crossref], [Google Scholar],9Switzer WM, Shaohua T, Ahuka-Mundeke Set al.Novel simian foamy virus infections from multiple monkey species in women from the Democratic Republic of Congo. Retrovirology2012;9:100. [Crossref], [Google Scholar] Additionally, the few longitudinal studies of SFV infected humans suggest that SFV is not transmitted from one person to another.10Boneva RS, Switzer WM, Spira TJet al.Clinical and virological characterization of persistent human infection with simian foamy viruses. AIDS Res Hum Retroviruses2007;23:1330–1337. [Crossref], [Web of Science ®], [Google Scholar] As such, characterization of the factors that influence zoonotic transmission of SFV may provide the best contemporary model for understanding how and why some NHP viruses are capable of causing sustained human-to-human transmission.Our group has studied the interface between humans and NHP in Bangladesh over the past 6 years, focusing on cross-species transmission of infectious agents.11Oberste MS, Feeroz MM, Maher Ket al.Characterizing the picornavirus landscape among synanthropic nonhuman primates in Bangladesh, 2007–2008. J Virol2013;87:558–571. [Crossref], [Google Scholar],12Oberste MS, Feeroz MM, Maher Ket al.Naturally acquired picornavirus infections in nonhuman primates at the Dhaka Zoo. J Virol2013;87:572–580. [Crossref], [Google Scholar],13Karlsson EA, Engel GA, Feeroz MMet al.Influenza virus infection in nonhuman primates. Emerg Infect Dis2012;18:1672–1675. [Crossref], [Web of Science ®], [Google Scholar] Bangladesh is one of the world’s most densely populated countries, with many urban areas located within or near NHP habitats. Several NHP taxa are native to Bangladesh, with the rhesus macaque (Macaca mulatta) the most numerous and widely distributed.14Hasan K, Aziz MA, Alam SMet al.Distribution of rhesus macaques (Macaca mulatta) in Bangladesh: inter-population variation in group size and composition. Primate Conserv2013;26:115–124. [Crossref], [Google Scholar] As forest habitat continues to dwindle, the small, ecologically flexible rhesus thrives in human-altered habitats, particularly in areas where cultural attitudes encourage their tolerance.15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] While contact between humans and rhesus macaques occurs primarily in communities where the macaques are free-ranging, it also occurs at religious shrines that have become refuges for macaques and through macaque pet ownership. In addition, a nomadic people, known variously as Bade, or Qalandar, have traveled the region for centuries, making a livelihood by staging performances with trained macaques. Unlike Africa, hunting of monkeys for bushmeat is rare in Bangladesh, being confined to indigenous peoples in the Chittagong Hill Tracts areas that border Myanmar.16Chowdhury MSH, Halim M, Miah Met al.Research communication: biodiversity use through harvesting faunal resources from forests by the Mro tribe in the Chittagong Hill Tracts, Bangladesh. Int J Biodiversity Sci Manage2007;3:56–62. [Taylor & Francis Online], [Google Scholar]NHP-to-human transmission of SFV is thought to occur most commonly through bites. SFV replicates actively in the oral mucosae of infected monkeys, achieving high concentrations in saliva, where genomic and mRNAs can readily be detected.17Murray SM, Picker LJ, Axthelm MK, Linial ML.Expanded tissue targets for foamy virus replication with simian immunodeficiency virus-induced immunosuppression. J Virol2006;80:663–670. [Crossref], [Google Scholar] However, relatively little is known concerning how host or viral characteristics influence SFV transmission. Even severe bites do not reliably result in infection.9Switzer WM, Shaohua T, Ahuka-Mundeke Set al.Novel simian foamy virus infections from multiple monkey species in women from the Democratic Republic of Congo. Retrovirology2012;9:100. [Crossref], [Google Scholar] Conversely, some humans with documented infection do not recall significant exposure to NHP or NHP tissue.Other important questions regarding zoonotic SFV transmission and infection remain unresolved. For example, while proviral DNA is detected persistently over decades in some SFV infected humans; other individuals develop antibodies to SFV without detectable proviral DNA.3Gessain A, Rua R, Betsem E, Turpin J, Mahieux R.HTLV-3/4 and simian foamy retroviruses in humans: discovery, epidemiology, cross-species transmission and molecular virology. Virology2013;435:187–199. [Crossref], [Google Scholar] It remains to be discovered whether some individuals clear infection, or whether certain viral strains are more capable of persistence. Further research is needed to determine if, analogous to SIV/HIV, in which recombination between retroviral strains in a novel host was a precursor event to the evolution of pandemic viruses, there is evidence that SFVs recombine in human hosts. Few studies have been conducted to determine whether or not the concentration of SFV in saliva influences zoonotic transmission, or how the human immune response facilitates or prevents infection or continued viremia (Soliven et al. and Matsen et al., unpublished). And while data collected in other geographic areas and other contexts of human–macaque contact have provided insight into how demographic variables and macaque and human behavior contribute to the likelihood of macaque-on-human aggression, relatively little is known about how these variables influence retroviral transmission.18Fuentes A, Gamerl S.Disproportionate participation by age/sex classes in aggressive interactions between long-tailed macaques (Macaca fascicularis) and human tourists at Padangtegal monkey forest, Bali, Indonesia. Am J Primatol2005;66:197–204. [Crossref], [Google Scholar]Here we present data describing human exposures to rhesus macaques and infection with SFV at several sites in Bangladesh and in performing monkey owners. Most other studies have focused on a region of the pol gene (integrase), which is highly conserved and thus may not reveal SFV variation found in infected individuals of a species. We have obtained multiple clones from whole blood and sequenced a portion of the SFV gag gene, chosen over pol to reveal greater SFV genetic variation, and used the sequence data for comparison of the virus found in humans to that found in macaques with which they interact. We show that humans can be concurrently infected by multiple SFV strains; some individuals are infected by both an autochthonous (originating where found) SFV strain as well as a strain similar to SFV found in macaques from other geographic areas. Increasing age was a significant risk factor for zoonotic transmission of SFV in humans. Female sex and Hindu religion may also be associated with increased risk. These data combined with our previous results15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] suggest that human-facilitated movement of macaques, multiple SFV infections and retroviral recombination all contribute to retroviral diversity in Bangladesh.MATERIALS AND METHODSStudy sites and populationsThe study population included 209 human subjects sampled at five community sites (Sylhet, Bormi, Dhamrai, Narayanganj and Charmaguria), as well as 13 performing monkey owners belonging to a nomadic group who stage performances featuring their trained macaques. The community sites were established in 2007 in locales where macaques range freely and where residents were willing to participate in longitudinal research on cross-species transmission of infectious agents. We sampled the performing monkey owners opportunistically, when we encountered them en route to or from our community study sites. An analysis of the SFV diversity among monkeys from Bangladesh is presented in our companion study.15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] The data from that study also form part of the macaque SFV data set investigated in the present study. Here we present a brief description of the field sites (Figure 1) included in this study. Zoonotic simian foamy virus in Bangladesh reflects diverse patterns of transmission and co-infectionAll authorsGregory A Engel, Christopher T Small, Khanh Soliven, Mostafa M Feeroz, Xiaoxing Wang, M Kamrul Hasan, Gunwha Oh, SM Rabiul Alam, Karen L Craig, Dana L Jackson, Frederick A Matsen IV, Maxine L Linial & Lisa Jones-Engelhttps://doi.org/10.1038/emi.2013.60Published online:25 January 2019Figure 1 Sampling locations and diversity of SFV strains detected in zoonotically infected human subjects. Human subjects were interviewed and sampled at five urban sites throughout Bangladesh where rhesus macaques were free-ranging. We have recently shown that at each of these sites a core strain(s) of SFV can be detected in the macaques.15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] The rhesus SFV core strains are color-coded on this map. For human subjects (denoted by ‘BGH’) who were found to be infected with SFV, we obtained multiple clones from whole blood and sequenced a portion of the SFV gag gene. The sequence data were used to compare the virus found in humans to that found in macaques from that site. The color of the circle(s) next to each of the BGH identifiers signifies the SFV strain that was detected in these humans. Note that four of the subjects were concurrently infected by multiple SFV strains and that some individuals are infected by both an autochthonous (originating where found) SFV strain as well as a strain similar to SFV found in macaques from other geographic areas. nks, no known source.Display full sizeFigure 1 Sampling locations and diversity of SFV strains detected in zoonotically infected human subjects. Human subjects were interviewed and sampled at five urban sites throughout Bangladesh where rhesus macaques were free-ranging. We have recently shown that at each of these sites a core strain(s) of SFV can be detected in the macaques.15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] The rhesus SFV core strains are color-coded on this map. For human subjects (denoted by ‘BGH’) who were found to be infected with SFV, we obtained multiple clones from whole blood and sequenced a portion of the SFV gag gene. The sequence data were used to compare the virus found in humans to that found in macaques from that site. The color of the circle(s) next to each of the BGH identifiers signifies the SFV strain that was detected in these humans. Note that four of the subjects were concurrently infected by multiple SFV strains and that some individuals are infected by both an autochthonous (originating where found) SFV strain as well as a strain similar to SFV found in macaques from other geographic areas. nks, no known source.SylhetLocated in the northeastern part of the country, within 35 km of the border with India’s Assam province, Sylhet is the third largest city in Bangladesh. A population of approximately 125 macaques is found at the Shah Jalal shrine in the center of city. Sylhet’s macaques range over the shrine site and into the immediately adjacent neighborhood. The macaques of Shah Jalal are regularly provisioned by the shrine staff and often receive food from visitors to the shrine. Fourteen human subjects who live close to the shrine participated in the study.BormiIn the town of Bormi, approximately 50 km north of Dhaka, more than 200 rhesus macaques range throughout the village, moving freely between Hindu and Muslim areas, often entering homes in search of food. Although some Bormi residents occasionally offer food to the macaques, there is no organized provisioning. A total of 105 human subjects from Bormi participated in this study.DhamraiThe town of Dhamrai is located 40 km southwest of Bormi. Approximately 75–100 macaques range freely through Dhamrai. The macaques favor areas with large shade and fruiting trees, which are more commonly found in Hindu neighborhoods. As in Bormi, there is no organized provisioning of macaques. Nine subjects from Dhamrai were included in this dataset.NarayanganjNarayanganj is a densely populated port city approximately 20 km south of Dhaka. The rhesus macaque population continues to decline in this area as urban development displaces green spaces. The estimated 60 macaques in this city are not provisioned and are not well habituated to humans. Thirty-six human subjects from Narayanganj participated in the study.CharmaguriaCharmaguria is located 150 km south/southwest of Dhaka along the Padma River. The >200 macaques in this area are provisioned by a local organization at a central location, and abundant green areas in this small town provide natural corridors along which the animals range widely. Forty-five human subjects from Charmaguria participated in the study.Recruitment, informed consent and sampling protocols, including follow-up of SFV positive individuals, have been approved by the University of Washington’s Human Subjects Review Board (23055) and an analogous review board at Jahangirnagar University, Bangladesh. Biological and demographic data from human subjects were coded with unique, anonymous identifiers that began with ‘BanGladesh Human’ (BGH). At each community site, Bangladeshi team members initiated contact with neighborhood leaders and identified a central location within the macaques’ range for data collection. Bangladeshi team members then partnered with community residents and fanned out on foot, requesting the participation of people who identified themselves as having been bitten or injured by macaques. Inclusion criteria for the subjects included age greater than 18, self-reported exposure to NHP and/or long-term (>20 years) residence in the village. Written informed consent was obtained from all subjects. Demographic data and data describing NHP exposures were acquired through a questionnaire administered by a team member in the local language, Bengali. Ten milliliters of whole blood was collected from each subject’s antecubital vein in a tube containing ethylene diamine tetraacetic acid, stored on ice for not more than 12 h, then centrifuged. Whole blood and plasma aliquots were stored at −80 °C until analyzed. Whatman Sterile Omni Swabs (Whatman, Piscataway, NJ, USA) were used to acquire buccal mucosal samples. Swabs were immediately placed in 500 µL of Qiagen Buffer RLT (Qiagen, Valencia, CA, USA) cooled on ice for not more than 12 h before being frozen at −80 °C until analyzed. These methods are essentially identical to those we used to acquire and store biological samples derived from macaques.15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar]Repeat sampling of confirmed SFV positive humansIf a human subject was confirmed SFV positive by polymerase chain reaction (PCR), the research team attempted to contact and resample the subject approximately 1 year after the initial sampling. The biological sample collection protocols for follow-up sampling of the SFV positive subjects were the same used during the initial sampling. In addition, SFV positive subjects received a brief physical exam and an in-depth medical history was collected.Rhesus macaque samplingOur companion manuscript for this project15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] provides all of the animal sampling and laboratory protocols. SFV gag sequences from a total of 184 rhesus macaques are included in the present study (Figure 2). We also include in this dataset sequences obtained from four rhesus, two from Dhamrai and two from Bormi, that we were able to resample two years after their initial sampling. Zoonotic simian foamy virus in Bangladesh reflects diverse patterns of transmission and co-infectionAll authorsGregory A Engel, Christopher T Small, Khanh Soliven, Mostafa M Feeroz, Xiaoxing Wang, M Kamrul Hasan, Gunwha Oh, SM Rabiul Alam, Karen L Craig, Dana L Jackson, Frederick A Matsen IV, Maxine L Linial & Lisa Jones-Engelhttps://doi.org/10.1038/emi.2013.60Published online:25 January 2019Figure 2 Blood strain classifications for monkeys from sampling populations. For each monkey, a color bar corresponding to the monkeys sampling population is presented in (A), and colored tiles corresponding to strains observed within that monkey’s blood sample shown in (B). The non-core strains are labeled according to recombinant relationships established with parental strains. Positive matches are defined by at least 90% likelihood for 200 or more contiguous base pairs in a cBrother analysis. Regions of the genome matching no parental strains above this threshold for at least 200 nt were marked as nks (no known source). This is a simplified version of a figure from the companion paper for this study,15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] but with updated strain classifications.Display full sizeFigure 2 Blood strain classifications for monkeys from sampling populations. For each monkey, a color bar corresponding to the monkeys sampling population is presented in (A), and colored tiles corresponding to strains observed within that monkey’s blood sample shown in (B). The non-core strains are labeled according to recombinant relationships established with parental strains. Positive matches are defined by at least 90% likelihood for 200 or more contiguous base pairs in a cBrother analysis. Regions of the genome matching no parental strains above this threshold for at least 200 nt were marked as nks (no known source). This is a simplified version of a figure from the companion paper for this study,15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] but with updated strain classifications.SFV detectionWestern blotRhesus macaque fibroblast Telo-RF cell line19Kirchoff V, Wong S, St JS, Pari GS.Generation of a life-expanded rhesus monkey fibroblast cell line for the growth of rhesus rhadinovirus (RRV). Arch Virol2002;147:321–333. [Crossref], [Google Scholar] was infected with SFV M. mulatta or M. fascicularis, or left uninfected. After 48 h, cells were lysed in 1× sodium dodecyl sulfate sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 0.01% bromophenol blue) and samples elec trophoresed through 10% polyacrylamide gels. After transfer of proteins to polyvinylidene difluoride membranes (Millipore Billerica, MA, USA), the membranes were blocked in phosphate-buffered saline+0.05% Tween-20 containing 2% non-fat dry milk, and rinsed with water and left to dry. Strips were made, each containing three lanes (molecular weight marker, uninfected Telo-RF and infected Telo-RF cell lysates) and primary antibody (human plasma) was added at a 1∶20 dilution and incubated overnight at 4 °C. Strips were washed three times in phosphate-buffered saline+0.05% Tween-20 before adding secondary goat anti-human horse radish peroxidase (Thermo Scientific and GE Healthcare Rochester, NY, USA) at a 1∶5000 dilution for 1 h at room temperature. Strips were washed three more times before rinsing with water and applying 3,3′,5,5′-tetramethylbenzidine stabilized substrate for horse radish peroxidase (Promega Madision, WI, USA) to visualize bands. Plasma from BGH4, a known SFV positive human20Jones-Engel L, May CC, Engel GAet al.Diverse contexts of zoonotic transmission of simian foamy viruses in Asia. Emerg Infect Dis2008;14:1200–1208. [Crossref], [Google Scholar] was used as a positive control.DNA isolation from whole bloodTotal genomic DNA was extracted from the whole blood of humans using the QIAamp DNA blood mini kit (Qiagen) according to manufacturer’s protocol. We extracted DNA from 200 µL of whole blood. The purified DNA was eluted in 200 µL of buffer AE.PCR amplification of gag and pol fragments from genomic DNAA total of 15 µL of the isolated DNA was used for PCR amplification of gag or pol fragments by a two round PCR. First round PCR was a multiplex reaction with both gag and pol primers (primer sequence: G1 (+)(nt 114–134, taken from SFV1 GeneBank sequence NC_001364 NC_001736) 5′-AGG ATG GTG GGG ACC AGC TA-3′; G2 (−) (nt 1451–1433) 5′-CAG GAA GAG GTA CTC GGG G-3′; P1 (+) (nt 5179–5200) 5′-GCC ACC CAA GGA AGT TAT GTG G-3′; P2 (−) (nt 5768–5749) 5′-GCT GCC CCT TGG TCA GAG TG)-3′. The reactions were denatured at 95 °C for 3 min, followed by two cycles of 95 °C for 30 s, 38 °C for 1 min, 72 °C for 1 min, then followed by 25 cycles of 95 °C for 30 s, 52 °C for 1 min and 72 °C for 1 min. Water was used as a negative control. 2 µL of the PCR product was used as template for the second round PCR. Either gag or pol primers (G3 (+) (nt 190–212) 5′-CAA CCT AGA TGG AGA GCT GAA GG-3′; G4 (−) (nt 1425–1406) 5′-GGG GAC AAA CTA CGG CTG GG-3′; P5 (+) (nt 5339–5361) 5′-TAA TCC TCC AGG GTA TCC AAA A-3′; P6 (−) (nt 5728–5707) 5′-ATA CCA TCG ACT ACT ACA AGG A-3′) were used to amplify individual fragments. The reactions were denatured at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 52 °C for 1 min and 72 °C for 1 min. The expected sizes of the PCR products for gag and pol are 1235 bps and 390 bps, respectively.Cloning and sequencing of gag fragmentsThe FV PCR products were gel purified using a QIAquick gel extraction kit (Qiagen) according to manufacturer’s protocol. The purified fragments were ligated into the TOPO-TA cloning vector (Invitrogen, Carlsbad, CA, USA) and transformed into Escherichia coli TOP10 competent cells (Invitrogen). Multiple single colonies for each gag sample were selected and plasmid DNA was purified and sequenced. Sequencing was carried out using M13 forward and reverse primers, as well as internal forward and reverse primers for gag by the University of Washington Genomics Facility or GeneWiz Inc. (Seattle, WA, USA). Viral strains are denoted using lowercase letters to distinguish them from the geographic sites where the humans or macaques were sampled (e.g., Dhamrai is a study site where the dhamrai core strain was detected.)Isolation of RNA from buccal swabsFrozen buccal swab samples in RLT buffer (Qiagen) were thawed at 37 °C for 15 min with 40 U of the RNaseOUT ribonuclease inhibitor (Invitrogen) and 1 U of RNase-free DNase I (Promega) immediately prior to RNA extraction. Total RNA was extracted according to the standard protocol using the RNeasy Mini Kit (Qiagen). RNA was treated again with 40 U of RNaseOUT and 1 U of RNase-free DNase I at 37 °C for 1 h. Samples were then incubated at 65 °C for 15 min to inactivate enzymes. RNA was stored at −20 °C for subsequent use.Reverse transcription-PCR (RT-PCR) of buccal swab RNART-PCR was performed to clone partial gag sequences (1235 bp) from buccal swab RNA. First-strand cDNA was synthesized using the Thermoscript RT-PCR system (Invitrogen) according to the manufacturer’s protocol using an Oligo(dT)18 primer. The RT reactions were inactivated at 85 °C for 5 min. The resulting cDNAs were used as template for two round PCR as described above for whole blood FV gag amplification.AnalysisClustering methods and hypermutation analysisSequences under examination in this study exhibited evidence of APOBEC3-mediated hypermutation. A thorough analysis of this activity is presented in Matsen et al. (unpublished). This analysis produced putatively hypermutation negative sequence alignments by removing columns suspected of hypermutation. These alignments were used for the remainder of the analyses in this study, in order to avoid conflation of hypermutation and genuine phylogenetic signal.Phylogenetic analysesA phylogenetic tree was constructed from the putatively hypermutation-free alignment using FastTree 2.1.7.21Price MN.DPAAP FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol2009;26:1641–1650. [Crossref], [Web of Science ®], [Google Scholar],22Price MN, Dehal PS, Arkin AP.FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE2010;5:e9490. [Crossref], [Web of Science ®], [Google Scholar] This tree was visualized using Archeopteryx (https://sites.google.com/site/cmzmasek/home/software/archaeopteryx). From the same alignment, SplitsTree 4.12.823Huson DH, Bryant D.Application of phylogenetic networks in evolutionary studies. Mol Biol Evol2006;23:254–267. [Crossref], [Web of Science ®], [Google Scholar] was used to produce a splits network.Strain analysisStrains were identified by passing the hypermutation-negative alignment through an iterative recentering clustering algorithm. It was found that a single application of UCLUST v1.124Edgar RC.Search and clustering orders of magnitude faster than BLAST. Bioinformatics2010;26:2460–2461. [Crossref], [Web of Science ®], [Google Scholar] produced poor clustering results due to the greedy nature of the algorithm. An iterative recentering algorithm, suggested by the UCLUST author at http://drive5.com/usearch/manual/recenter.html, was implemented which dealt well with this issue. For each round of clustering, consensus sequences from the prior round were added to the top of an ungapped alignment, in order of cluster size from greatest to least. For this iteration, clustering was carried out using cluster_smallmem, producing new consensus sequences and clusters. This process was repeated twice. Cluster assignments were further fine-tuned by a script that found the true centroid of each cluster, as defined by the sequence with minimal average normalized Hamming distance to every other sequence in the cluster. The script was then checked to make sure that each sequence clustered with the centroid to which it was closest (again, as defined by normalized Hamming distance), making reassignments as necessary.This iterative algorithm was applied using a threshold of 97.77%. This threshold, while slightly higher than that used in our companion study,15Feeroz MM, Soliven K, Small CTet al.Population dynamics of rhesus macaques and associated foamy virus in Bangladesh. Emerg Microbes Infect2013;2:e29. [Taylor & Francis Online], [Google Scholar] better reflects the somewhat lower amount of sequence diversity as a result of removing columns suspected of hypermutation. As mentioned above, viral strains are denoted using lowercase letters to distinguish them from the geographic sites where the humans or macaques were sampled (e.g., Dhamrai is a study site where the dhamrai core strain was primarily found.)Statistical methodsStatistical analysis of demographic variables was performed using the JMP (SAS Institute, Cary, NC, USA) statistical software package; analysis of contingency tables was performed with the R fisher.test function.25 R Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing, 2012. [Google Scholar]RESULTSDemographic data including NHP exposuresOver a 5-year period, 2007–2011, biological samples were collected from 222 people. The majority of the subjects (n=209) were sampled from five urban sites and a sixth group (n=13), comprised of nomadic people who travel the country with their performing monkeys, were sampled opportunistically as the research team traveled between site

Leave a message 

Message List