Development of an integrative database with 499 novel microsatellite markers for Macaca fascicularis

Background

Cynomolgus macaques (Macaca fascicularis) are a valuable resource for linkage studies of genetic disorders, but their microsatellite markers are not sufficient. In genetic studies, a prerequisite for mapping genes is development of a genome-wide set of microsatellite markers in target organisms. A whole genome sequence and its annotation also facilitate identification of markers for causative mutations. The aim of this study is to establish hundreds of microsatellite markers and to develop an integrative cynomolgus macaque genome database with a variety of datasets including marker and gene information that will be useful for further genetic analyses in this species.

Results

We investigated the level of polymorphisms in cynomolgus monkeys for 671 microsatellite markers that are covered by our established Bacterial Artificial Chromosome (BAC) clones. Four hundred and ninety-nine (74.4%) of the markers were found to be polymorphic using standard PCR analysis. The average number of alleles and average expected heterozygosity at these polymorphic loci in ten cynomolgus macaques were 8.20 and 0.75, respectively.

Conclusion

BAC clones and novel microsatellite markers were assigned to the rhesus genome sequence and linked with our cynomolgus macaque cDNA database (QFbase). Our novel microsatellite marker set and genomic database will be valuable integrative resources in analyzing genetic disorders in cynomolgus macaques.     

An initial genetic linkage map of the rhesus macaque (Macaca mulatta) genome using human microsatellite loci

Rhesus macaques (Macaca mulatta) are the most widely used nonhuman primate species in biomedical research. To create new opportunities for genetic and genomic studies using rhesus monkeys, we constructed a genetic linkage map of the rhesus genome. This map consists of 241 microsatellite loci, all previously mapped in the human genome. These polymorphisms were genotyped in five pedigrees of rhesus monkeys totaling 865 animals. The resulting linkage map covers 2048 cM including all 20 rhesus autosomes, with average spacing between markers of 9.3 cM. Average heterozygosity among those markers is 0.73. This linkage map provides new comparative information concerning locus order and interlocus distances in humans and rhesus monkeys. The map will facilitate whole-genome linkage screens to locate quantitative trait loci (QTLs) that influence individual variation in phenotypic traits related to basic primate anatomy, physiology, and behavior, as well as QTLs relevant to risk factors for human disease.     

Molecular Evidence for Distinct Genotypes of Monkey B Virus (Herpesvirus Simiae) Which Are Related to the Macaque Host Species

Although monkey B virus (herpesvirus simiae; BV) is common in all macaque species, fatal human infections appear to be associated with exposure to rhesus macaques (Macaca mulatta), suggesting that BV isolates from rhesus monkeys may be more lethal to nonmacaques than are BV strains indigenous to other macaque species. To determine if significant differences that would support this supposition exist among BV isolates, we compared multiple BV strains isolated from rhesus, cynomolgus, pigtail, and Japanese macaques. Antigenic analyses indicated that while the isolates were very closely related to one another, there are some antigenic determinants that are specific to BV isolates from different macaque species. Restriction enzyme digest patterns of viral DNA revealed marked similarities between rhesus and Japanese macaque isolates, while pigtail and cynomolgus macaque isolates had distinctive cleavage patterns. To further compare genetic diversity among BV isolates, DNA sequences from two regions of the viral genome containing genes that are conserved (UL27 and US6) and variable (US4 and US5) among primate alphaherpesviruses, as well as from two noncoding intergenic regions, were determined. From these sequence data and a phylogenetic analysis of them it was evident that while all isolates were closely related strains of BV, there were three distinct genotypes. The three BV genotypes were directly related to the macaque species of origin and were composed of (i) isolates from rhesus and Japanese macaques, (ii) cynomolgus monkey isolates, and (iii) isolates from pigtail macaques. This study demonstrates the existence of different BV genotypes which are related to the macaque host species and thus provides a molecular basis for the possible existence of BV isolates which vary in their levels of pathogenicity for nonmacaque species.     

Use of Genome-wide Heterospecific Single-Nucleotide Polymorphisms to Estimate Linkage Disequilibrium in Rhesus and Cynomolgus Macaques

Rhesus and cynomolgus macaques are frequently used in biomedical research, and the availability of their reference genomes now provides for their use in genome-wide association studies. However, little is known about linkage disequilibrium (LD) in their genomes, which can affect the design and success of such studies. Here we studied LD by using 1781 conserved single-nucleotide polymorphisms (SNPs) in 183 rhesus macaques (Macaca mulatta), including 97 purebred Chinese and 86 purebred Indian animals, and 96 cynomolgus macaques (M. fascicularis fascicularis). Correlation between loci pairs decayed to 0.02 at 1146.83, 2197.92, and 3955.83 kb for Chinese rhesus, Indian rhesus, and cynomolgus macaques, respectively. Differences between the observed heterozygosity and minor allele frequency (MAF) of pairs of these 3 taxa were highly statistically significant. These 3 nonhuman primate taxa have significantly different genetic diversities (heterozygosity and MAF) and rates of LD decay. Our study confirms a much lower rate of LD decay in Indian than in Chinese rhesus macaques relative to that previously reported. In contrast, the especially low rate of LD decay in cynomolgus macaques suggests the particular usefulness of this species in genome-wide association studies. Although conserved markers, such as those used here, are required for valid LD comparisons among taxa, LD can be assessed with less bias by using species-specific markers, because conserved SNPs may be ancestral and therefore not informative for LD.Abbreviations: GWAS, genome-wide association study; LD, linkage disequilibrium; MAF, minor allele frequencyContributing to the widespread use of nonhuman primates in biomedical research, captive-breeding programs such as those of the National Primate Research Center system in the United States were established initially by using animals imported from Asia. The 2 most commonly used primates are rhesus macaques (Macaca mulatta) and long-tailed or cynomolgus macaques (M. fascicularis fascicularis).After humans, rhesus macaques are the most widely distributed primate species.^37,38 This species is found throughout mainland Asia, ranging from Afghanistan to India and eastward through Thailand and southern China to the Yellow Sea.^31,34 In addition to their significant morphological differences,⁹ rhesus macaques of Indian and Chinese origins have been demonstrated to exhibit significant phenotypic differences that are directly relevant to their use as biomedical models in experimental studies.^2,23,42 Cynomolgus macaques are found south of the subtropical and temperate geographic distributions of rhesus macaques, in the south and southeast Indo-Malayan regions.^8,10The 2 species share a common ancestor that lived 1 to 2 million years ago.^3,13,25 This ancestral population of rhesus macaques diverged from a fascicularis-like ancestor shared in common with both rhesus and cynomolgus macaques after cynomolgus macaques expanded from their homeland in Indonesia.³⁶ For this reason, genetic markers present in Indian rhesus macaques are either highly derived or are conserved as ancestral markers shared with Chinese rhesus macaques. The interspecific boundaries of rhesus and cynomolgus macaques are delineated by a narrow zone of parapatry in northern Indochina,^7,8,10 within which male-biased gene flow^37,39 and relatively high, but highly variable, levels of introgression of genes³² have occurred from rhesus to cynomolgus macaque groups.^37,39 Because cynomolgus macaques originated in Indonesia³⁶ and because rhesus macaques probably diverged from cynomolgus macaques in southwestern China,¹¹ genetic markers shared between Indonesian cynomolgus macaques and Chinese rhesus macaques comprise a unique set of markers that are conserved in both macaque species.The wide assortment of morphometric differences^8,9 and the broad geographic distribution of these 2 macaque species foster an expectation of high genetic diversity within and between them that could be exploited for mapping genes responsible for phenotypic differences between taxa. A better understanding of linkage disequilibrium (LD) in these nonhuman primate species can lead to a more informed selection of study subjects for, and more efficient conduct of, genome-wide association studies (GWAS) of particular diseases that macaques share in common with humans. LD is the nonrandom association of alleles at 2 or more adjacent loci that descend from single, ancestral chromosomes.²⁹ LD plays a critical role in gene mapping, both as a tool for fine mapping of complex disease genes and in GWAS-based approaches. GWAS facilitate the identification of genes associated with complex and common traits or diseases by examining LD estimates among large numbers of common genetic variants, typically single-nucleotide polymorphisms (SNPs), between pairs of different groups of subjects to determine whether any variant is associated with a trait or disease of interest. LD data make tightly linked variants strongly correlated to produce successful association studies. For instance, LD reduces the number of markers and sample size of study subjects required to map genes influencing phenotypes to the genome because markers in LD are linked and inherited together.¹³ In addition, differences in LD can be used to identify orthologs for detecting the signatures of selective sweeps,²¹ as defined by d_N/d_S ratios obtained through the McDonald–Kreitman neutrality test.²⁴ Furthermore, LD assessments can provide a more complete understanding of genome structure by defining the boundaries of haplotype blocks, within which recombination is rare or absent and which are separated by recombination ‘hotspots,’ in genomes.⁴³Evidence from a study based on 1476 SNPs identified in ENCODE regions of the Indian rhesus macaque genome¹³ indicated that the rate of LD decay is higher in Chinese than in Indian rhesus macaques due to an hypothesized genetic bottleneck experienced by Indian rhesus macaques after diverging from the eastern subspecies, and, therefore, that Indian rhesus macaques, having higher LD, may be more useful for GWAS than Chinese rhesus macaques. In that study,¹³ only 33% of the SNPs were shared in common between the 2 subspecies, with Chinese rhesus macaques contributing to more than 60% of the remaining rhesus SNPs. Conversely, another study⁴¹ reported a slower rate of decay of LD in 25 Chinese than in 25 Indian rhesus macaques on the basis of 4040 SNPs, only 2% of which fell in coding regions, but 68% of those SNPs were shared between the 2 subspecies, with Indian rhesus macaques contributing almost 60% of the remaining SNPs. The marked disparity between the 2 studies in the proportions of shared SNPs used, the subspecies with the most genetic diversity, the sample size of Chinese rhesus macaques, the proportions of SNPs located in or near coding regions that are subject to functional constraints, and the greater disparity in LD decay between the 2 subspecies of rhesus macaques might reflect biases in either or both studies. For example, the use of markers whose frequencies are uncharacteristically low in one subspecies relative to the other can underestimate the rate of LD decay because lower frequency alleles, on average, are younger and have experienced less time for recombination.²⁶ To avoid the influence of such ascertainment biases, comparisons of LD between 2 taxa should involve only SNPs conserved in both taxa. Moreover, because 2 points do not provide a phylogenetic or cladistic analysis to assign specific SNPs to origin on one phylogenetic line or another, comparing just the Indian and Chinese rhesus macaques without an additional primate taxon makes it is difficult to establish polarity and distinguish between derived and conserved SNPs. This limitation likely led to the contradictory conclusions of the 2 previously cited studies^13,41 regarding the rate of LD decay in Chinese and Indian rhesus macaques.Because rhesus and cynomolgus macaques share a common fascicularis-like ancestor, a comparison of heterospecific SNPs among cynomolgus, Indian rhesus, and Chinese rhesus macaques would likely be fundamental to inferences regarding genome-wide LD estimates. The objective of the present study was to evaluate the conclusions of previous studies^13,41 by using our panel of 1781 autosomal SNPs that are conserved in both rhesus and cynomolgus macaques to estimate the rates at which genome-wide LD decays in Indian and Chinese rhesus macaques and cynomolgus macaques, the species ancestral to rhesus macaques, and to evaluate the suitability of these populations for GWAS.     

Identifying human-rhesus macaque gene orthologs using heterospecific SNP probes

We genotyped a Chinese and an Indian-origin rhesus macaque using the Affymetrix Genome-Wide Human SNP Array 6.0 and cataloged 85,473 uniquely mapping heterospecific SNPs. These SNPs were assigned to rhesus chromosomes according to their probe sequence alignments as displayed in the human and rhesus reference sequences. The conserved gene order (synteny) revealed by heterospecific SNP maps is in concordance with that of the published human and rhesus macaque genomes.Using these SNPs' original human rs numbers, we identified 12,328 genes annotated in humans that are associated with these SNPs, 3674 of which were found in at least one of the two rhesus macaques studied. Due to their density, the heterospecific SNPs allow fine-grained comparisons, including approximate boundaries of intra- and extra-chromosomal rearrangements involving gene orthologs, which can be used to distinguish rhesus macaque chromosomes from human chromosomes.     

Whole-genome sequencing and analysis of the Malaysian cynomolgus macaque (Macaca fascicularis) genome

ABSTRACT: BACKGROUND: The genetic background of the cynomolgus macaque (Macaca fascicularis) is made complex by the high genetic diversity, population structure, and gene introgression from the closely related rhesus macaque (Macaca mulatta). Herein we report the whole-genome sequence of a Malaysian cynomolgus macaque male with more than 40-fold coverage, which was determined using a resequencing method based on the Indian rhesus macaque genome. RESULTS: We identified approximately 9.7 million single nucleotide variants (SNVs) between the Malaysian cynomolgus and the Indian rhesus macaque genomes. Compared with humans, a smaller nonsynonymous/synonymous SNV ratio in the cynomolgus macaque suggests more effective removal of slightly deleterious mutations. Comparison of two cynomolgus (Malaysian and Vietnamese) and two rhesus (Indian and Chinese) macaque genomes, including previously published macaque genomes, suggests that Indochinese cynomolgus macaques have been more affected by gene introgression from rhesus macaques. We further identified 60 nonsynonymous SNVs that completely differentiated the cynomolgus and rhesus macaque genomes, and that could be important candidate variants for determining species-specific responses to drugs and pathogens. The demographic inference using the genome sequence data revealed that Malaysian cynomolgus macaques have experienced at least three population bottlenecks. CONCLUSIONS: This list of whole-genome SNVs will be useful for many future applications, such as an array-based genotyping system for macaque individuals. High-quality whole-genome sequencing of the cynomolgus macaque genome may aid studies on finding genetic differences that are responsible for phenotypic diversity in macaques and may help control genetic backgrounds among individuals.     

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed V alpha 24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with alpha-galactosylceramide (alpha-GalCer) and interleukin-2. We found that rhesus macaques possess V alpha 24TCR+ T cells, suggesting that recognition of alpha-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V-J junction for the V alpha 24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d alpha1-alpha2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.     

Differential recombination dynamics within the MHC of macaque species

A panel of 15 carefully selected microsatellites (short tandem repeats, STRs) has allowed us to study segregation and haplotype stability in various macaque species. The STRs span the major histocompatibility complex (MHC) region and map in more detail from the centromeric part of the Mhc-A to the DR region. Two large panels of Indian rhesus and Indonesian/Indochinese cynomolgus macaques have been subjected to pedigree analysis, allowing the definition of 161 and 36 different haplotypes and the physical mapping of 10 and 5 recombination sites, respectively. Although most recombination sites within the studied section of the Indian rhesus monkey MHC are situated between the Mhc-A and Mhc-B regions, the resulting recombination rate for this genomic segment is low and similar to that in humans. In contrast, in Indonesian/Indochinese macaques, two recombination sites, which appear to be absent in rhesus macaques, map between the class III and II regions. As a result, the mean recombination frequency of the core MHC, Mhc-A to class II, is higher in Indonesian/Indochinese cynomolgus than in Indian rhesus macaques, but as such is comparable to that in humans. The present communication demonstrates that the dynamics of recombination ‘hot/cold spots’ in the MHC, as well as their frequencies, may differ substantially between highly related macaque species.     

Unprecedented polymorphism of Mhc-DRB region configurations in rhesus macaques

The rhesus macaque is an important model in preclinical transplantation research and for the study of chronic and infectious diseases, and so extensive knowledge of its MHC (MhcMamu) is needed. Nucleotide sequencing of exon 2 allowed the detection of 68 Mamu-DRB alleles. Although most alleles belong to loci/lineages that have human equivalents, identical Mhc-DRB alleles are not shared between humans and rhesus macaques. The number of -DRB genes present per haplotype can vary from two to seven in the rhesus macaque, whereas it ranges from one to four in humans. Within a panel of 210 rhesus macaques, 24 Mamu-DRB region configurations can be distinguished differing in the number and composition of loci. None of the Mamu-DRB region configurations has been described for any other species, and only one of them displays major allelic variation giving rise to a total of 33 Mamu-DRB haplotypes. In the human population, only five HLA-DRB region configurations were defined, which in contrast to the rhesus macaque exhibit extensive allelic polymorphism. In comparison with humans, the unprecedented polymorphism of the Mamu-DRB region configurations may reflect an alternative strategy of this primate species to cope with pathogens. Because of the Mamu-DRB diversity, nonhuman primate colonies used for immunological research should be thoroughly typed to facilitate proper interpretation of results. This approach will minimize as well the number of animals necessary to conduct experiments.     

Molecular phylogeny of macaques: implications of nucleotide sequences from an 896-base pair region of mitochondrial DNA

We determined the nucleotide sequences of an 896-base pair region of mitochondrial DNA (mtDNA) from 20 primates representing 13 species of macaques, a baboon, and a patas. We compared these sequences and the homologous sequences from four macaques and a human against each other and deduced the phylogenetic relationships of macaques. The results from the phylogenetic analyses revealed five groups among the macaques: (1) Barbary macaque, (2) two species of Sulawesi macaques, (3) Japanese, rhesus, Taiwanese, crab-eating, and stump-tailed macaques, (4) toque, pig-tailed, and lion-tailed macaques, and (5) Assamese and bonnet macaques. The phylogenetic position of Tibetan macaque remains ambiguous as to whether it belongs to the fourth or fifth group. Phylogenetic trees revealed that Barbary macaque diverged first from the other Asian macaques. Subsequently, the four groups of Asian macaques diverged from one another in a relatively short period of time. Within each group, most of the species diverged in a relatively short period of time following the divergence of the groups. Assuming that the Asian macaques diverged from the outgroup Barbary macaque three million years ago (MYA), the divergence times among groups of Asian macaques were estimated at 2.1-2.5 MYA and within groups at 1.4- 2.2 MYA. The intraspecific nucleotide diversity observed among three rhesus macaques was so large that they did not form a monophyletic cluster in the phylogenetic trees. Instead, one of them formed a cluster with Japanese and Taiwanese macaques, whereas the other two formed a separate cluster. This implies that either polymorphisms of mtDNA sequences that existed before the divergence of these three species (ca. 700,000 years ago) have been retained in rhesus macaques or introgression has occurred among the three species.      

Nineteen new microsatellite DNA polymorphisms in pigtailed macaques (Macaca nemestrina)

Microsatellite loci known to be polymorphic in baboons (Papio hamadryas) and/or humans were tested in pigtailed macaques (Macaca nemestrina) from the Washington Regional Primate Research Center. Nineteen polymorphisms were identified in the macaques, with an average of 9.2 alleles per locus and an average heterozygosity of 0.76. Seven loci were analyzed using radiolabelled PCR primers and standard gel electrophoresis. Twelve loci were studied using fluorescently labelled primers and the Perkin-Elmer ABI 377 genotyping system. Of these 19 pigtailed macaque polymorphisms, 12 were used to perform paternity testing among captive animals. In a set of 15 infants, this panel of 12 genetic polymorphisms was sufficient to establish paternity in all cases. The number of alleles per locus in pigtailed macaques was compared with the number of alleles in a sample of baboons, and no significant correlation was observed. This indicates that population genetic processes such as genetic drift and recurrent mutation act rapidly enough on these loci to eliminate any relationship in levels of polymorphism across those two species. These 19 loci will be valuable for a range of genetic studies in pigtailed macaques, including paternity testing, analysis of population structure and differentiation among wild populations, and genetic linkage mapping.     

Sex-Specific Heritability of Spontaneous Lipid Levels in an Extended Pedigree of Indian-Origin Rhesus Macaques (Macaca mulatta)

The rhesus macaque is an important model for human atherosclerosis but genetic determinants of relevant phenotypes have not yet been investigated in this species. Because lipid levels are well-established and heritable risk factors for human atherosclerosis, our goal was to assess the heritability of lipoprotein cholesterol and triglyceride levels in a single, extended pedigree of 1,289 Indian-origin rhesus macaques. Additionally, because increasing evidence supports sex differences in the genetic architecture of lipid levels and lipid metabolism in humans and macaques, we also explored sex-specific heritability for all lipid measures investigated in this study. Using standard methods, we measured lipoprotein cholesterol and triglyceride levels from fasted plasma in a sample of 193 pedigreed rhesus macaques selected for membership in large, paternal half-sib cohorts, and maintained on a low-fat, low cholesterol chow diet. Employing a variance components approach, we found moderate heritability for total cholesterol (h²=0.257, P=0.032), LDL cholesterol (h²=0.252, P=0.030), and triglyceride levels (h²=0.197, P=0.034) in the full sample. However, stratification by sex (N=68 males, N=125 females) revealed substantial sex-specific heritability for total cholesterol (0.644, P=0.004, females only), HDL cholesterol (0.843, P=0.0008, females only), VLDL cholesterol (0.482, P=0.018, males only), and triglyceride levels (0.705, P=0.001, males only) that was obscured or absent when sexes were combined in the full sample. We conclude that genes contribute to spontaneous variation in circulating lipid levels in the Indian-origin rhesus macaque in a sex-specific manner, and that the rhesus macaque is likely to be a valuable model for sex-specific genetic effects on lipid risk factors for human atherosclerosis. These findings are a first-ever report of heritability for cholesterol levels in this species, and support the need for expanded analysis of these traits in this population.     

Human–wildlife conflict: Proximate predictors of aggression between humans and rhesus macaques in India

Macaques live in close contact with humans across South and Southeast Asia, and direct interaction is frequent. Aggressive contact is a concern in many locations, particularly among populations of rhesus and longtail macaques that co‐inhabit urbanized cities and towns with humans. We investigated the proximate factors influencing the occurrence of macaque aggression toward humans as well as human aggression toward macaques to determine the extent to which human behavior elicits macaque aggression and vice versa. We conducted a 3‐month study of four free‐ranging populations of rhesus macaques in Dehradun, India from October–December 2012, using event sampling to record all instances of human‐macaque interaction (N = 3120). Our results show that while human aggression was predicted by the potential for economic losses or damage, macaque aggression was influenced by aggressive or intimidating behavior by humans as well as recent rates of conspecific aggression. Further, adult female macaques participated in aggression more frequently than expected, whereas adult and subadult males participated as frequently as expected. Our analyses demonstrate that neither human nor macaque aggression is unprovoked. Rather, both humans and macaques are responding to one another's behavior. Mitigation of human‐primate conflict, and indeed other types of human‐wildlife conflict in such coupled systems, will require a holistic investigation of the ways in which each participant is responding to, and consequently altering, the behavior of the other. Am J Phys Anthropol 156:286–294, 2015. © 2014 Wiley Periodicals, Inc.     

A Diffusion-Tensor-Based White Matter Atlas for Rhesus Macaques

Atlases of key white matter (WM) structures in humans are widely available, and are very useful for region of interest (ROI)-based analyses of WM properties. There are histology-based atlases of cortical areas in the rhesus macaque, but none currently of specific WM structures. Since ROI-based analysis of WM pathways is also useful in studies using rhesus diffusion tensor imaging (DTI) data, we have here created an atlas based on a publicly available DTI-based template of young rhesus macaques. The atlas was constructed to mimic the structure of an existing human atlas that is widely used, making results translatable between species. Parcellations were carefully hand-drawn on a principle-direction color-coded fractional anisotropy image of the population template. The resulting atlas can be used as a reference to which registration of individual rhesus data can be performed for the purpose of white-matter parcellation. Alternatively, specific ROIs from the atlas may be warped into individual space to be used in ROI-based group analyses. This atlas will be made publicly available so that it may be used as a resource for DTI studies of rhesus macaques.     

Modulation of antigen-specific humoral responses in rhesus macaques by using cytokine cDNAs as DNA vaccine adjuvants

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.     

Niche separation of sympatric macaques, Macaca assamensis and M. mulatta, in limestone habitats of Nonggang, China

Comparative studies of sympatric species are essential in understanding those species’ behavioral and ecological adaptations as well as the mechanisms that can reduce resource competition enough to allow coexistence. We collected data on diet, activity budget and habitat use from two sympatric macaque species, the Assamese macaque (Macaca assamensis) and the rhesus macaque (M. mulatta), in a limestone seasonal rainforest of Nonggang Nature Reserve, southwestern Guangxi, China. Our results show that the two sympatric macaques differ in diet, activity budget, and habitat use: (1) out of the 131 plant species that were used by both macaque species as food over the year, only 15 plant species (11 %) were shared. Rhesus macaques used more plant species as major foods, and had higher dietary diversity and evenness indexes than Assamese macaques. (2) Assamese macaques fed predominantly on leaves, whereas rhesus macaques fed more selectively on fruits. The rhesus macaques’ diet varied according to season, and was significantly correlated to season fluctuation in fruit availability. (3) Assamese macaques devoted more time to resting, and less time to feeding than rhesus macaques (4) Assamese macaques were present mostly on the cliff, and tended to stay on the ground, whereas rhesus macaques were present mostly on the hillside, and showed preference to lower and middle canopy. The observed differences in diet and habitat use between the two macaque species represent behavioral patterns enabling their coexistence.     

Genome-wide DNA methylation analyses in the brain reveal four differentially methylated regions between humans and non-human primates

ABSTRACT: BACKGROUND: The highly improved cognitive function is the most significant change in human evolutionary history. Recently, several large-scale studies reported the evolutionary roles of DNA methylation; however, the role of DNA methylation on brain evolution is largely unknown. RESULTS: To test if DNA methylation has contributed to the evolution of human brain, with the use of MeDIP-Chip and SEQUENOM MassARRAY, we conducted a genome-wide analysis to identify differentially methylated regions (DMRs) in the brain between humans and rhesus macaques. We first identified a total of 150 candidate DMRs by the MeDIP-Chip method, among which 4DMRs were confirmed by the MassARRAY analysis. All 4 DMRs are within or close to the CpG islands, and a MIR3 repeat element was identified in one DMR, but no repeat sequence was observed in the other 3 DMRs. For the 4 DMR genes, their proteins tend to be conserved and two genes have neural related functions. Bisulfite sequencing and phylogenetic comparison among human, chimpanzee, rhesus macaque and rat suggested several regions of lineage specific DNA methylation, including a human specific hypomethylated region in the promoter of K6IRS2 gene. CONCLUSIONS: Our study provides a new angle of studying human brain evolution and understanding the evolutionary role of DNA methylation in the central nervous system. The results suggest that the patterns of DNA methylation in the brain are in general similar between humans and nonhuman primates, and only a few DMRs were identified.     

Pandemic Swine-Origin H1N1 Influenza Virus Replicates to Higher Levels and Induces More Fever and Acute Inflammatory Cytokines in Cynomolgus versus Rhesus Monkeys and Can Replicate in Common Marmosets

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.     

A rapid quantitative real-time PCR-based DNA quantification assay coupled with species--assignment capabilities for two hybridizing Macaca species

Regional populations of rhesus and long-tailed macaques exhibit fundamental differences in mitochondrial DNA, short tandem repeat and single nucleotide polymorphism variation between mainland and insular Southeast Asian populations. Some studies have revealed genetic admixture between these species due to natural hybridization and human-assisted intercrosses. A quantitative real-time PCR (qPCR) assay was developed to efficiently determine the species of origin of a macaque biological sample, and to quantify the species-specific template DNA. Prior knowledge of species identity and DNA concentrations are crucial for maintaining cost-effective methods and accurate DNA analysis. DNA from 109 regionally representative rhesus and long-tailed macaques was qPCR amplified to determine the species and template quantities. Of the 19 Vietnamese long-tailed macaques, 3 samples were discovered to be hybrids.