Simian-Human Immunodeficiency Virus (SHIV) Containing the nef/Long Terminal Repeat Region of the Highly Virulent SIVsmmPBj14 Causes PBj-Like Activation of Cultured Resting Peripheral Blood Mononuclear Cells, but the Chimera Showed No Increase in Virulence

SIV_smmPBj14 is a highly pathogenic lentivirus which causes acute diarrhea, rash, massive lymphocyte proliferation predominantly in the gastrointestinal tract, and death within 7 to 14 days. In cell culture, the virus has mitogenic effects on resting macaque T lymphocytes. In contrast, SIV_mac239 causes AIDS in rhesus macaques, generally within 2 years after inoculation. In a previous study, replacement of amino acid residues 17 and 18 of the Nef protein of SIV_mac239 with the corresponding amino acid residues of the Nef protein of SIV_smmPBj14 yielded a PBj-like virus that caused extensive activation of resting T lymphocytes in cultures and acute PBj-like disease when inoculated into pig-tailed macaques. This study suggested that nef played a major role in both processes. In this study, we replaced the nef/long terminal repeat (LTR) region of a nonpathogenic simian-human immunodeficiency virus (SHIV), SHIV_PPc, with the corresponding region from SIV_smmPBj14 and examined the biological properties of the resultant virus. Like SIV_smmPBj14, SHIV_PPcPBjnef caused massive stimulation of resting peripheral blood mononuclear cells (PBMC), which then produced virus in the absence of extraneous interleukin 2. However, when inoculated into macaques, the virus failed to replicate productively or cause disease. Thus, while these results confirmed that the nef/LTR region of SIV_smmPBj14 played a major role in the activation of resting PBMC, duplication of the cellular activation process in macaques may require a further interaction between nef and the envelope glycoprotein of simian immunodeficiency virus because SHIV, containing the envelope of human immunodeficiency virus type 1, failed to cause activation in vivo.     

Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIV_smm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIV_mac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIV_smm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.     

Rhesus Macaques Infected with Macrophage-Tropic Simian Immunodeficiency Virus (SIVmacR71/17E) Exhibit Extensive Focal Segmental and Global Glomerulosclerosis

We previously showed that inoculation of rhesus macaques with molecularly cloned lymphocytetropic simian immunodeficiency virus (SIV_mac239) results in SIV-associated nephropathy (SIVAN) and that the glomerulosclerotic lesions were associated with the selection of macrophagetropic (M-tropic) variants (V. H. Gattone et al., AIDS Res. Hum. Retroviruses 14:1163–1180, 1998). In the present study, seven rhesus macaques were inoculated with M-tropic SIV_macR71/17E, and the renal pathology was examined at necropsy. All SIV_macR71/17E-infected macaques developed AIDS, and most developed other systemic complications, including SIV-induced encephalitis and lentivirus interstitial pneumonia. There was no correlation between the length of infection (42 to 97 days), circulating CD4⁺ T-cell counts, and renal disease. Of the seven macaques inoculated with SIV_macR71/17E, five developed significant mesangial hyperplasia and expansion of matrix and four were clearly azotemic (serum urea nitrogen concentration of 40 to 112 mg/dl). These same five macaques developed focal segmental to global glomerulosclerotic lesions. Increased numbers of glomerular CD68⁺ cells (monocytes/macrophages) were found in glomeruli but not the tubulointerstitium of the macaques inoculated with SIV_macR71/17E. All macaques had glomerular deposits of immunoglobulin G (IgG), IgM, and tubuloreticular inclusions, and six of seven had IgA deposition. However, there was no correlation between the presence of circulating anti-SIV_mac antibodies, immunoglobulin deposition, and glomerular disease. Tubulointerstitial infiltrates were mild, with little or no correlation to azotemia, while microcystic tubules were evident in those with glomerulosclerosis or azotemia. The four most severely affected macaques were positive for diffuse glomerular immunostaining for viral core p27 antigen, and there was intense staining in the glomeruli of the two macaques with the most severe glomerulosclerosis. Viral sequences were isolated from glomerular and tubulointerstitial fractions from macaques with severe glomerulosclerosis but only from the tubulointerstitial compartment of those that did not develop glomerulosclerosis. Interviral recombinant viruses generated with env sequences isolated from glomeruli confirmed the M-tropic nature of the virus found in the glomeruli. The correlation between the increased number of CD68⁺ cells (monocytes/macrophages) in the glomeruli, the localization of p27 antigen in the glomeruli, and the glomerular pathology confirms and extends our previous observations of an association between glomerular infection and infiltration by M-tropic virus and SIVAN.     

Differences in viral distribution and cell adhesion molecule expression in the intestinal tract of rhesus macaques infected with pathogenic and nonpathogenic SIV

We investigated SIV infection and expression of adhesion molecules in the small intestine of rhesus macaques infected with pathogenic SIV (SIV_mac) or nonpathogenic clone (SIV_1A11). There was a wider dissemination and marked difference in tissue localization of SIV_mac relative to SIV_1A11. Our results also indicate that viral pathogenicity is associated with increased migration of inflammatory cells expressing VLA-α4, LFA-1α, Mac-1α, ICAM-1, and β2 integrin into the intestinal mucosa.     

Early helper T-cell dysfunction in simian immunodeficiency virus but not in human immunodeficiency virus type-2-infected macaques

Both naive and vaccinated macaques acquired a virus-specific proliferative helper T-cell reactivity in response to infection with the nonpathogenic human immunodeficiency virus type 2 (HIV-2). In contrast, macaques infected with the pathogenic simian immunodeficiency virus of the macaque strain (SIV_mac) did not develop a helper T-cell response. Furthermore, a vaccine-induced preexisting T-cell reactivity was abrogated after SIV_mac infection in vaccine failures. These differences may reflect the different pathogenicity of the two closely related viruses.     

The most common Chinese rhesus macaque MHC class I molecule shares peptide binding repertoire with the HLA-B7 supertype

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques.     

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.     

Identification of MHC class I sequences in Chinese-origin rhesus macaques

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population.     

Comprehensive identification of MHC class II alleles in a cohort of Chinese rhesus macaques

Rhesus macaque is a very important animal model for various human diseases, especially for AIDS and vaccine research. The susceptibility and/or resistance to some of these diseases are related to the major histocompatibility complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, Mamu-DPB1, Mamu-DQB1, and Mamu-DRB alleles were investigated in 30 Chinese rhesus macaques through gene cloning and sequencing. A total of 66 alleles were identified in this study, including 14 Mamu-DPB1, 20 Mamu-DQB1, and 30 Mamu-DRB alleles as well as 2 high-frequency Mamu-DPB1 alleles. Interestingly, one of the high-frequency Mamu-DPB1 alleles had been undocumented in earlier studies. Eleven of the other alleles, including four Mamu-DPB1, three Mamu-DQB1, and four Mamu-DRB alleles were also novel. Importantly, like MHC-DRB, more than two Mamu-DPB1 sequences per animal were detected in 13 monkeys, which suggested that they might represent gene duplication. Our data also indicated quite a few differences in the distribution of MHC class II alleles between the Chinese rhesus macaques and the previously reported Indian rhesus macaques. To our knowledge, our results revealed comprehensively the combination of MHC II alleles. This information will not only promote the understanding of Chinese rhesus macaque MHC polymorphism but will also facilitate the use of Chinese rhesus macaques in studies of human disease.     

A Large‐Scale SNP‐Based Genomic Admixture Analysis of the Captive Rhesus Macaque Colony at the California National Primate Research Center

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Center's (CNPRC) super‐Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian‐origin animals. The results also reveal no evidence of recent gene flow from long‐tailed and pig‐tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long‐tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long‐tailed, and pig‐tailed macaques likely predate the divergence among these groups. Am. J. Primatol. 74:747‐757, 2012. © 2012 Wiley Periodicals, Inc.     

CD8+ T cells from SIV elite controller macaques recognize Mamu-B*08-bound epitopes and select for widespread viral variation

Background

It is generally accepted that CD8⁺ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8⁺ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8⁺ cells, implicating that these cells actively suppress viral replication in ECs.

Methods and Findings

Here we show that three ECs in that study made at least seven robust CD8⁺ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8⁺ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques.

Conclusions

Together, our data suggest that Mamu-B*08-restricted CD8⁺ T cell responses effectively control replication of pathogenic SIV_mac239. All seven regions encoding Mamu-B*08-restricted CD8⁺ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8⁺ T cell responses. SIV_mac239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8⁺ T cell-mediated control of HIV replication in humans.     

Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) provide increasingly common models for infectious disease research. Several geographically distinct populations of these macaques from Southeast Asia and the Indian Ocean island of Mauritius are available for pathogenesis studies. Though host genetics may profoundly impact results of such studies, similarities and differences between populations are often overlooked. In this study we identified 47 full-length MHC class I nucleotide sequences in 16 cynomolgus macaques of Filipino origin. The majority of MHC class I sequences characterized (39 of 47) were unique to this regional population. However, we discovered eight sequences with perfect identity and six sequences with close similarity to previously defined MHC class I sequences from other macaque populations. We identified two ancestral MHC haplotypes that appear to be shared between Filipino and Mauritian cynomolgus macaques, notably a Mafa-B haplotype that has previously been shown to protect Mauritian cynomolgus macaques against challenge with a simian/human immunodeficiency virus, SHIV_89.6P. We also identified a Filipino cynomolgus macaque MHC class I sequence for which the predicted protein sequence differs from Mamu-B*17 by a single amino acid. This is important because Mamu-B*17 is strongly associated with protection against simian immunodeficiency virus (SIV) challenge in Indian rhesus macaques. These findings have implications for the evolutionary history of Filipino cynomolgus macaques as well as for the use of this model in SIV/SHIV research protocols. Kevin J. Campbell and Ann M. Detmer contributed equally to this work.     

Functional analysis of frequently expressed Chinese rhesus macaque MHC class I molecules Mamu-A1*02601 and Mamu-B*08301 reveals HLA-A2 and HLA-A3 supertypic specificities

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques?? major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC?Cpeptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.     

Identification of Mamu-DPA1, Mamu-DQA1, and Mamu-DRA alleles in a cohort of Chinese rhesus macaques

Rhesus macaques have long been used as animal models for various human diseases; the susceptibility and/or resistance to some of these diseases are related to the major histocompatibility complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, Mamu-DPA1, Mamu-DQA1, and Mamu-DRA alleles were investigated in 30 Chinese rhesus macaques by gene cloning and sequencing. A total of 14 Mamu-DPA1, 17 Mamu-DQA1, and 9 Mamu-DRA alleles were identified in this study. Of these alleles, 22 novel sequences have not been documented in earlier studies, including nine Mamu-DPA1, ten Mamu-DQA1, and three Mamu-DRA alleles. Interestingly, like Mafa-DQA1 and Mafa-DPA1, more than two Mamu-DQA1 and Mamu-DPA1 alleles were detected in one animal in this study, which suggested that they might represent gene duplication. If our findings can be validated by other studies, it will further increase the number of known Mamu-DPA1 and Mamu-DQA1 polymorphisms. Our data also indicated significant differences in MHC class II allele distribution among the Chinese rhesus macaques, Vietnamese cynomolgus macaques, and the previously reported rhesus macaques, which were mostly of Indian origin. This information will not only promote the understanding of Chinese rhesus macaque MHC diversity and polymorphism but will also facilitate the use of Chinese rhesus macaques in studies of human disease.     

SIVMAC vaccine studies using whole inactivated virus antigen sequentially depleted of viral proteins

Groups of four rhesus monkeys were immunised at 0, 1, 2, and 13 months with whole inactivated SIV_mac32H, SIV_mac depleted of the outer envelope glycoprotein gp130, virus cores depleted of the lipid membrane (and hence transmembrane glycoproteins), or purified gag protein. These macaques plus controls were challenged with either the homologous SIV_mac251–32H. grown in human cells or the same virus passed once through monkey cells. None of those challenged with monkey-grown virus were protected, whereas all in the whole and gp130-depleted virus groups, and one in the core group resisted challenge with human-grown virus. As the only difference between the challenge viruses was a single in vitro passage in monkey cells it can be concluded that protection was solely due to human cell components. Finally, passive transfer of high titer IgG from monkeys infected with the homologous challenge virus failed to protect monkeys from infection despite the presence of circulating neutralising antibodies.     

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.     

猕猴MHC-DPB1基因外显子2多态性研究

猕猴(Macaca mulatta)是最理想的医学实验灵长类动物, 且为国家二级保护动物。为了解中国猕猴主要组织相容复合体(Major histocompatibility complex, MHC)基因的遗传多态性背景, 为它们在生物医学研究中的应用及其遗传资源的保护提供一定的科学依据, 文章采用变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和克隆测序技术分析了106个四川野生猕猴MHC-DPB1基因的exon 2, 共检测到21个Mamu-DPB1等位基因, 其中有15个为本研究中首次发现的新等位基因; 从整个大的猕猴群体(106个个体)来看, 等位基因频率最高的是Mamu-DPB1*30(0.1120); 单独从不同地理群体来看, 最高等位基因频率分别为: 小金-DPB1*30 (0.1120), 黑水-DPB1*04 (0.1702), 巴中-DPB1*32 (0.1613), 汉源-DPB1*30(0.1120), 九龙-DPB1*04(0.1139); 氨基酸序列比对发现, 猕猴Mamu-DPB1等位基因编码的氨基酸序列中, 有12个氨基酸残基变异位点表现出物种特异性, 其中有9个位于新发现的15个Mamu-DPB1等位基因氨基酸序列中; 不同物种来源的DPB1等位基因系统发生树表明, 猕猴与其近缘物种食蟹猴(Macaca fascicularis)的DPB1等位基因间存在着跨种多态(Trans-species polymorphism)现象。研究还表明, MHC-DPB1等位基因在中国猕猴群体和先前为主要研究对象的印度猕猴群体间具有较大的差异。     

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.     

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.