Relation between phylogeny of African green monkey CD4 genes and their respective simian immunodeficiency virus genes

Abstract: An apparent species-specific relatedness of SIV_agm suggests a coevolution with their natural hosts. However, the exact species or subspecies classification of African green monkeys, AGM, is uncertain because current classification schemes rely on phenotype markers, while more definitive genetic data are lacking. In this study, the CD4 protein involved in tissue type recognition was genetically cloned and sequenced from PBMC RNA from all AGM species, including Barbados green monkeys (BGM). Phylogenetic trees were constructed that also included genomic CD4 nucleotide sequences from patas, sooty mangabeys, rhesus and pig-tail macaques, chimpanzees, and humans. Chimpanzees and humans consistently clustered together. Monkeys within the Cercopithecus genus formed a separate cluster which included pata monkeys, supporting its grouping as a member of Cercopithecus. Surprisingly, sooty mangabeys were genetically more closely related to Asian macaques than to other African species, which might explain why macaques are more susceptible to infection by the SIV_sm group than to infection by SIV_agm or HIV-1 and why patas, on the other hand, are highly susceptible to SIV_agm infection. Based on CD4 genetic data, tantalus, vervets, grivets, and sabaeus formed separate subgroups with BGM grouping closely with vervets. The branching order of the AGM species was related to that of their respective SIV_agm env sequences. The study suggests a strong correlation between CD4 phylogeny and the susceptibility of the host species to infection by a specific lentivirus and supports the assumption of a coevolution of SIV_agm and AGM. CD4 sequencing is suggested as a relevant method for genetic determination of primate species.     

Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium

 In humans, the 30 000 M _r Rhesus (Rh) polypeptide D (RhD) is a dominant antigen (Ag) of the Rh blood group system. To date, an Rh-like protein has been found in chimpanzees, gorillas, gibbons, and rhesus monkeys. Related to the 30 000 M _r Rh Ag protein are two polypeptides of 50 000 M _r , the human 50 000 M _r Rh Ag and the RhD-like protein from Caenorhabditis elegans. The function of all these proteins is not sufficiently known. Here we characterize a cDNA clone (GCRH) encoding a putative 57 000 M _r polypeptide from the marine sponge Geodia cydonium, which shares sequence similarity both to the RhD Ag and the Rh50 glycoprotein. The sponge Rh-like protein comprises 523 aa residues; hydropathy analysis hints at the presence of ten transmembrane domains. An N-terminal hydrophobic cleavage signal sequence is missing, suggesting that the first membrane-spanning domain of the sponge Rh-like protein acts as a signal-anchor sequence. The sponge Rh-like protein, like the human Rh50, lacks the CLP motif which is characteristic of the 30 000 M _r RhD. In addition, the hydropathy profile of the sponge Rh-like protein is of a similar size and shape as that of human Rh50. This data indicates that the RhD and its structurally related Rh50 glycoprotein, which are highly immunogenic in humans, share a common ancestral molecule with the G. cydonium Rh-like protein. Received: 9 April 1997 / Revised: 29 May 1997     

Amino acids in the plasma of fasting Japanese monkeys (Macaca fuscata fuscata andM. f. yakui)

The plasma levels of 26 amino acids and related compounds were determined in five male and five female adult members of each of the two subspecies of Japanese monkeys,Macaca fuscata fuscata andM. f. yakui. Activities of L-asparaginase and histaminase in plasma were also measured.Numerous differences in amino acid levels between the sexes in the subspeciesfuscata were noted, with the female consistently exhibiting lower values. Few differences were observed between the sexes of the subspeciesyakui or between the two subspecies of Japanese monkeys. These monkeys were similar to other previously studied nonhuman primates in exhibiting measurable levels of 3-methylhistidine in plasma. There were numerous quantitative differences among Japanese monkeys and stump-tailed macaques, rhesus monkeys, chimpanzees, and man, with the Japanese monkeys usually exhibiting higher plasma levels.L-asparaginase activity was not detectable in these Japanese monkeys. Histaminase activity was similar to that previously measured in pig-tailed macaques and chimpanzees, lower than that in rhesus monkeys and stump-tailed macaques, and higher than that in man.     

Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever

Bartonella quintana is a bacterium that causes a broad spectrum of diseases in humans including trench fever. Humans were previously considered to be the primary, if not the only, reservoir hosts for B. quintana. To identify the animal reservoir and extend our understanding of the ecological and evolutionary history of B. quintana, we examined blood samples from macaques and performed multilocus sequence typing (MLST) analysis. We demonstrated the prevalence of B. quintana infection was common in macaques from main primate centres in mainland China. Overall, 18.0% (59/328) of rhesus macaques and 12.7% (39/308) of cynomolgus macaques were found to be infected with B. quintana by blood culture and/or polymerase chain reaction. The infection was more frequently identified in juvenile and young monkeys compared with adult animals. In contrast with the relatively low level of sequence divergence of B. quintana reported in humans, our investigation revealed much higher genetic diversity in nonhuman primates. We identified 44 new nucleotide variable sites and 14 novel sequence types (STs) among the B. quintana isolates by MLST analysis. Some STs were found only in cynomolgus macaques, while some others were detected only in rhesus macaques, suggesting evidence of host‐cospeciation, which were further confirmed by phylogenetic analysis and Splits decomposition analysis. Our findings suggest that trench fever may primarily be a zoonotic disease with macaques as the natural hosts.     

Interspecies pair housing of macaques in a research facility

The eighth edition of The Guide for the Care and Use of Laboratory Animals establishes social housing as the 'default' for social species including non-human primates. The advantages of social housing for primates have been well established, but small research facilities housing few primates in indoor cages have struggled with social housing as a result of limitations on appropriate housing and availability of compatible monkeys. Here, we report a novel approach to pair housing macaques - crossing species. We have successfully pair housed an intact male rhesus macaque with an intact male cynomolgus macaque, and an adult female rhesus macaque with numerous subadult female cynomolgus macaques. Monkeys in these pairs established dominant-subordinate relationships similar to same-species pairs. Rhesus and cynomolgus macaques can be successfully paired for the purpose of social housing in facilities with limited numbers of monkeys.     

Comparative genetics of a highly divergent DRB microsatellite in different macaque species

The DRB region of the major histocompatibility complex (MHC) of cynomolgus and rhesus macaques is highly plastic, and extensive copy number variation together with allelic polymorphism makes it a challenging enterprise to design a typing protocol. All intact DRB genes in cynomolgus monkeys (Mafa) appear to possess a compound microsatellite, DRB-STR, in intron 2, which displays extensive length polymorphism. Therefore, this STR was studied in a large panel of animals, comprising pedigreed families as well. Sequencing analysis resulted in the detection of 60 Mafa-DRB exon 2 sequences that were unambiguously linked to the corresponding microsatellite. Its length is often allele specific and follows Mendelian segregation. In cynomolgus and rhesus macaques, the nucleotide composition of the DRB-STR is in concordance with the phylogeny of exon 2 sequences. As in humans and rhesus monkeys, this protocol detects specific combinations of different DRB-STR lengths that are unique for each haplotype. In the present panel, 22 Mafa-DRB region configurations could be defined, which exceeds the number detected in a comparable cohort of Indian rhesus macaques. The results suggest that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new haplotypes are generated by recombination-like events. Although both macaque species are known to share several identical DRB exon 2 sequences, the lengths of the corresponding microsatellites often differ. Thus, this method allows not only fast and accurate DRB haplotyping but may also permit discrimination between highly related macaque species.     

Diversity of TRIM5α and TRIMCyp sequences in cynomolgus macaques from different geographical origins

The TRIM5α restriction factor can protect some species of monkeys, but not humans, from HIV infection. It has also emerged that some monkeys have a cyclophilin A domain retrotransposed into the TRIM5 locus resulting in the expression of a TRIMCyp protein with anti-retroviral activity. A high degree of sequence variation in the primate TRIM5 gene has been reported that varies between populations of rhesus macaques, a widely used non-human primate model of HIV/AIDS, and recently shown to correlate with susceptibility to simian immunodeficiency viruses in this species. Cynomolgus macaques are also used widely in HIV research. A non-indigenous population on Mauritius has highly restricted genetic diversity compared with macaques from Indonesia. The relative allelic diversity of TRIM5α and TRIMCyp within these two sub-populations may impact on the susceptibility of the macaques to simian immunodeficiency virus thereby influencing the outcome of studies using these monkeys. We sought to establish the genetic diversity of these alleles in cynomolgus macaques. We identified seven TRIM5α alleles in Indonesian macaques, three of which are novel, but only three in the Mauritian-origin macaques. Strikingly, 87% of Indonesian, but none of the Mauritian macaques, possessed a retrotransposed Cyp domain. A splice acceptor site single-nucleotide polymorphism that allows formation of a TRIMCyp protein was absent for the TRIM5α alleles found in the Mauritian macaques. The level of allelic diversity reported here is greater than previously proposed for cynomolgus macaque species.     

Pathogenicity and mucosal transmissibility of the R5-tropic simian/human immunodeficiency virus SHIV(AD8) in rhesus macaques: implications for use in vaccine studies

There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIV_AD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIV_AD8-CE8J, SHIV_AD8-CK15, and SHIV_AD8-CL98) exhibited robust replication in vivo and caused marked depletion of CD4⁺ T cells affecting both memory and naïve CD4⁺ T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIV_AD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections (Pneumocystis, Candida, and Mycobacterium). A single but unique founder virus, also present in the SHIV_AD8-CE8J swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIV_AD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4⁺ T lymphocytes in vivo, and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments.     

Nonhuman primate intestinal villous M-like cells: an effective poliovirus entry site

Humans and some Old World monkeys, chimpanzees, and cynomolgus macaques, are susceptible to oral poliovirus (PV) infection. Interestingly, rhesus macaques, although sensitive to injected PV, are not susceptible to gut infection. Not much is known about the initial event of gut infection by PV in rhesus macaques so far. Here, we show that PV can efficiently enter the lamina propria (LP) by penetrating across intestinal villous M-like cells in rhesus macaques. We found by immunofluorescence analysis that PV effectively invades LP rather than germinal centers (GCs) in rhesus macaques despite expressing PV receptor CD155 on cells within GCs and LP. Furthermore, energy dispersive X-ray spectroscopy demonstrated that gold-labeled PV is spatiotemporally internalized into villous M-like cells and engulfed by macrophage-like cells in LP. These results suggest that rhesus macaques may be resistant to productive gut PV infection owing to a defective translocation of PV to GCs.     

Transmission of Sarcocystis suihominis from humans to swine to nonhuman primates (Pan troglodytes, Macaca mulatta, Macaca irus).

Sporocysts of Sarcocystis suihominis obtained from human feces were used to infect swine. Heart, tongue, and skeletal muscle from experimentally infected and noninfected control swine were fed via stomach tube to nonhuman primates including chimpanzees (Pan troglodytes), rhesus monkeys (Macaca mulatta), and cynomolgus monkeys (Macaca irus). All primates fed infected swine tissues shed sporocysts beginning 13 to 15 days postinfection and were still shedding sporocysts at the conclusion of the experiment, 30 days postinfection. Rhesus and cynomolgus monkeys were fed infected swine tissues a second time and shed sporocysts. All primates remained in good health throughout both experiments and exhibited no unusual clinical signs as a result of infection.     

Divergence and diversity of ULBP2 genes in rhesus and cynomolgus macaques

Non-human primates such as rhesus macaque and cynomolgus macaque are important animals for medical research fields and they are classified as Old World monkey, in which genome structure is characterized by gene duplications. In the present study, we investigated polymorphisms in two genes for ULBP2 molecules that are ligands for NKG2D. A total of 15 and 11 ULBP2.1 alleles and 11 and 10 ULBP2.2 alleles were identified in rhesus macaques and cynomolgus macaques, respectively. Nucleotide sequences of exons for extra cellular domain were highly polymorphic and more than 70 % were non-synonymous variations in both ULBP2.1 and ULBP2.2. In addition, phylogenetic analyses revealed that the ULBP2.2 was diverged from a branch of ULBP2.1 along with ULBP2s of higher primates. Moreover, when 3D structural models were constructed for the rhesus ULBP2 molecules, residues at presumed contact sites with NKG2D were polymorphic in ULBP2.1 and ULBP2.2 in the rhesus macaque and cynomolgus macaque, respectively. These observations suggest that amino acid replacements at the interaction sites with NKG2D might shape a specific nature of ULBP2 molecules in the Old World monkeys.     

Serological survey for two simian retroviruses in macaques and African green monkeys

Colonies of nonhuman primates at the Bowman Gray School of Medicine (BGSM) were tested for antibodies to two retroviruses associated with immunodeficiency by indirect immunofluorescence (IFA) and western blot. A total of 471 cynomolgus macaques (Macaca fascicularis), 144 rhesus monkeys (M. mulatta) and 67 stumptail monkey M. arctoides) were tested for SRV-1, and 152 African green monkeys (Cercopithecus aethiops) were tested for SIV. Of the macaques tested, 170 (36%) cynomolgus, 5 (3%) rhesus and 8 (12%) stumptails were positive for SRV-1 antibodies by IFA. Of the African green monkeys, 54 (36%) were IFA positive for SIV antibodies. A total of 143 African green monkeys tested by IFA also were tested by western blot. In the African green monkeys, the IFA had a positive predictive value of 98% and a negative predictive value of 96%. Of 176 IFA positive macaque sera tested by western blot, 49 (28%) were positive, 55 (31%) were considered equivocal (only one band, usually to p27 core protein), and 72 (41%) were negative.     

Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis

Abstract: Cross-reactivity between several commercially available mouse antihuman monoclonal antibodies (mAbs), conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC) fluorochromes, and peripheral blood leukocyte surface antigens, has been established in infant cynomolgus (Macaca fascicularis) monkeys using whole blood lysis, and two-color, PE and FITC flow cytometric analysis. With the exception of the CD8 marker, the bivariate dot-plot patterns for all other markers were similar in infant monkeys and in humans. For the CD8 marker, however, a CD8⁺CD2^? population of cells was observed in the majority of monkeys tested (10 out of 12). The number of CD8⁺CD2^? cells was higher (13%) in infant monkeys compared to the 3% reported for adult human blood. The mean percentage and absolute numbers for the cell surface markers identified with the human mAbs CD2 (FITC, Ortho, Paritan, NJ), CD4 (PE, B-D, Mountain View, CA), and CD8 (PE, B-D) when these were combined with a series of PE- or FITC-labelled human mAbs were similar across all combinations tested. Statistically significant differences were observed between male and female monkeys for the mean percentage levels of CD4 (females > males) and for the CD4/CD8 ratio (females > males). Such gender differences need to be taken into consideration when infant cynomolgus monkeys are used as models for chemical-induced immunotoxicity studies. Measurement of the interleukin-2 (IL-2) and transferrin proved to be useful in monitoring in vitro cellular activation in infant cynomolgus and possibly in rhesus (M. mulatta) monkeys.     

Some Evolutionary Aspects of the Food-Procuring Group Behavior in Phylogenesis of Primates

The peculiarities were studied of the intragroup competitive and cooperative behavior of primates differing by the level of phylogenetic development, beginning from low-organized species of the higher primates to high-organized anthropoid monkeys (Callithrix jacchus, Cebus apella, Macaca mulatta, Papio hamaofrijas, Pan troglodytes). It has been shown that in all monkeys, under conditions of competitive feeding situation, there were changes of the complex behavioral forms observed at individual learning. Under conditions of the activity in pairs, the females of platyrrhine (New World) and lower narrow-nose (Old World) monkeys lost the possibility to realize the skill and food needs, so the programmed tasks were solved predominantly by the males. In chimpanzees, the change of indices of instrumental activity involved to the equal degree the male and female, and solution of the alternation task was higher in individual experiments. The age differences were revealed in the competitive behavior of chimpanzees in group. The realization of formed skills depended on the complex of factors including the requirements dominating in the given situation and the age and typological peculiarities, as well as the social rank of each monkey in the community. In rhesus macaques the capability for cooperative interaction was revealed under conditions of complex operant activity.     

Pathogenicity of simian-human immunodeficiency virus SHIV-89.6P and SIVmac is attenuated in cynomolgus macaques and associated with early T-lymphocyte responses

Because most studies of AIDS pathogenesis in nonhuman primates have been performed in Indian-origin rhesus macaques (Macaca mulatta), little is known about lentiviral pathogenicity and control of virus replication following infection of alternative macaque species. Here, we report the consequences of simian-human immunodeficiency virus SHIV-89.6P and SIVmac251 infection in cynomolgus (Macaca fascicularis) and rhesus macaques of Chinese origin. Compared to the pathogenicity of the same viruses in Indian rhesus macaques, both cynomolgus and Chinese rhesus macaques showed lower levels of plasma virus. By 9 to 10 months after infection, both viruses became undetectable in plasma more frequently in cynomolgus than in either Chinese or Indian rhesus macaques. Furthermore, after SHIV-89.6P infection, CD4+ T-cell numbers declined less and survival was longer in cynomolgus and Chinese rhesus macaques than in Indian rhesus macaques. This attenuated pathogenicity was associated with gamma interferon ELISPOT responses to Gag and Env that were generated earlier and of higher frequency in cynomolgus than in Indian rhesus macaques. Cynomolgus macaques also developed higher titer neutralizing antibodies against SHIV-89.6 at 10 and 20 weeks postinoculation than Indian rhesus macaques. These studies demonstrate that the pathogenicity of nonhuman primate lentiviruses varies markedly based on the species or geographic origin of the macaques infected and suggest that the cellular immune responses may contribute to the control of pathogenicity in cynomolgus macaques. While cynomolgus and Chinese rhesus macaques provide alternative animal models of lentiviral infection, the lower levels of viremia in cynomolgus macaques limit the usefulness of infection of this species for vaccine trials that utilize viral load as an experimental endpoint.     

Biologic data of Macaca mulatta, Macaca fascicularis, and Saimiri sciureus used for research at the Fiocruz primate center

Physiological parameters of laboratory animals used for biomedical research is crucial for following several experimental procedures. With the intent to establish baseline biologic parameters for non-human primates held in closed colonies, hematological and morphometric data of captive monkeys were determined. Data of clinically healthy rhesus macaques (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis), and squirrel monkeys (Saimiri sciureus) were collected over a period of five years. Animals were separated according to sex and divided into five age groups. Hematological data were compared with those in the literature by Student's t test. Discrepancies with significance levels of 0.1, 1 or 5% were found in the hematological studies. Growth curves showed that the sexual dimorphism of rhesus monkeys appeared at an age of four years. In earlier ages, the differences between sexes could not be distinguished (p < 0.05). Sexual dimorphism in both squirrel monkeys and cynomolgus monkeys occurred at an age of about 32 months. Data presented in this paper could be useful for comparative studies using primates under similar conditions.     

Comparison of allele O sequences of the human and non-human primate ABO system

 Like humans, non-human primates express the antigens A and B of the ABO histoblood group system. In chimpanzees, only A and O types are found, while the types A, B, AB, and O are found in macaques. The sequences of exons 6 and 7 of two chimpanzee O alleles (O ^del and O ^x ), two macaque species O alleles (rhesus monkey and crab-eating macaque), and sequences of exon 7 of two major chimpanzee A alleles (A ^1ch and A ^2ch ) were established. The sequences of cDNAs corresponding to the chimpanzee and rhesus monkey O alleles were characterized from exon 1 to 7 and from exon 4 to 7, respectively. A comparison of our results with ABO gene sequences already published by others demonstrates that human and non-human primate O alleles are species-specific and result from independent silencing mutations. These observations reinforce the hypothesis that the maintenance of the ABO gene polymorphism in primates reflects convergent evolution more than transpecies inheritance of ancestor alleles. Received: 30 July 1998 / Revised: 12 December 1998     

High frequency of triplicatedα-globin genes in tropical primates,crab-eating macaques (Macaca fascicularis), chimpanzees (Pan troglodytes), and orang-utans (Pongo pygmaeus)

Chimpanzees and orang-utans had triplicatedα-globin gene haplotypes in the frequency of 0.80 and 0.20, respectively. Homozygous duplicated haplotypes could not be found in any of the 44 chimpanzees examined. Chimpanzees having homozygous triplicated haplotypes have greater numbers of red blood cells than those chimpanzees heterozygous for the duplicate and triplicate haplotype. Crab-eating macaques in Malayan peninsula of Thailand had triplicated haplotypes occurring in frequencies ranging from 0.13 to 0.50. On the other hand, triplicated haplotypes occurred in very low frequencies (0–0.07) in crab-eating macaques in the northern and eastern part of Thailand as well as in rhesus macaques from India and China, and in Japanese macaques.