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Trypanosoma cruzinaturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure ofT. cruziinfection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and non-human primates (NHP). Collectively these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ∼2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment forT. cruziinfection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Supplementary Data from Treatment Strategies and Mechanisms Associated with the Prevention of NASH-Associated HCC by a Toll-like Receptor 4 Inhibitor

We evaluated the cancer preventive efficacy of TAK-242, an inhibitor of Toll-like receptor 4 (TLR4), in a mouse model of hepatocellular carcinoma (HCC) occurring in the context of nonalcoholic steatohepatitis (NASH). We also assessed the cellular events associated with the preventive treatment efficacy. We tested oral administration of TAK-242, at clinically relevant but toxicity-reducing doses and scheduling, in mice with hepatocyte-specific deletion of Pten (HepPten−). The optimal dose and oral gavage formulation of TAK-242 were determined to be 30 mg/kg in 5% DMSO in 30% 2-hydroxypropyl-β-cyclodextrin. Daily oral administration of 30 mg/kg TAK-242 over 18 weeks was well tolerated and resulted in reduced development of tumors (lesions > 7.5 mm3) in HepPten− mice. This effect was accompanied by reduced macrovesicular steatosis and serum levels of alanine aminotransferase. In addition, 30 mg/kg TAK-242 daily treatment of small preexisting adenomas (lesions < 7.5 mm3) over 18 weeks, significantly reduced their progression to HCC. RNA sequencing identified 220 hepatic genes significantly altered upon TAK-242 treatment, that significantly correlated with tumor burden. Finally, cell deconvolution analysis revealed that TAK-242 treatment resulted in reduced hepatic populations of endothelial cells and myeloid-derived immune cells (Kupffer cells, Siglec-H high dendritic cells, and neutrophilic granule protein high neutrophils), while the proportion of mt-Nd4 high hepatocytes significantly increased, suggesting a decrease in hepatic inflammation and concomitant increase in mitochondrial function and oxidative phosphorylation upon TLR4 inhibition. In conclusion, this study identified treatment strategies and novel molecular and cellular mechanisms associated with the prevention of HCC in the context of NASH that merit further investigations.Prevention Relevance:Means to prevent development of HCC or progression of small adenomas to HCC in patients with NASH are urgently needed to reduce the growing mortality due to HCC. We characterized the chemopreventive effect of oral administration of the TLR4 inhibitor TAK-242 in a model of NASH-associated HCC.

Supplementary Figure from Treatment Strategies and Mechanisms Associated with the Prevention of NASH-Associated HCC by a Toll-like Receptor 4 Inhibitor

To better understand the pattern of primate genome structural variation, we sequenced and assembled using multiple long-read sequencing technologies the genomes of eight nonhuman primate species, including New World monkeys (owl monkey and marmoset), Old World monkey (macaque), Asian apes (orangutan and gibbon), and African ape lineages (gorilla, bonobo, and chimpanzee). Compared to the human genome, we identified 1,338,997 lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. Across 50 million years of primate evolution, we estimate that 819.47 Mbp or ~27% of the genome has been affected by SVs based on analysis of these primate lineages. We identify 1,607 structurally divergent regions (SDRs) wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (CARDs,ABCD7,OLAH) and new lineage-specific genes are generated (e.g.,CKAP2,NEK5) and have become targets of rapid chromosomal diversification and positive selection (e.g.,RGPDs). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species for the first time.

We evaluated the cancer preventive efficacy of TAK-242, an inhibitor of Toll-like receptor 4 (TLR4), in a mouse model of hepatocellular carcinoma (HCC) occurring in the context of nonalcoholic steatohepatitis (NASH). We also assessed the cellular events associated with the preventive treatment efficacy. We tested oral administration of TAK-242, at clinically relevant but toxicity-reducing doses and scheduling, in mice with hepatocyte-specific deletion of Pten (HepPten−). The optimal dose and oral gavage formulation of TAK-242 were determined to be 30 mg/kg in 5% DMSO in 30% 2-hydroxypropyl-β-cyclodextrin. Daily oral administration of 30 mg/kg TAK-242 over 18 weeks was well tolerated and resulted in reduced development of tumors (lesions > 7.5 mm3) in HepPten− mice. This effect was accompanied by reduced macrovesicular steatosis and serum levels of alanine aminotransferase. In addition, 30 mg/kg TAK-242 daily treatment of small preexisting adenomas (lesions < 7.5 mm3) over 18 weeks, significantly reduced their progression to HCC. RNA sequencing identified 220 hepatic genes significantly altered upon TAK-242 treatment, that significantly correlated with tumor burden. Finally, cell deconvolution analysis revealed that TAK-242 treatment resulted in reduced hepatic populations of endothelial cells and myeloid-derived immune cells (Kupffer cells, Siglec-H high dendritic cells, and neutrophilic granule protein high neutrophils), while the proportion of mt-Nd4 high hepatocytes significantly increased, suggesting a decrease in hepatic inflammation and concomitant increase in mitochondrial function and oxidative phosphorylation upon TLR4 inhibition. In conclusion, this study identified treatment strategies and novel molecular and cellular mechanisms associated with the prevention of HCC in the context of NASH that merit further investigations. Prevention Relevance: Means to prevent development of HCC or progression of small adenomas to HCC in patients with NASH are urgently needed to reduce the growing mortality due to HCC. We characterized the chemopreventive effect of oral administration of the TLR4 inhibitor TAK-242 in a model of NASH-associated HCC.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

The mammary gland contains adult stem cells that are capable of self-renewal. Although these cells hold an important role in the biology and pathology of the breast, the studies of mammary stem cells are few due to the difficulty of acquiring and expanding undifferentiated adult stem cell populations. In this study, we developed mammosphere cultures from frozen mammary cells of nulliparous cynomolgus macaques (Macaca fascicularis) as a culture system to enrich mammary stem cells. Small samples of mammary tissues were collected by surgical biopsy; cells were cultured in epithelial cell growth medium and cryopreserved. Cryopreserved cells were cultured into mammospheres, and the expression of markers for stemness was evaluated by using quantitative PCR analysis. Cells were further differentiated by using 2D and 3D approaches to evaluate morphology and organoid budding, respectively. The study showed that mammosphere culture resulted in an increase in the expression of mammary stem cell markers with each passage. In contrast, markers for epithelial cells and pluripotency decreased across multiple passages. The 2D differentiation of the cells showed heterogeneous morphology, whereas 3D differentiation allowed for organoid formation. The results indicate that mammospheres can be successfully developed from frozen mammary cells derived from breast tissue collected from nulliparous cynomolgus macaques through surgical biopsy. Because mammosphere cultures allow for the enrichment of a mammary stem cell population, this refined method provides a model for the in vitro or ex vivo study of mammary stem cells.

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with βIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with “long-hauler” symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A variety of immunological assays are performed on the phenotype and function of peripheral blood mononuclear cells (PBMCs) in a group of squirrel monkeys that were transported by road for approximately 10 hours from one facility to another. Using a panel of human antibodies and a set of standardized human immune assays, we evaluated the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-g by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time require for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on female squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A group of squirrel monkeys we transported, by truck, for approximately 10 hours. Peripheral blood mononuclear cells (PBMCs) were assayed in order to evaluate the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-γ by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time required for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.

CD36 is a multifunctional scavenger receptor and lipid transporter implicated in metabolic and inflammatory pathologies, as well as cancer progression. CD36 is known to be expressed by adipocytes and monocytes/macrophages, but its expression by T cells is not clearly established. We found that CD4 and CD8 T cells in adipose tissue and liver of humans, monkeys, and mice upregulated CD36 expression (ranging from ~5–40% CD36+), whereas little to no CD36 was expressed by T cells in blood, spleen, and lymph nodes. CD36 was expressed predominantly by resting CD38-, HLA.DR-, and PD-1- adipose tissue T cells in monkeys, and increased during high-fat feeding in mice. Adipose tissue and liver promote a distinct phenotype in resident T cells characterized by CD36 upregulation.

Chagas disease is a zoonotic vector-borne disease caused by infection with the protozoan parasite Trypanosoma cruzi. T. cruzi is found in Latin America and the Southern United States, where it infects many species, including humans and nonhuman primates (NHPs). NHPs are susceptible to natural infection and can develop clinical symptoms consistent with human disease, including Chagasic cardiomyopathy, gastrointestinal disease and transplacental transmission, leading to congenital infection. Due to evidence of Chagas transmission in Texas, this study hypothesized T. cruzi infection was present in a closed, outdoor-housed breeding colony of rhesus macaques (Macaca mulatta) located at a biomedical research facility in Central Texas. In addition, we questioned whether seropositive female rhesus macaques might experience reproductive complications consistent with maternal-fetal Chagas disease. The seroprevalence of T. cruzi infection in the colony was assessed using an Enzyme Linked Immunosorbant Assay (ELISA) to detect antibodies against Tc24 antigen as a screening assay, and a commercially available immunochromatographic test (Chagas Stat Pak) as a confirmatory assay. Retrospective serologic analysis was performed to confirm the status of all T. cruzi-infected animals between the years 2012 to 2016. The medical history of all seropositive and seronegative breeding females within the colony from 2012 to 2016 was reviewed to determine each animals' level of reproductive fitness. The percentage of T. cruzi-seropositive animals ranged from 6.7% to 9.7% in adult animals and 0% to 0.44% in juveniles or weanling animals, depending on the year. An overall 3.9% seroprevalence of T. cruzi infection was found in the total population. No significant differences in any measure of reproductive outcomes were identified between seropositive and seronegative females from 2012 to 2016. The lack of significant adverse reproductive outcomes reported here may help inform future management decisions regarding seropositive female rhesus macaques within breeding colonies.

Natural infection of captive nonhuman primates (NHPs) with Trypanosoma cruzi (agent of Chagas disease) is an increasingly recognized problem in facilities across the southern USA, with negative consequences for NHP health and biomedical research. We explored a central Texas NHP facility as a nidus of transmission by characterizing parasite discrete typing units (DTU) in seropositive rhesus macaques (Macaca mulatta), identifying the wildlife reservoirs, and characterizing vector infection. In seropositive NHPs, we documented low and intermittent concentrations of circulating T. cruzi DNA, with two DTUs in equal proportions, TcI and TcIV. In contrast, consistently high concentrations of T. cruzi DNA were found in wild mesomammals at the facility, yet rodents were PCR-negative. Strong wildlife host associations were found in which raccoons (Procyon lotor) harbored TcIV and opossums (Didelphis virginiana) harbored TcI, while skunks (Mephitis mephitis) were infected with both DTUs. Active and passive vector surveillance yielded three species of triatomines from the facility and in proximity to the NHP enclosures, with 17% T. cruzi infection prevalence. Interventions to protect NHP and human health must focus on interrupting spillover from the robust sylvatic transmission in the surrounding environment.

The establishment of a sylvatic reservoir of Zika virus (ZIKV) in the Americas is dependent on the susceptibility of primates of sufficient population density, the duration and magnitude of viremia, and their exposure to the human mosquito-borne transmission cycle. To assess the susceptibility of squirrel (Saimiri sp.) and owl monkeys (Aotus sp.) to infection, we inoculated four animals of each species with ZIKV from the current epidemic. Viremia in the absence of detectible disease was observed in both species and seroconversion occurred by day 28. ZIKV was detected in the spleen of three owl monkeys: one at 7 days postinoculation (dpi) and two at 14 dpi. This study confirms the susceptibility to ZIKV infection of two Neotropical primate species that live in close proximity to humans in South America, suggesting that they could support a widespread sylvatic ZIKV cycle there. Collectively, establishment of a ZIKV sylvatic transmission cycle in South America would imperil eradication efforts and could provide a mechanism for continued exposure of humans to ZIKV infection and disease.

The mammary gland contains adult stem cells that are capable of self-renewal and are likely target for neoplastic transformation leading to breast cancer. In this study, we developed a cell culture derived from the mammary glands of cynomolgus monkeys ( Macaca fascicularis) (MfMC) and furthermore identified the expression of markers for stemness and estrogen receptor-associated activities. We found that the primary culture can be successfully subcultured to at least 3 passages, primarily epithelial-like in morphology, the cultured cells remained heterogenous in phenotype as they expressed epithelial cell markers CD24 , CK18 , and marker for fibroblast S1004A . Importantly, the cell population also consistently expressed the markers of mammary stem cells ( ITGB1 or CD29 and ITGA6 or CD49f), mesenchymal stem cells ( CD73 and CD105 ) and pluripotency ( NANOG, OCT4, SOX2 ). In addition to this, the cells were also positive for Estrogen Receptor (ER), and ER-activated marker Trefoil Factor 1, suggesting an estrogen responsiveness of the culture model. These results indicate that our cell culture model is a reliable model for acquiring a population of cells with mammary stem cell properties and that these cultures may also serve as a reservoir from which more purified populations of stem cell populations can be isolated in the future.

Pandemic HIV-1 (group M) emerged following the cross-species transmission of a simian immunodeficiency virus from chimpanzees (SIVcpz) to humans. Primate lentiviruses (HIV/SIV) require the T cell receptor CD4 to enter into target cells. By surveying the sequence and function of CD4 in 50 chimpanzee individuals, we find that all chimpanzee CD4 alleles encode a fixed, chimpanzee-specific substitution (34T) that creates a glycosylation site on the virus binding surface of the CD4 receptor. Additionally, a single nucleotide polymorphism (SNP) has arisen in chimpanzee CD4 (68T) that creates a second glycosylation site on the same virus-binding interface. This substitution is not yet fixed, but instead alleles containing this SNP are still circulating within chimpanzee populations. Thus, all allelic versions of chimpanzee CD4 are singly glycosylated at the virus binding surface, and some allelic versions are doubly glycosylated. Doubly glycosylated forms of chimpanzee CD4 reduce HIV-1 and SIVcpz infection by as much as two orders of magnitude. Full restoration of virus infection in cells bearing chimpanzee CD4 requires reversion of both threonines at sites 34 and 68, destroying both of the glycosylation sites, suggesting that the effects of the glycans are additive. Differentially glycosylated CD4 receptors were biochemically purified and used in neutralization assays and microscale thermophoresis to show that the glycans on chimpanzee CD4 reduce binding affinity with the lentiviral surface glycoprotein, Env. These glycans create a shield that protects CD4 from being engaged by viruses, demonstrating a powerful form of host resistance against deadly primate lentiviruses.

American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Eight male M. fascicularis between 8 and 10 years of age of Chinese origin were infected through oral administration of T. cruzi-infected triatomine bugs and followed over 6 weeks. The purpose of this study was to define cellular immunological responses in T. Cruzi challenged M. fascicularis. We measured immunofluorescent staining and multifunctional T. cruzi antigen specific T-cell responses in experimental infected M. fascicularis. Significant increases in the CD4+ T-cell population and polyfunctional T cell responses were observed in all experimental-infected animals compared with the uninfected time points. We observed a decrease in absolute number of lymphocytes, T cells and T cell subsets, in early phase of infection but recovered in later period. T. cruzi antigen specific IFN-g and IL-12p40 ELISPOT showed no significant infection specific responses during the study period. Circulating cytokines measurement in plasma revealed a significant increase IFN-g and IL-1ra during infection period. In conclusion, our present findings provide the first evidence demonstrating the Trypanosoma cruzi infection by oral route.

Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys ( Aotus vociferans ) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys ( Aotus azara e and Aotus nancymaae ) also serve as functional entry receptors for early-stage isolates of HIV-1. IMPORTANCE Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.

Cellular immune responses were tested to determine the effect of fenbendazole on the function of lymphocytes from Bolivian squirrel monkeys (Samiri boliviensis boliviensis). Giardia-infected squirrel monkeys were treated with commercially available fenbendazole (FBZ)-medicated monkey chow. Immune responses were compared between historical controls (Giardia naive, untreated with FBZ (control animals)) and Giardia-infected, FBZ-treated squirrel monkeys (study animals). Peripheral blood lymphocytes from study monkeys had significantly lower stimulation indices compared to control animals when cultured in vitro with concanavalin A (Con A) (p

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 receptor of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates., The current animal model for HIV, the macaque, encodes a CD4 receptor that is non-permissive for HIV entry. This paper reveals that six primate species encode CD4 receptors compatible with HIV infection, potentially making them powerful tools for the study of HIV biology. Furthermore, weak CD4 binding is a nearly constant, and apparently selected, property of HIV circulating in the human bloodstream.

We describe the pathologic features of mural arterial dissection involving brain-supplying arteries in a 31-y-old female chimpanzee (Pan troglodytes). Several hours after examination for a possible respiratory tract infection, the chimpanzee became unresponsive, developed seizures, and died within 18 h. At necropsy, the occipital cortex of the brain had a small area of congestion, and the cerebellar cortex contained a small necrotic area. Histologic evaluation confirmed the cortical lesions and revealed an additional necrotic area in the medulla oblongata characterized by mural dissection of the brain-supplying vertebral and basilar arteries and subsequent branches. Lesions in the cortices and medulla were within areas supplied by the vertebrobasilar system. Dissection of brain-supplying arteries has been described in humans but not previously in chimpanzees (or any other NHP), suggesting that these species might be useful in understanding this condition in humans. In addition, the lesion should be added to the NHP clinician's and pathologist's differential diagnosis list for similar presentations in this species.

Chronic pain and distress are universally accepted conditions that may adversely affect an animal's quality of life (QOL) and lead to the humane euthanasia of an animal. At most research institutions and zoological parks in the USA, a veterinarian, who has physically examined the animal and reviewed the clinical records, ultimately decides when an animal has reached a humane endpoint. To aid in the difficult process of interpreting pain and distress, we have developed specific behavioural guidelines, in addition to standard clinical information, to help define unique characteristics and traits of primates to assess and promote discussion of an individual primate's QOL, and thereby, to assist in the decision-making process regarding euthanasia. These guidelines advocate the creation of a QOL team when the animal is diagnosed with a life-threatening or debilitating chronic condition, or at the time the animal is entered into a terminal study. The team compiles a list of characteristics unique to that individual animal by utilising a questionnaire and a behavioural ethogram. This list enables the team to quantitatively assess any deviations from the established normal behavioural repertoire of that individual. Concurrently, the QOL team determines the number of behavioural deviations that are needed to trigger an immediate discussion of the necessity for humane euthanasia of the animal. The team remains intact once created, and revisits the animal's condition as frequently as deemed necessary. This process improves animal welfare by continuing the quest to optimally define QOL for captive primates, and potentially for all captive animals.

Background A 4-year-old rhesus macaque presented with acute, progressive paresis of the extremities. Methods A complete blood count, serum biochemical analysis, neurologic exam and necropsy were performed. Results The clinical, histopathological, and immunohistochemical findings confirmed a high-grade intramedullary glial tumor of the spinal cord that was most consistent with an ependymoma. Conclusions We describe a case of a naturally occurring spontaneous spinal cord neoplasia in a non-human primate.

SUMMARY Mammals that hibernate (hibernators) exhibit a circannual rhythm of food intake and body mass. In the laboratory during the winter hibernation period, many hibernators enter a series of multi-day torpor bouts, dropping their body temperature to near ambient, and cease to feed even if food is present in their cage. The mechanism(s) that regulates food intake in hibernators is unclear. Recently, AMP-activated protein kinase (AMPK) has been shown to play a key role in the central regulation of food intake in mammals. We hypothesized that infusing an AMPK activator, 5-aminoimidazole-4-carboxamide 1 B-D-ribofuranoside (AICAR), intracerebroventricularly (ICV) into the third ventricle of the hypothalamus would stimulate yellow-bellied marmots (Marmota flaviventris) to feed during their hibernation season. Infusion of AICAR ICV into marmots at an ambient temperature of 22°C caused a significant (P

Evolutionary analyses have revealed that most host-encoded restriction factors against HIV have experienced virus-driven selection during primate evolution. However, HIV also depends on the function of many human proteins, called host factors, for its replication. It is not clear whether virus-driven selection shapes the evolution of host factor genes to the extent that it is known to shape restriction factor genes. We show that five out of 40 HIV host factor genes (13%) analyzed do bear strong signatures of positive selection. Some of these genes (CD4, NUP153, RANBP2/NUP358) have been characterized with respect to the HIV lifecycle, while others (ANKRD30A/NY-BR-1 and MAP4) remain relatively uncharacterized. One of these, ANKRD30A, shows the most rapid evolution within this set of genes and is induced by interferon stimulation. We discuss how evolutionary analysis can aid the study of host factors for viral replication, just as it has the study of host immunity systems.

Background The maintenance of chromosomal integrity is an essential task of every living organism and cellular repair mechanisms exist to guard against insults to DNA. Given the importance of this process, it is expected that DNA repair proteins would be evolutionarily conserved, exhibiting very minimal sequence change over time. However, BRCA1, an essential gene involved in DNA repair, has been reported to be evolving rapidly despite the fact that many protein-altering mutations within this gene convey a significantly elevated risk for breast and ovarian cancers. Results To obtain a deeper understanding of the evolutionary trajectory of BRCA1, we analyzed complete BRCA1 gene sequences from 23 primate species. We show that specific amino acid sites have experienced repeated selection for amino acid replacement over primate evolution. This selection has been focused specifically on humans and our closest living relatives, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). After examining BRCA1 polymorphisms in 7 bonobo, 44 chimpanzee, and 44 rhesus macaque (Macaca mulatta) individuals, we find considerable variation within each of these species and evidence for recent selection in chimpanzee populations. Finally, we also sequenced and analyzed BRCA2 from 24 primate species and find that this gene has also evolved under positive selection. Conclusions While mutations leading to truncated forms of BRCA1 are clearly linked to cancer phenotypes in humans, there is also an underlying selective pressure in favor of amino acid-altering substitutions in this gene. A hypothesis where viruses are the drivers of this natural selection is discussed.