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Cardiac ventricular pressure overload affects patients with congenital heart defects and can cause cardiac insufficiency. Grafts of stem cell–derived cardiomyocytes are proposed as a complementary treatment to surgical repair of the cardiac defect, aiming to support ventricular function. Here, we report successful engraftment of human induced pluripotent stem cell–derived cardiac lineage cells into the heart of immunosuppressed rhesus macaques with a novel surgical model of right ventricular pressure overload. The human troponin+ grafts were detected in low-dose (2 × 10 6 cells/kg) and high-dose (10 × 10 6 cells/kg) treatment groups up to 12 weeks post-injection. Transplanted cells integrated and progressively matched the organization of the surrounding host myocardium. Ventricular tachycardia occurred in five out of 16 animals receiving cells, with episodes of incessant tachycardia observed in two animals; ventricular tachycardia events resolved within 19 days. Our results demonstrate that grafted cardiomyocytes mature and integrate into the myocardium of nonhuman primates modeling right ventricular pressure overload.

Genetically diverse simian arteriviruses (simarteriviruses) naturally infect geographically and phylogenetically diverse monkeys, and cross-species transmission and emergence are of considerable concern. Characterization of most simarteriviruses beyond sequence analysis has not been possible because the viruses fail to propagate in the laboratory. We attempted to isolate 4 simarteriviruses, Kibale red colobus virus 1, Pebjah virus, simian hemorrhagic fever virus, and Southwest baboon virus 1, by inoculating an immortalized grivet cell line (known to replicate simian hemorrhagic fever virus), primary macaque cells, macrophages derived from macaque induced pluripotent stem cells, and mice engrafted with macaque CD34+-enriched hematopoietic stem cells. The combined effort resulted in successful virus isolation; however, no single approach was successful for all 4 simarteriviruses. We describe several approaches that might be used to isolate additional simarteriviruses for phenotypic characterization. Our results will expedite laboratory studies of simarteriviruses to elucidate virus-host interactions, assess zoonotic risk, and develop medical countermeasures.

Barbiturates and benzodiazepines are GABAA-receptor agonists and potent antiseizure medications. We reported that exposure of neonatal macaques to combination of phenobarbital and midazolam (Pb/M) for 24 h, at clinically relevant doses and plasma levels, causes widespread apoptosis affecting neurons and oligodendrocytes. Notably, the extent of injury was markedly more severe compared to shorter (8 h) exposure to these drugs. We also reported that, in the infant macaque, mild hypothermia ameliorates the apoptosis response to the anesthetic sevoflurane. These findings prompted us explore whether mild hypothermia might protect infant nonhuman primates from neuro- and gliotoxicity of Pb/M. Since human infants with seizures may receive combinations of benzodiazepines and barbiturates for days, we opted for 24 h treatment with Pb/M.Neonatal rhesus monkeys received phenobarbital intravenously, followed by midazolam infusion over 24 h under normothermia (T > 36.5 °C-37.5 °C; n = 4) or mild hypothermia (T = 35 °C-36.5 °C; n = 5). Medication doses and blood levels measured were comparable to those in human infants. Animals were euthanized at 36 h and brains examined immunohistochemically and stereologically.Treatment was well tolerated. Extensive degeneration of neurons and oligodendrocytes was seen at 36 h in both groups within neocortex, basal ganglia, hippocampus and brainstem. Mild hypothermia over 36 h (maintained until terminal perfusion) conferred no protection against the neurotoxic and gliotoxic effects of Pb/M. This is in marked contrast to our previous findings that mild hypothermia is protective in the context of a 5 h-long exposure to sevoflurane in infant macaques.These findings demonstrate that brain injury caused by prolonged exposure to Pb/M in the neonatal primate cannot be ameliorated by mild hypothermia.

α-Synuclein (α-syn) proteinopathy in the neurons of the Enteric Nervous System (ENS) is proposed to have a critical role in Parkinson’s disease (PD) onset and progression. Interestingly, the ENS of the human appendix harbors abundant α-syn and appendectomy has been linked to a decreased risk and delayed onset of PD, suggesting that the appendix may influence PD pathology. Common marmosets and rhesus macaques lack a distinct appendix (a narrow closed-end appendage with a distinct change in diameter at the junction with the cecum), yet the cecal microanatomy of these monkeys is similar to the human appendix. Sections of human appendix (n = 3) and ceca from common marmosets (n = 4) and rhesus macaques (n = 3) were evaluated to shed light on the microanatomy and the expression of PD-related proteins. Analysis confirmed that the human appendix and marmoset and rhesus ceca present thick walls comprised of serosa, muscularis externa, submucosa, and mucosa plus abundant lymphoid tissue. Across all three species, the myenteric plexus of the ENS was located within the muscularis externa with nerve fibers innervating all layers of the appendix/ceca. Expression of α-syn and tau in the appendix/cecum was present within myenteric ganglia and along nerve fibers of the muscularis externa and mucosa in all species. In the myenteric ganglia α-syn, p-α-syn, tau and p-tau immunoreactivities (ir) were not significantly different across species. The percent area above threshold of α-syn-ir and tau-ir in the nerve fibers of the muscularis externa and mucosa were greater in the human appendix than in the NHP ceca (α-syn-ir p

In this study, a traumatic spinal cord injury (TSCI) classification system is proposed using a convolutional neural network (CNN) technique with automatically learned features from electromyography (EMG) signals for a non-human primate (NHP) model. A comparison between the proposed classification system and a classical classification method (k-nearest neighbors, kNN) is also presented. Developing such an NHP model with a suitable assessment tool (i.e., classifier) is a crucial step in detecting the effect of TSCI using EMG, which is expected to be essential in the evaluation of the efficacy of new TSCI treatments. Intramuscular EMG data were collected from an agonist/antagonist tail muscle pair for the pre- and post-spinal cord lesion from five Macaca fasicularis monkeys. The proposed classifier is based on a CNN using filtered segmented EMG signals from the pre- and post-lesion periods as inputs, while the kNN is designed using four hand-crafted EMG features. The results suggest that the CNN provides a promising classification technique for TSCI, compared to conventional machine learning classification. The kNN with hand-crafted EMG features classified the pre- and post-lesion EMG data with an F-measure of 89.7% and 92.7% for the left- and right-side muscles, respectively, while the CNN with the EMG segments classified the data with an F-measure of 89.8% and 96.9% for the left- and right-side muscles, respectively. Finally, the proposed deep learning classification model (CNN), with its learning ability of high-level features using EMG segments as inputs, shows high potential and promising results for use as a TSCI classification system. Future studies can confirm this finding by considering more subjects.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) and is primarily transmitted by Aedes species mosquitoes; however, ZIKV can also be sexually transmitted. During the initial epidemic and in places where ZIKV is now considered endemic, it is difficult to disentangle the risks and contributions of sexual versus vector-borne transmission to adverse pregnancy outcomes. To examine the potential impact of sexual transmission of ZIKV on pregnancy outcome, we challenged three rhesus macaques (Macaca mulatta) three times intravaginally with 1 x 107 PFU of a low passage, African lineage ZIKV isolate (ZIKV-DAK) in the first trimester (~30 days gestational age). Samples were collected from all animals initially on days 3 through 10 post challenge, followed by twice, and then once weekly sample collection; ultrasound examinations were performed every 3-4 days then weekly as pregnancies progressed. All three dams had ZIKV RNA detectable in plasma on day 3 post-ZIKV challenge. At approximately 45 days gestation (17-18 days post-challenge), two of the three dams were found with nonviable embryos by ultrasound. Viral RNA was detected in recovered tissues and at the maternal-fetal interface (MFI) in both cases. The remaining viable pregnancy proceeded to near term (~155 days gestational age) and ZIKV RNA was detected at the MFI but not in fetal tissues. These results suggest that sexual transmission of ZIKV may represent an underappreciated risk of pregnancy loss during early gestation.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life.Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain.Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically.Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners.These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.

Minimal myelination is proposed to be a contributing factor to the preferential nigral neuronal loss in Parkinson’s disease (PD). Similar to nigral dopaminergic neurons, sympathetic neurons innervating the heart have long, thin axons which are unmyelinated or minimally myelinated. Interestingly, cardiac sympathetic loss in PD is heterogeneous across the heart, yet the spatial relationship between myelination and neurodegeneration is unknown. Here, we report the mapping of myelin basic protein (MBP) expression across the left ventricle of normal rhesus macaques (n = 5) and animals intoxicated with systemic 6-OHDA (50 mg/kg iv) to model parkinsonian cardiac neurodegeneration (n = 10). A subset of 6-OHDA-treated rhesus received daily dosing of pioglitazone (5 mg/kg po; n = 5), a PPARγ agonist with neuroprotective properties. In normal animals, MBP-immunoreactivity (-ir) was identified surrounding approximately 14% of axonal fibers within nerve bundles of the left ventricle, with more myelinated nerve fibers at the base level of the left ventricle than the apex p

Infants exposed to Zika virus (ZIKV) prenatally may develop birth defects, developmental deficits, or remain asymptomatic. It is unclear why some infants are more affected than others, although enhancement of maternal ZIKV infection via immunity to an antigenically similar virus, dengue virus (DENV), may play a role. We hypothesized that DENV immunity may worsen prenatal ZIKV infection and developmental deficits in offspring. We utilized a translational macaque model to examine how maternal DENV immunity influences ZIKV-exposed infant macaque neurodevelopment in the first month of life. We inoculated eight macaques with prior DENV infection with ZIKV, five macaques with ZIKV, and four macaques with saline. DENV/ZIKV-exposed infants had significantly worse visual orientation skills than ZIKV-exposed infants whose mothers were DENV-naive, with no differences in motor, sensory or state control development. ZIKV infection characteristics and pregnancy outcomes did not individually differ between dams with and without DENV immunity, but when multiple factors were combined in a multivariate model, maternal DENV immunity combined with ZIKV infection characteristics and pregnancy parameters predicted select developmental outcomes. We demonstrate that maternal DENV immunity exacerbates visual orientation and tracking deficits in ZIKV-exposed infant macaques, suggesting that human studies should evaluate how maternal DENV immunity impacts long-term neurodevelopment.

Listeria monocytogenes (Lm) is a common environmental bacterium that thrives on vegetation and soil matter, but can infect humans if contaminated food products are ingested, resulting in severe disease in immunosuppressed populations, including pregnant women and newborns. To better understand how the unique immunological milieu of pregnancy increases susceptibility to infection, we study listeriosis in cynomolgus macaques, a non-human primate that closely resembles humans in placentation and in the physiology, and immunology of pregnancy. Non-human primates are naturally susceptible to Lm infection, and spontaneous abortions due to listeriosis are known to occur in outdoor macaque colonies, making them ideal models to understand the disease pathogenesis and host-pathogen relationship of listeriosis. We have previously shown that Lm infection in the first trimester has a high rate of miscarriage. This study expands on our previous findings by assessing how the quantity of Lm as well as stage of pregnancy at the time of exposure may influence disease susceptibility. In the current study we inoculated a cohort of macaques with a lower dose of Lm than our previous study and although this did not result in fetal demise, there was evidence of in utero inflammation and fetal distress. Animals that were reinfected with an equivalent or higher dose of the same strain of Lm resulted in approximately half of cases continuing to term and half ending in fetal demise. These cases had inconsistent bacterial colonization of the fetal compartment, suggesting that Lm does not need to directly infect the placenta to cause adverse pregnancy outcomes. Timed surgical collection of tissues following inoculation demonstrated that transmission from mother to fetus can occur as soon as 5 days post-inoculation. Lastly, third trimester inoculation resulted in pregnancy loss in 3 out of 4 macaques, accompanied by characteristic pathology and Lm colonization. Collectively, our studies demonstrate that common laboratory culture tests may not always recover Lm despite known maternal ingestion. Notably, we also find it is possible for maternal infection to resolve in some cases with no discernible adverse outcome; however, such cases had evidence of a sterile intrauterine inflammatory response, with unknown consequences for fetal development.

Animal models of infection with Zika virus (ZIKV) are urgently needed for a better understanding of pathogenesis and for testing potential therapies. Here, the authors describe infection of rhesus macaques with an Asian-lineage ZIKV strain as a relevant animal model for studying ZIKV pathogenesis.

From 2010 to 2015, 73 common marmosets (Callithrix jacchus) housed at the Wisconsin National Primate Research Center (WNPRC) were diagnosed postmortem with lymphocytic enterocolitis. We used unbiased deep-sequencing to screen the blood of deceased enterocolitis-positive marmosets for viruses. In five out of eight common marmosets with lymphocytic enterocolitis, we discovered a novel pegivirus not present in ten matched, clinically normal controls. The novel virus, which we named Southwest bike trail virus (SOBV), is most closely related (68% nucleotide identity) to a strain of simian pegivirus A isolated from a three-striped night monkey (Aotus trivirgatus). We screened 146 living WNPRC common marmosets for SOBV, finding an overall prevalence of 34% (50/146). Over four years, 85 of these 146 animals died or were euthanized. Histological examination revealed 27 SOBV-positive marmosets from this cohort had lymphocytic enterocolitis, compared to 42 SOBV-negative marmosets, indicating no association between SOBV and disease in this cohort (p = 0.0798). We also detected SOBV in two of 33 (6%) clinically normal marmosets screened during transfer from the New England Primate Research Center, suggesting SOBV could be exerting confounding influences on comparisons of common marmoset studies from multiple colonies.

ABSTRACT Infection with Listeria monocytogenes during pregnancy is associated with miscarriage, preterm birth, and neonatal complications, including sepsis and meningitis. While the risk of these conditions is thought to be greatest during the third trimester of pregnancy, the determinants of fetoplacental susceptibility to infection, the contribution of gestational age, and the in vivo progression of disease at the maternal-fetal interface are poorly understood. We developed a nonhuman primate model of listeriosis to better understand antecedents of adverse pregnancy outcomes in early pregnancy. Four pregnant cynomolgus macaques (Macaca fascicularis) received a single intragastric inoculation between days 36 and 46 of gestation with 107 CFU of an L. monocytogenes strain isolated from a previous cluster of human listeriosis cases that resulted in adverse pregnancy outcomes. Fecal shedding, maternal bacteremia, and fetal demise were consistently noted within 7 to 13 days. Biopsy specimens of maternal liver, spleen, and lymph node displayed variable inflammation and relatively low bacterial burden. In comparison, we observed greater bacterial burden in the decidua and placenta and the highest burden in fetal tissues. Histopathology indicated vasculitis, fibrinoid necrosis, and thrombosis of the decidual spiral arteries, acute chorioamnionitis and villitis in the placenta, and hematogenous infection of the fetus. Vascular pathology suggests early impact of L. monocytogenes infection on spiral arteries in the decidua, which we hypothesize precipitates subsequent placentitis and fetal demise. These results demonstrate that L. monocytogenes tropism for the maternal reproductive tract results in infection of the decidua, placenta, and the fetus itself during the first trimester of pregnancy. IMPORTANCE Although listeriosis is known to cause significant fetal morbidity and mortality, it is typically recognized in the third trimester of human pregnancy. Its impact on early pregnancy is poorly defined. Here we provide evidence that exposure to L. monocytogenes in the first trimester poses a greater risk of fetal loss than currently appreciated. Similarities in human and nonhuman primate placentation, physiology, and reproductive immunology make this work highly relevant to human pregnancy. We highlight the concept that the maternal immune response that protects the mother from serious disease is unable to protect the fetus, a concept relevant to classic TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes) infections and newly illuminated by current Zika virus outbreaks. Studies with this model, using the well-understood organism L. monocytogenes, will permit precise analysis of host-pathogen interactions at the maternal-fetal interface and have broad significance to both recognized and emerging infections in the setting of pregnancy.

In 2021, the world marked 10 years free from rinderpest. The United Nations Food and Agriculture Organization and World Organisation for Animal Health have since made great strides in consolidating, sequencing, and destroying stocks of rinderpest virus–containing material, currently kept by only 14 known institutions. This progress must continue.

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated for ZIKV using RT-qPCR, in situ hybridization, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days after infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of paraplacental vertical ZIKV transmission through the chorionic membrane, the outer layer of the fetal membranes.

In the 2016 Zika virus (ZIKV) pandemic, a previously unrecognized risk of birth defects surfaced in babies whose mothers were infected with Asian-lineage ZIKV during pregnancy. Less is known about the impacts of gestational African-lineage ZIKV infections. Given high human immunodeficiency virus (HIV) burdens in regions where African-lineage ZIKV circulates, we evaluated whether pregnant rhesus macaques infected with simian immunodeficiency virus (SIV) have a higher risk of African-lineage ZIKV-associated birth defects. Remarkably, in both SIV+ and SIV- animals, ZIKV infection early in the first trimester caused a high incidence (78%) of spontaneous pregnancy loss within 20 days. These findings suggest a significant risk for early pregnancy loss associated with African-lineage ZIKV infection and provide the first consistent ZIKV-associated phenotype in macaques for testing medical countermeasures.

BackgroundCongenital Zika virus (ZIKV) infection can result in birth defects, including malformations in the fetal brain and visual system. There are two distinct genetic lineages of ZIKV: African and Asian. Asian-lineage ZIKVs have been associated with adverse pregnancy outcomes in humans; however, recent evidence from experimental models suggests that African-lineage viruses can also be vertically transmitted and cause fetal harm.Methodology/principal findingsTo evaluate the pathway of vertical transmission of African-lineage ZIKV, we inoculated nine pregnant rhesus macaques (Macaca mulatta) subcutaneously with 44 plaque-forming units of a ZIKV strain from Senegal, (ZIKV-DAK). Dams were inoculated either at gestational day 30 or 45. Following maternal inoculation, pregnancies were surgically terminated seven or 14 days later and fetal and maternal-fetal interface tissues were collected and evaluated. Infection in the dams was evaluated via plasma viremia and neutralizing antibody titers pre- and post- ZIKV inoculation. All dams became productively infected and developed strong neutralizing antibody responses. ZIKV RNA was detected in maternal-fetal interface tissues (placenta, decidua, and fetal membranes) by RT-qPCR and in situ hybridization. In situ hybridization detected ZIKV predominantly in the decidua and revealed that the fetal membranes may play a role in ZIKV vertical transmission. Infectious ZIKV was detected in the amniotic fluid of three pregnancies and one fetus had ZIKV RNA detected in multiple tissues. No significant pathology was observed in any fetus; and ZIKV did not have a substantial effect on the placenta.Conclusions/significanceThis study demonstrates that a very low dose of African-lineage ZIKV can be vertically transmitted to the macaque fetus during pregnancy. The low inoculating dose used in this study suggests a low minimal infectious dose for rhesus macaques. Vertical transmission with a low dose in macaques further supports the high epidemic potential of African ZIKV strains.

Countermeasures against Zika virus (ZIKV), including vaccines, are frequently tested in nonhuman primates (NHP). Macaque models are important for understanding how ZIKV infections impact human pregnancy due to similarities in placental development. The lack of consistent adverse pregnancy outcomes in ZIKV-affected pregnancies poses a challenge in macaque studies where group sizes are often small (4-8 animals). Studies in small animal models suggest that African-lineage Zika viruses can cause more frequent and severe fetal outcomes. No adverse outcomes were observed in macaques exposed to 1x104 PFU (low dose) of African-lineage ZIKV at gestational day (GD) 45. Here, we exposed eight pregnant rhesus macaques to 1x108 PFU (high dose) of African-lineage ZIKV at GD 45 to test the hypothesis that adverse pregnancy outcomes are dose-dependent. Three of eight pregnancies ended prematurely with fetal death. ZIKV was detected in both fetal and placental tissues from all cases of early fetal loss. Further refinements of this exposure system (e.g., varying the dose and timing of infection) could lead to an even more consistent, unambiguous fetal loss phenotype for assessing ZIKV countermeasures in pregnancy. These data demonstrate that high-dose exposure to African-lineage ZIKV causes pregnancy loss in macaques and also suggest that ZIKV-induced first trimester pregnancy loss could be strain-specific.

There are currently no approved drugs to treat Zika virus (ZIKV) infection during pregnancy. Hyperimmune globulin products such as VARIZIG and WinRho are FDA-approved to treat conditions during pregnancy such as Varicella Zoster virus infection and Rh-incompatibility. We administered ZIKV-specific human immune globulin as a treatment in pregnant rhesus macaques one day after subcutaneous ZIKV infection. All animals controlled ZIKV viremia following the treatment and generated robust levels of anti-Zika virus antibodies in their blood. No adverse fetal or infant outcomes were identified in the treated animals, yet the placebo control treated animals also did not have signs related to congenital Zika syndrome (CZS). Human immune globulin may be a viable prophylaxis and treatment option for ZIKV infection during pregnancy, however, more studies are required to fully assess the impact of this treatment to prevent CZS.

α-Synuclein (α-syn) proteinopathy in the neurons of the Enteric Nervous System (ENS) is proposed to have a critical role in Parkinson's disease (PD) onset and progression. Interestingly, the ENS of the human appendix harbors abundant α-syn and appendectomy has been linked to a decreased risk and delayed onset of PD, suggesting that the appendix may influence PD pathology. Common marmosets and rhesus macaques lack a distinct appendix (a narrow closed-end appendage with a distinct change in diameter at the junction with the cecum), yet the cecal microanatomy of these monkeys is similar to the human appendix. Sections of human appendix (n = 3) and ceca from common marmosets (n = 4) and rhesus macaques (n = 3) were evaluated to shed light on the microanatomy and the expression of PD-related proteins. Analysis confirmed that the human appendix and marmoset and rhesus ceca present thick walls comprised of serosa, muscularis externa, submucosa, and mucosa plus abundant lymphoid tissue. Across all three species, the myenteric plexus of the ENS was located within the muscularis externa with nerve fibers innervating all layers of the appendix/ceca. Expression of α-syn and tau in the appendix/cecum was present within myenteric ganglia and along nerve fibers of the muscularis externa and mucosa in all species. In the myenteric ganglia α-syn, p-α-syn, tau and p-tau immunoreactivities (ir) were not significantly different across species. The percent area above threshold of α-syn-ir and tau-ir in the nerve fibers of the muscularis externa and mucosa were greater in the human appendix than in the NHP ceca (α-syn-ir p

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.

Apoptosis is triggered in the developing mammalian brain by sedative, anesthetic or antiepileptic drugs during late gestation and early life. Whether human children are vulnerable to this toxicity mechanism remains unknown, as there are no imaging techniques to capture it. Apoptosis is characterized by distinct structural features, which affect the way damaged tissue scatters ultrasound compared to healthy tissue. We evaluated whether apoptosis, triggered by the anesthetic sevoflurane in the brains of neonatal rhesus macaques, can be detected using quantitative ultrasound (QUS).Neonatal (n = 15) rhesus macaques underwent 5 h of sevoflurane anesthesia. QUS images were obtained through the sagittal suture at 0.5 and 6 h. Brains were collected at 8 h and examined immunohistochemically to analyze apoptotic neuronal and oligodendroglial death.Significant apoptosis was detected in white and gray matter throughout the brain, including the thalamus. We measured a change in the effective scatterer size (ESS), a QUS biomarker derived from ultrasound echo signals obtained with clinical scanners, after sevoflurane-anesthesia in the thalamus. Although initial inclusion of all measurements did not reveal a significant correlation, when outliers were excluded, the change in the ESS between the pre- and post-anesthesia measurements correlated strongly and proportionally with the severity of apoptotic death.We report for the first time in vivo changes in QUS parameters, which may reflect severity of apoptosis in the brains of infant nonhuman primates. These findings suggest that QUS may enable in vivo studies of apoptosis in the brains of human infants following exposure to anesthetics, antiepileptics and other brain injury mechanisms.

Cardiac dysautonomia is a common nonmotor symptom of Parkinson's disease (PD) associated with loss of sympathetic innervation to the heart and decreased plasma catecholamines. Disease-modifying strategies for PD cardiac neurodegeneration are not available, and biomarkers of target engagement are lacking. Systemic administration of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) recapitulates PD cardiac dysautonomia pathology. We recently used positron emission tomography (PET) to visualize and quantify cardiac sympathetic innervation, oxidative stress, and inflammation in adult male rhesus macaques (Macaca mulatta; n = 10) challenged with 6-OHDA (50mg/kg; i.v.). Twenty-four hours post-intoxication, the animals were blindly and randomly assigned to receive daily doses of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist pioglitazone (n = 5; 5mg/kg p.o.) or placebo (n = 5). Quantification of PET radioligand uptake showed increased oxidative stress and inflammation one week after 6-OHDA which resolved to baseline levels by twelve weeks, at which time pioglitazone-treated animals showed regionally preserved sympathetic innervation. Here we report post mortem characterization of heart and adrenal tissue in these animals compared to age and sex matched normal controls (n = 5). In the heart, 6-OHDA-treated animals showed a significant loss of sympathetic nerve fibers density (tyrosine hydroxylase (TH)-positive fibers). The anatomical distribution of markers of sympathetic innervation (TH) and inflammation (HLA-DR) significantly correlated with respective in vivo PET findings across left ventricle levels and regions. No changes were found in alpha-synuclein immunoreactivity. Additionally, CD36 protein expression was increased at the cardiomyocyte intercalated discs following PPARγ-activation compared to placebo and control groups. Systemic 6-OHDA decreased adrenal medulla expression of catecholamine producing enzymes (TH and aromatic L-amino acid decarboxylase) and circulating levels of norepinephrine, which were attenuated by PPARγ-activation. Overall, these results validate in vivo PET findings of cardiac sympathetic innervation, oxidative stress, and inflammation and illustrate cardiomyocyte CD36 upregulation as a marker of PPARγ target engagement.

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

Sedatives and anesthetics can injure the developing brain. They cause apoptosis of neurons and oligodendrocytes, impair synaptic plasticity, inhibit neurogenesis and trigger long-term neurocognitive deficits. The projected vulnerable period in humans extends from the third trimester of pregnancy to the third year of life. Despite all concerns, there is no ethically and medically acceptable alternative to the use of sedatives and anesthetics for surgeries and painful interventions. Development of measures that prevent injury while allowing the medications to exert their desired actions has enormous translational value.Here we investigated protective potential of hypothermia against histological toxicity of the anesthetic sevoflurane in the developing nonhuman primate brain.Neonatal rhesus monkeys underwent sevoflurane anesthesia over 5 h. Body temperature was regulated in the normothermic (>36.5 °C), mild hypothermic (35–36.5 °C) and moderately hypothermic (

Congenital Zika virus (ZIKV) infection was first linked to birth defects during the American outbreak in 2015/2016. It has been proposed that mutations unique to the Asian/American-genotype explain, at least in part, the ability of Asian/American ZIKV to cause congenital Zika syndrome (CZS). Recent studies identified mutations in ZIKV infecting humans that arose coincident with the outbreak in French Polynesia and were stably maintained during subsequent spread to the Americas. Here we show that African ZIKV can infect and harm fetuses and that the S139N substitution that has been associated with the American outbreak is not essential for fetal harm. Our findings, in a vertical transmission mouse model, suggest that ZIKV will remain a threat to pregnant women for the foreseeable future, including in Africa, Southeast Asia, and the Americas. Additional research is needed to better understand the risks associated with ZIKV infection during pregnancy, both in areas where the virus is newly endemic and where it has been circulating for decades.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a "synthetic swarm" whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.

Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis-as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses-suggesting that HPgV's protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses-an understudied group of viruses with a high prevalence in the global human population-and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals in a previous study contemporary with the current report clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection. The results may also suggest that maternal-fetal ZIKV transmission in human pregnancy may be more frequent than currently appreciated.

Glial cell line derived neurotrophic factor (GDNF) is a neurotrophic factor that has neuroprotective effects in animal models of Parkinson's disease (PD) and has been proposed as a PD therapy. GDNF does not cross the blood brain barrier (BBB), and requires direct intracerebral delivery to be effective. Trojan horse technology, in which GDNF is coupled to a monoclonal antibody (mAb) against the human insulin receptor (HIR), has been proposed to allow GDNF BBB transport (ArmaGen Technologies Inc.). In this study we tested the feasibility of HIRMAb-GDNF to induce neuroprotection in parkinsonian monkeys, as well as its tolerability and safety. Adult rhesus macaques were assessed throughout the study with a clinical rating scale, a computerized fine motor skills task and general health evaluations. Following baseline measurements, the animals received a unilateral intracarotid artery MPTP injection. Seven days later the animals were evaluated, matched according to disability and blindly assigned to receive twice a week i.v. treatments (vehicle, 1 or 5 mg/kg HIRmAb-GDNF) for a period of three months. HIRmAb-GDNF did not improve parkinsonian motor symptoms and induced a dose-dependent hypersensitivity reaction. Quantification of dopaminergic striatal optical density and stereological nigral cell counts did not demonstrate differences between treatment groups. Focal pancreatic acinar to ductular metaplasia (ADM) was noted in four of seven animals treated with 1 mg/kg HIRmAb-GDNF; two of four with ADM also had focal pancreatic intraepithelial neoplasia 1B (PanIN-1B) lesions. Minimal to mild, focal to multifocal, nonsuppurative myocarditis was noted in all animals in the 5 mg/kg treatment group. Our results demonstrate that HIRmAb-GDNF dosing in a monkey model of PD is not an effective neuroprotective strategy and may present serious health risks that should be considered when planning future use of the IR antibody as a carrier, or of any systemic treatment of a GDNF-containing molecule.