Insights into the Geographical Origins of the Cabo Verde Green Monkey.

The green monkey Chlorocebus sabaeus, L. 1766, native to West Africa, was introduced to the Cabo Verde Archipelago in the 16th century. Historical sources suggest that, due to the importance of Cabo Verde as a commercial entrepôt in the Atlantic slave trade, establishing the precise place of origin of this introduced species is challenging. Non-invasive fecal samples were collected from feral and captive green monkey individuals in Cabo Verde. Two mitochondrial fragments, HVRI and cyt b, were used to confirm the taxonomic identification of the species and to tentatively determine the geographic origin of introduction to the archipelago from the African continent. By comparing the new sequences of this study to previously published ones, it was shown that Cabo Verde individuals have unique haplotypes in the HVRI, while also showing affinities to several populations from north-western coastal Africa in the cyt b, suggesting probable multiple sources of introduction and an undetermined most probable origin. The latter is consistent with historical information, but may also have resulted from solely using mtDNA as a genetic marker and the dispersal characteristics of the species. The limitations of the methodology are discussed and future directions of research are suggested.

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences.

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.

The history, taxonomy, and geographic origins of an introduced African monkey in the southeastern United States.

The origins and taxonomy of the introduced vervet monkey population in Dania Beach, Florida has been unconfirmed due to a lack of documentation and genetic research. Our goal was to determine the introduction history, species identification, and geographic origins of the monkeys. Through interviews, historical archives, and popular media, we traced the monkeys to an escape from the Dania Chimpanzee Farm in 1948. The facility imported primates from Africa for medical research purposes. Historical archives suggest the monkeys were caught in Sierra Leone. We tested the hypothesis of West African origins using three genetic markers: one mitochondrial DNA gene (cytochrome b) and two fragments from the Y-chromosome, the sex-determining gene and the zinc-finger gene. We ran Bayesian and maximum-likelihood analyses to reconstruct phylogenetic trees. Results from all loci confirmed the species identification is Chlorocebus sabaeus. We found no variation among the sampled individuals and found the cytochrome b haplotype to be a complete match to a C. sabaeus sample from Senegal. Phylogenetic analyses showed strong support for the Dania Beach mitochondrial and Y-chromosome lineages to group within a monophyletic C. sabaeus clade endemic to West Africa. Our study provides critical baseline information to the scientific community about a little-known population of Chlorocebus monkeys that have adapted to a novel environment in the southeastern United States.

Analysis and annotation of DNA methylation in two nonhuman primate species using the Infinium Human Methylation 450K and EPIC BeadChips.

Aim: Nonhuman primates are essential for research on many human diseases. The Infinium Human Methylation450/EPIC BeadChips are popular tools for the study of the methylation state across the human genome at affordable cost. Methods: We performed a precise evaluation and re-annotation of the BeadChip probes for the analysis of genome-wide DNA methylation patterns in rhesus macaques and African green monkeys through in silico analyses combined with functional validation by pyrosequencing. Results: Up to 165,847 of the 450K and 261,545 probes of the EPIC BeadChip can be reliably used. The annotation files are provided in a format compatible with a variety of standard bioinformatic pipelines. Conclusion: Our study will facilitate high-throughput DNA methylation analyses in Macaca mulatta and Chlorocebus sabaeus.

DNA methylation changes in metabolic and immune-regulatory pathways in blood and lymph node CD4 + T cells in response to SIV infections.

The molecular mechanisms underlying HIV-induced inflammation, which persists even during effective long-term treatment, remain incompletely defined. Here, we studied pathogenic and nonpathogenic simian immunodeficiency virus (SIV) infections in macaques and African green monkeys, respectively. We longitudinally analyzed genome-wide DNA methylation changes in CD4 + T cells from lymph node and blood, using arrays. DNA methylation changes after SIV infection were more pronounced in lymph nodes than blood and already detected in primary infection. Differentially methylated genes in pathogenic SIV infection were enriched for Th1-signaling (e.g., RUNX3, STAT4, NFKB1) and metabolic pathways (e.g., PRKCZ). In contrast, nonpathogenic SIVagm infection induced DNA methylation in genes coding for regulatory proteins such as LAG-3, arginase-2, interleukin-21 and interleukin-31. Between 15 and 18% of genes with DNA methylation changes were differentially expressed in CD4 + T cells in vivo. Selected identified sites were validated using bisulfite pyrosequencing in an independent cohort of uninfected, viremic and SIV controller macaques. Altered DNA methylation was confirmed in blood and lymph node CD4 + T cells in viremic macaques but was notably absent from SIV controller macaques. Our study identified key genes differentially methylated already in primary infection and in tissues that could contribute to the persisting metabolic disorders and inflammation in HIV-infected individuals despite effective treatment.

Pseudorabies virus production using a serum-free medium in fixed-bed bioreactors with low cell inoculum density.

Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.

Unusual mtDNA Control Region Length Heteroplasmy in the COS-7 Cell Line.

The COS-7 cell line is a workhorse of virology research. To expand this cell line's utility and to enable studies on mitochondrial DNA (mtDNA) transcription and replication, we determined the complete nucleotide sequence of its mitochondrial genome by Sanger sequencing. In contrast to other available mtDNA sequences from Chlorocebus aethiops, the mtDNA of the COS-7 cell line was found to contain a variable number of perfect copies of a 108 bp unit tandemly repeated in the control region. We established that COS-7 cells are heteroplasmic with at least two variants being present: with four and five repeat units. The analysis of the mitochondrial genome sequences from other primates revealed that tandem repeats are absent from examined mtDNA control regions of humans and great apes, but appear in lower primates, where they are present in a homoplasmic state. To our knowledge, this is the first report of mtDNA length heteroplasmy in primates.

A Fine-Scale Genetic Map for Vervet Monkeys.

Despite its important biological role, the evolution of recombination rates remains relatively poorly characterized. This owes, in part, to the lack of high-quality genomic resources to address this question across diverse species. Humans and our closest evolutionary relatives, anthropoid apes, have remained a major focus of large-scale sequencing efforts, and thus recombination rate variation has been comparatively well studied in this group-with earlier work revealing a conservation at the broad- but not the fine-scale. However, in order to better understand the nature of this variation, and the time scales on which substantial modifications occur, it is necessary to take a broader phylogenetic perspective. I here present the first fine-scale genetic map for vervet monkeys based on whole-genome population genetic data from ten individuals and perform a series of comparative analyses with the great apes. The results reveal a number of striking features. First, owing to strong positive correlations with diversity and weak negative correlations with divergence, analyses suggest a dominant role for purifying and background selection in shaping patterns of variation in this species. Second, results support a generally reduced broad-scale recombination rate compared with the great apes, as well as a narrower fraction of the genome in which the majority of recombination events are observed to occur. Taken together, this data set highlights the great necessity of future research to identify genomic features and quantify evolutionary processes that are driving these rate changes across primates.

Human immunodeficiency virus type-1 Tat protein induces secretory leukocyte protease inhibitor expression in African green monkey but not human cells.

African monkeys are resistant to HIV-1 infection due to intrinsic restriction mechanisms found in their cells. However, although they can be infected by monkey-adapted modified HIV-1 particles that are designed to overcome known restriction factors, virus numbers drop to undetectable levels in immunocompetent animals. These results indicate the possibility of the presence of yet unidentified factor(s) that restrict HIV-1 in old-world monkey (OWM) cells after integration of the viral genome into the host cell chromosome. In the light of these findings, we hypothesized that OWMs might have evolved resistance mechanism(s) against HIV-1 by switching specific gene(s) on in response to the synthesis of viral proteins in infected cells. In an attempt to mimic post-infection status, we expressed HIV-1 Tat gene in African green monkey cells and compared the whole proteome with normal cells and identified secretory leukocyte protease inhibitor (SLPI), a protein with known extracellular anti-HIV-1 activity, as an over-expressed protein in the presence of HIV-1 Tat protein by 2D-PAGE and mass spectrometry analysis. We also showed that overexpression of SLPI in the presence of HIV-1 Tat was specific to monkey cells. Our results also suggest that SLPI had a previously undiscovered intracellular anti-HIV activity in addition to its extracellular activity.

Macrophage-associated wound healing contributes to African green monkey SIV pathogenesis control.

Natural hosts of simian immunodeficiency virus (SIV) avoid AIDS despite lifelong infection. Here, we examined how this outcome is achieved by comparing a natural SIV host, African green monkey (AGM) to an AIDS susceptible species, rhesus macaque (RM). To asses gene expression profiles from acutely SIV infected AGMs and RMs, we developed a systems biology approach termed Conserved Gene Signature Analysis (CGSA), which compared RNA sequencing data from rectal AGM and RM tissues to various other species. We found that AGMs rapidly activate, and then maintain, evolutionarily conserved regenerative wound healing mechanisms in mucosal tissue. The wound healing protein fibronectin shows distinct tissue distribution and abundance kinetics in AGMs. Furthermore, AGM monocytes exhibit an embryonic development and repair/regeneration signature featuring TGF-ß and concomitant reduced expression of inflammatory genes compared to RMs. This regenerative wound healing process likely preserves mucosal integrity and prevents inflammatory insults that underlie immune exhaustion in RMs.

Signature MicroRNA expression profile is associated with lipid metabolism in African green monkey.

BACKGROUND: Non-human primates (NHPs) are important models of medical research on obesity and cardiovascular diseases. As two of the most commonly used NHPs, cynomolgus macaque (CM) and African green monkey (AGM) own different capacities in lipid metabolism of which the mechanism is unknown. This study investigated the expression profiles of lipid metabolism-related microRNAs (miRNAs) in CM and AGM and their possible roles in controlling lipid metabolism-related gene expression. METHODS: By small RNA deep sequencing, the plasma miRNA expression patterns of CM and AGM were compared. The lipid metabolism-related miRNAs were validated through quantitative reverse-transcription (RT) polymerase chain reaction (PCR). Related-target genes were predicted by TargetScan and validated in Vero cells. RESULTS: Compared to CM, 85 miRNAs were upregulated with over 1.5-fold change in AGM of which 12 miRNAs were related to lipid metabolism. miR-122, miR-9, miR-185, miR-182 exhibited the greatest fold changes(fold changes are 51.2, 3.8, 3.7, 3.3 respectively; all P < 0.01). And 77 miRNAs were downregulated with over 1.5-fold change in AGM of which 3, miR-370, miR-26, miR-128 (fold changes are 9.3, 1.8, 1.7 respectively; all P < 0.05) were related to lipid metabolism. The lipid metabolism-related gene targets were predicted by TargetScan and confirmed in the Vero cells. CONCLUSION: We report for the first time a circulating lipid metabolism-related miRNA profile for CM and AGM, which may add to knowledge of differences between these two non-human primate species and miRNAs' roles in lipid metabolism.

Assessment of Male Reproductive Skew via Highly Polymorphic STR Markers in Wild Vervet Monkeys, Chlorocebus pygerythrus.

Male reproductive strategies have been well studied in primate species where the ability of males to monopolize reproductive access is high. Less is known about species where males cannot monopolize mating access. Vervet monkeys (Chlorocebus pygerythrus) are interesting in this regard as female codominance reduces the potential for male monopolization. Under this condition, we assessed whether male dominance rank still influences male mating and reproductive success, by assigning paternities to infants in a population of wild vervets in the Eastern Cape, South Africa. To determine paternity, we established microsatellite markers from noninvasive fecal samples via cross-species amplification. In addition, we evaluated male mating and reproductive success for 3 groups over 4 mating seasons. We identified 21 highly polymorphic microsatellites (number of alleles = 7.5 ± 3.1 [mean ± SD], observed heterozygosity = 0.691 ± 0.138 [mean ± SD]) and assigned paternity to 94 of 97 sampled infants (96.9%) with high confidence. Matings pooled over 4 seasons were significantly skewed across 3 groups, although skew indices were low (B index = 0.023-0.030) and mating success did not correlate with male dominance. Paternities pooled over 4 seasons were not consistently significantly skewed (B index = 0.005-0.062), with high-ranking males siring more offspring than subordinates only in some seasons. We detected 6 cases of extra-group paternity (6.4%) and 4 cases of natal breeding (4.3%). Our results suggest that alternative reproductive strategies besides priority of access for dominant males are likely to affect paternity success, warranting further investigation into the determinants of paternity among species with limited male monopolization potential.

A substrate-trapping strategy for protein phosphatase PP1 holoenzymes using hypoactive subunit fusions.

The protein Ser/Thr phosphatase PP1 catalyzes an important fraction of protein dephosphorylation events and forms highly specific holoenzymes through an association with regulatory interactors of protein phosphatase one (RIPPOs). The functional characterization of individual PP1 holoenzymes is hampered by the lack of straightforward strategies for substrate mapping. Because efficient substrate recruitment often involves binding to both PP1 and its associated RIPPO, here we examined whether PP1-RIPPO fusions can be used to trap substrates for further analysis. Fusions of an hypoactive point mutant of PP1 and either of four tested RIPPOs accumulated in HEK293T cells with their associated substrates and were co-immunoprecipitated for subsequent identification of the substrates by immunoblotting or MS analysis. Hypoactive fusions were also used to study RIPPOs themselves as substrates for associated PP1. In contrast, substrate trapping was barely detected with active PP1-RIPPO fusions or with nonfused PP1 or RIPPO subunits. Our results suggest that hypoactive fusions of PP1 subunits represent an easy-to-use tool for substrate identification of individual holoenzymes.

Characterisation of the hepatitis B virus cross-species transmission pattern via Na+/taurocholate co-transporting polypeptides from 11 New World and Old World primate species.

The hepatic Na+/taurocholate co-transporting polypeptide (NTCP in man, Ntcp in animals) is the high-affinity receptor for the hepatitis B (HBV) and hepatitis D (HDV) viruses. Species barriers for human HBV/HDV within the order Primates were previously attributed to Ntcp sequence variations that disable virus-receptor interaction. However, only a limited number of primate Ntcps have been analysed so far. In the present study, a total of 11 Ntcps from apes, Old and New World monkeys were cloned and expressed in vitro to characterise their interaction with HBV and HDV. All Ntcps showed intact bile salt transport. Human NTCP as well as the Ntcps from the great apes chimpanzee and orangutan showed transport-competing binding of HBV derived myr-preS1-peptides. In contrast, all six Ntcps from the group of Old World monkeys were insensitive to HBV myr-preS1-peptide binding and HBV/HDV infection. This is basically predetermined by the amino acid arginine at position 158 of all studied Old World monkey Ntcps. An exchange from arginine to glycine (as present in humans and great apes) at this position (R158G) alone was sufficient to achieve full transport-competing HBV myr-preS1-peptide binding and susceptibility for HBV/HDV infection. New World monkey Ntcps showed higher sequence heterogeneity, but in two cases with 158G showed transport-competing HBV myr-preS1-peptide binding, and in one case (Saimiri sciureus) even susceptibility for HBV/HDV infection. In conclusion, amino acid position 158 of NTCP/Ntcp is sufficient to discriminate between the HBV/HDV susceptible group of humans and great apes (158G) and the non-susceptible group of Old World monkeys (158R). In the case of the phylogenetically more distant New World monkey Ntcps amino acid 158 plays a significant, but not exclusive role.

Direct estimate of the spontaneous germ line mutation rate in African green monkeys.

Here, I provide the first direct estimate of the spontaneous mutation rate in an Old World monkey, using a seven individual, three-generation pedigree of African green monkeys. Eight de novo mutations were identified within ∼1.5 Gbp of accessible genome, corresponding to an estimated point mutation rate of 0.94 × 10-8 per site per generation, suggesting an effective population size of ∼12000 for the species. This estimation represents a significant improvement in our knowledge of the population genetics of the African green monkey, one of the most important nonhuman primate models in biomedical research. Furthermore, by comparing mutation rates in Old World monkeys with the only other direct estimates in primates to date-humans and chimpanzees-it is possible to uniquely address how mutation rates have evolved over longer time scales. While the estimated spontaneous mutation rate for African green monkeys is slightly lower than the rate of 1.2 × 10-8 per base pair per generation reported in chimpanzees, it is similar to the lower range of rates of 0.96 × 10-8 -1.28 × 10-8 per base pair per generation recently estimated from whole genome pedigrees in humans. This result suggests a long-term constraint on mutation rate that is quite different from similar evidence pertaining to recombination rate evolution in primates.

Ancient hybridization and strong adaptation to viruses across African vervet monkey populations.

Vervet monkeys are among the most widely distributed nonhuman primates, show considerable phenotypic diversity, and have long been an important biomedical model for a variety of human diseases and in vaccine research. Using whole-genome sequencing data from 163 vervets sampled from across Africa and the Caribbean, we find high diversity within and between taxa and clear evidence that taxonomic divergence was reticulate rather than following a simple branching pattern. A scan for diversifying selection across taxa identifies strong and highly polygenic selection signals affecting viral processes. Furthermore, selection scores are elevated in genes whose human orthologs interact with HIV and in genes that show a response to experimental simian immunodeficiency virus (SIV) infection in vervet monkeys but not in rhesus macaques, suggesting that part of the signal reflects taxon-specific adaptation to SIV.

Genetic variation and gene expression across multiple tissues and developmental stages in a nonhuman primate.

By analyzing multitissue gene expression and genome-wide genetic variation data in samples from a vervet monkey pedigree, we generated a transcriptome resource and produced the first catalog of expression quantitative trait loci (eQTLs) in a nonhuman primate model. This catalog contains more genome-wide significant eQTLs per sample than comparable human resources and identifies sex- and age-related expression patterns. Findings include a master regulatory locus that likely has a role in immune function and a locus regulating hippocampal long noncoding RNAs (lncRNAs), whose expression correlates with hippocampal volume. This resource will facilitate genetic investigation of quantitative traits, including brain and behavioral phenotypes relevant to neuropsychiatric disorders.

The Demographic and Adaptive History of the African Green Monkey.

Relatively little is known about the evolutionary history of the African green monkey (genus Chlorocebus) due to the lack of sampled polymorphism data from wild populations. Yet, this characterization of genetic diversity is not only critical for a better understanding of their own history, but also for human biomedical research given that they are one of the most widely used primate models. Here, I analyze the demographic and selective history of the African green monkey, utilizing one of the most comprehensive catalogs of wild genetic diversity to date, consisting of 1,795,643 autosomal single nucleotide polymorphisms in 25 individuals, representing all five major populations: C. a. aethiops, C. a. cynosurus, C. a. pygerythrus, C. a. sabaeus, and C. a tantalus. Assuming a mutation rate of 5.9 × 10-9 per base pair per generation and a generation time of 8.5 years, divergence time estimates range from 523 to 621 kya for the basal split of C. a. aethiops from the other four populations. Importantly, the resulting tree characterizing the relationship and split-times between these populations differs significantly from that presented in the original genome paper, owing to their neglect of within-population variation when calculating between population-divergence. In addition, I find that the demographic history of all five populations is well explained by a model of population fragmentation and isolation, rather than novel colonization events. Finally, utilizing these demographic models as a null, I investigate the selective history of the populations, identifying candidate regions potentially related to adaptation in response to pathogen exposure.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

Localized population divergence of vervet monkeys (Chlorocebus spp.) in South Africa: Evidence from mtDNA.

OBJECTIVE: Vervet monkeys are common in most tree-rich areas of South Africa, but their absence from grassland and semi-desert areas of the country suggest potentially restricted and mosaic local population patterns that may have relevance to local phenotype patterns and selection. A portion of the mitochondrial DNA control region was sequenced to study patterns of genetic differentiation. METHODS: DNA was extracted, and mitochondrial DNA sequences were obtained from 101 vervet monkeys at 15 localities, which represent both an extensive (widely across the distribution range) and intensive (more than one troop at most of the localities) sampling strategy. Analyses utilized Arlequin 3.1, MEGA 6, BEAST v1.5.2, and Network V3.6.1. RESULTS: The dataset contained 26 distinct haplotypes, with six populations fixed for single haplotypes. Pairwise P-distance among population pairs showed significant differentiation among most population pairs, but with nonsignificant differences among populations within some regions. Populations were grouped into three broad clusters in a maximum likelihood phylogenetic tree and a haplotype network. These clusters correspond to i) north-western, northern, and north-eastern parts of the distribution range as well as the northern coastal belt; ii) central areas of the country; and iii) southern part of the Indian Ocean coastal belt and adjacent inland areas. CONCLUSIONS: Apparent patterns of genetic structure correspond to current and past distribution of suitable habitat, geographic barriers to gene flow, geographic distance, and female philopatry. However, further work on nuclear markers and other genomic data are necessary to confirm these results.