The architecture of the simian varicella virus transcriptome.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed nanopore direct RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Dramatic transcriptomic differences in Macaca mulatta and Macaca fascicularis with Plasmodium knowlesi infections.

Plasmodium knowlesi, a model malaria parasite, is responsible for a significant portion of zoonotic malaria cases in Southeast Asia and must be controlled to avoid disease severity and fatalities. However, little is known about the host-parasite interactions and molecular mechanisms in play during the course of P. knowlesi malaria infections, which also may be relevant across Plasmodium species. Here we contrast P. knowlesi sporozoite-initiated infections in Macaca mulatta and Macaca fascicularis using whole blood RNA-sequencing and transcriptomic analysis. These macaque hosts are evolutionarily close, yet malaria-naïve M. mulatta will succumb to blood-stage infection without treatment, whereas malaria-naïve M. fascicularis controls parasitemia without treatment. This comparative analysis reveals transcriptomic differences as early as the liver phase of infection, in the form of signaling pathways that are activated in M. fascicularis, but not M. mulatta. Additionally, while most immune responses are initially similar during the acute stage of the blood infection, significant differences arise subsequently. The observed differences point to prolonged inflammation and anti-inflammatory effects of IL10 in M. mulatta, while M. fascicularis undergoes a transcriptional makeover towards cell proliferation, consistent with its recovery. Together, these findings suggest that timely detection of P. knowlesi in M. fascicularis, coupled with control of inflammation while initiating the replenishment of key cell populations, helps contain the infection. Overall, this study points to specific genes and pathways that could be investigated as a basis for new drug targets that support recovery from acute malaria.

Transcriptomic landscape of persistent diarrhoea in rhesus macaques and comparison with humans and mouse models with inflammatory bowel disease.

Diarrhoea is a widespread disease in captive rhesus macaques (Macaca mulatta) and a small proportion of individuals may experience persistent diarrhoea. Persistent diarrhoea can lead to a compromised immune system, intestinal inflammation and malnutrition. We analyzed the blood transcriptomes of 10 persistent diarrhoeal and 12 healthy rhesus macaques to investigate the gene expression differences between the two groups. We identified 330 DEGs between persistent diarrhoeal and healthy rhesus macaques. The 211 up-regulated DEGs in the diarrhoeal group were mainly enriched in immune-related and interleukin-related categories. Among them, three interleukin (IL) 18 related DEGs (IL18, IL18R1, and IL18BP) played important roles in actively regulating pro-inflammatory responses. Interestingly, the up- and down-regulated DEGs were both enriched in the same immune-related categories. Thus, we applied a new method to examine the distribution of DEGs in all child categories. We found that interleukin and T cell related categories were mainly occupied by up-regulated DEGs, while immunoglobulin production and B cell related categories were enriched by down-regulated DEGs. We also compared rhesus macaque DEGs with the DEGs of inflammatory bowel disease (IBD) humans and IBD mouse models and found that 30-40% of macaque DEGs were shared with IBD humans and mouse models. In conclusion, our results showed that there were significant immune differences between persistent diarrhoeal rhesus macaques and healthy macaques, which was similar to the expression differences in IBD patients and mouse models.

PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys.

Mutations of PTEN-induced kinase I (PINK1) cause early-onset Parkinson's disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients. This suggests that generating PINK1 disease models in non-human primates (NHPs) that are close to humans is essential to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both of which can reduce off-target effects without compromising on-target editing, are two optimized strategies in the CRISPR/Cas9 system for establishing disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target the PINK1 gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA. However, western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts. We further reprogramed mutant fibroblasts into induced pluripotent stem cells (iPSCs), which showed similar ability to differentiate into dopamine (DA) neurons. Taken together, our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.

Distinct transcriptional responses to fatal Ebola virus infection in cynomolgus and rhesus macaques suggest species-specific immune responses.

Ebola virus (EBOV) is a negative single-stranded RNA virus within the Filoviridae family and the causative agent of Ebola virus disease (EVD). Nonhuman primates (NHPs), including cynomolgus and rhesus macaques, are considered the gold standard animal model to interrogate mechanisms of EBOV pathogenesis. However, despite significant genetic similarity (>90%), NHP species display different clinical presentation following EBOV infection, notably a ∼1-2 days delay in disease progression. Consequently, evaluation of therapeutics is generally conducted in rhesus macaques, whereas cynomolgus macaques are utilized to determine efficacy of preventative treatments, notably vaccines. This observation is in line with reported differences in disease severity and host responses between these two NHP following infection with simian varicella virus, influenza A and SARS-CoV-2. However, the molecular underpinnings of these differential outcomes following viral infections remain poorly defined. In this study, we compared published transcriptional profiles obtained from cynomolgus and rhesus macaques infected with the EBOV-Makona Guinea C07 using bivariate and regression analyses to elucidate differences in host responses. We report the presence of a shared core of differentially expressed genes (DEGs) reflecting EVD pathology, including aberrant inflammation, lymphopenia, and coagulopathy. However, the magnitudes of change differed between the two macaque species. These findings suggest that the differential clinical presentation of EVD in these two species is mediated by altered transcriptional responses.

Biological implications and limitations of a cynomolgus monkey with naturally occurring Parkinson's disease.

We recently identified a cynomolgus monkey with naturally occurring Parkinson's disease (PD), indicating that PD may not be a uniquely human disease (Li et al, 2020). In our previous study, four lines of evidence, including typical PD clinical symptoms, pharmacological responses, pathological hallmarks, and genetic mutations, strongly supported the identification of a monkey with spontaneous PD (Figure 1). To the best of our knowledge, this is the first reported case of naturally developed PD in animals. This suggests that PD is not a disease restricted to humans, with its existence in a non-human primate providing a novel evolutionary angle for understanding PD. As a close relative to humans (Buffalo et al, 2019; Phillips et al, 2014; Yan et al, 2011), this rare case of PD in another primate species provides solid evidence that monkeys are ideal candidates for the development of a genuine "animal version of PD", with conserved etiology and pathogenesis (Li et al, 2020). Furthermore, it allows us to compare similarities and differences in PD development between species and to understand PD pathogenesis from an evolutionary point of view.

Spontaneous KRT5 Gene Mutation in Rhesus Macaques (Macaca mulatta): A Novel Nonhuman Primate Model of Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder characterized by increased skin and mucous membrane fragility. Most cases are caused by mutations in keratin 5 (KRT5) and keratin 14 (KRT14). Mutations of these genes result in cytoskeletal disruption of the basal keratinocytes. Gross and histopathologic findings of 2 clinically affected homozygous rhesus macaques with an insertion variant mutation in KRT5 are described and compared with 6 deceased phenotypically normal animals that were heterozygous for the KRT5 insertion variant. Animals that were homozygous for the KRT5 insertion variant were stillborn and had widespread loss of the epidermis. Microscopic examination confirmed severe ulceration and basal cell vacuolation with basilar vesicle formation in the remaining intact epidermis. Immunohistochemistry for cytokeratin 5 demonstrated lack of epidermal immunoreactivity in homozygotes. DNA sequencing identified a 34-base pair insertion variant in exon 5 of the KRT5 gene. To our knowledge, this is the first report of epidermolysis bullosa in rhesus macaques.

Mutational analysis of BFSP1, CRYBB1, GALK1, and GJA8 in captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: Congenital cataract has been reported in a colony of captive-bred vervet monkeys (Chlorocebus aethiops). METHODS: Molecular tools such as genotyping and gene expression were used to identify mutations associated with congenital cataract in this vervet colony. Beaded filament structural protein 1 (BFSP1), beta-crystallin B1 (CRYBB1), galactokinase1 (GALK1), and gap junction alpha-8 protein (GJA8) were screened, sequenced, and analyzed for mutations in 24 vervet monkeys (control and cataract). RESULTS: Five missense sequence variants were identified (V147E, A167P, L212F, N55K, and T247A), three of which were found to be potentially disease-causing. Furthermore, downregulation was observed in BFSP1, CRYBB1, and GALK1 genes. CONCLUSION: This study reports two cases of incomplete penetrance and/or uniparental disomy (L212F and T247A) in BSFP1. Mutations in BSFP1 together with three mutations in GALK1 and GJA8 were predicted to be disease-causing.

Outer retinal degeneration in two closely related Goeldi's monkeys (Callimico goeldii).

This case report comprises studies of four Goeldi's monkeys (Callimico goeldii) from the same enclosure. Globe samples from two related C goeldii (the female C goeldii and her male offspring) were available for a histopathological evaluation. Both cases presented histopathologically evident outer retinal degeneration with differences in severity. There was marked outer retinal atrophy characterized by loss of the outer and inner photoreceptor segments, and depletion of the outer retinal nuclear layer. Furthermore, we report a reduction in the thickness of the outer retinal plexiform, inner retinal nuclear layer, and inner retinal plexiform layer in these C goeldii monkeys. To the authors' knowledge, these findings have not yet been reported in wild- or captive-bred population of C goeldii.

Occurrence of Selected Zoonotic Fecal Pathogens and First Molecular Identification of Hafnia paralvei in Wild Taihangshan Macaques (Macaca mulatta tcheliensis) in China.

Rhesus macaques (Macaca mulatta) are hosts to a range of zoonotic and potentially zoonotic pathogens. The present study firstly provides a broader investigation of the presence and prevalence of zoonotic fecal pathogens in wild Taihangshan macaques, a subspecies of rhesus macaque in China. A total of 458 fecal samples were collected between September 2015 and November 2016. Fourteen genera of intestinal parasites (four genera of protozoans and ten genera of helminths) and twelve genera of bacteria were tested for using PCR amplification. The overall samples prevalence of parasitic infection was 98.25%. Entamoeba spp. (89.96%), Balantidium coli (70.09%), and Isospora spp. (28.38%) were the most prevalent protozoa, whereas the predominant prevalent helminths were Trichuris sp. (93.23%), Strongyloides spp. (73.36%), and Oesophagostomum sp. (31.66%). Ten genera of intestinal bacteria were detected in samples of rhesus macaques, including Shigella (31.66%), Escherichia coli (29.91%), Klebsiella pneumoniae (28.38%), Leptospira (26.64%), Campylobacter jejuni (18.34%), Salmonella (13.32%), etc. Eight samples (1.75%) were tested Hafnia-positive based on sequences analysis of 16S rRNA and ampC gene. This is the first molecular characterization of Hafnia infection in NHPs. Our cross-sectional prevalence study provides important information for monitoring the potential transmission of zoonotic infections from wild rhesus macaques.

Nonketotic hyperglycinemia in captive-bred Vervet monkeys (Chlorocebus aethiops) with cataracts.

BACKGROUND: Nonketotic hyperglycinemia (NKH) is a rare metabolic disorder that is characterized by high levels of glycine in plasma and cerebrospinal fluid in humans. In this study, total congenital cataract captive-bred Vervet monkeys (Chlorocebus aethiops) that are hyperglycinemic were screened to identify mutations in Bola type 3 (BOLA3), glutaredoxin 5 (GLRX5), and lipoate synthase (LIAS) genes. METHODS: Twenty-four Vervet monkeys (12 hyperglycinemic and 12 healthy controls) were selected for mutation analysis using polymerase chain reaction (PCR), Sanger sequencing, and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Novel sequence variants were identified in BOLA3 (R23H and Q38R) and LIAS (R369I and A371A), and gene expression in the control group was significantly lower compared to the hyperglycinemic group (P < 0.05). CONCLUSION: The data obtained from this study will contribute to generation of new knowledge regarding the involvement of these genes in NKH development.

Assessment of DNA Methylation Patterns in the Bone and Cartilage of a Nonhuman Primate Model of Osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) affects humans and several other animals. Thus, the mechanisms underlying this disorder, such as specific skeletal tissue DNA methylation patterns, may be evolutionary conserved. However, associations between methylation and OA have not been readily studied in nonhuman animals. Baboons serve as important models of disease and develop OA at rates similar to those in humans. Therefore, this study investigated the associations between methylation and OA in baboons to advance the evolutionary understanding of OA. DESIGN: Trabecular bone and cartilage was collected from the medial condyles of adult female baboon femora, 5 with and 5 without knee OA. The Infinium HumanMethylation450 BeadChip (450K array) was used to identify DNA methylation patterns in these tissues. RESULTS: Approximately 44% of the 450K array probes reliably align to the baboon genome, contain a CpG site of interest, and maintain a wide distribution throughout the genome. Of the 2 filtering methods tested, both identified significantly differentially methylated positions (DMPs) between healthy and OA individuals in cartilage tissues, and some of these patterns overlap with those previously identified in humans. Conversely, no DMPs were found between tissue types or between disease states in bone tissues. CONCLUSIONS: Overall, the 450K array can be used to measure genome-wide DNA methylation in baboon tissues and identify significant associations with complex traits. The results of this study indicate that some DNA methylation patterns associated with OA are evolutionarily conserved, while others are not. This warrants further investigation in a larger and more phylogenetically diverse sample set.

Developmental and family history-based analysis of congenital fused labia phenotype in the captive common marmoset (Callithrix jacchus).

BACKGROUND: Congenital fused labia (CFL) is defined as a failure or significant delay in the opening of the juvenile sealed labia majora. This phenotype is known to be variably common in adult captive female marmosets but has never been investigated in detail before. MATERIALS AND METHODS: Here, we define, describe and quantify the variations in the degree of closure of the vulva in 122 captive marmosets (Callithrix jacchus) from 1.2 to 42 months old and include colony analysis. RESULTS: There was a negative correlation between the degree of labial fusion and animal age after prepubertal period (P < 0.05). CFL females had higher number CFL relatives (4.3 ± 0.6 vs 2.4 ± 0.5 for non-CFL, P < 0.05) and more external ancestors compared to non-CFL (P < 0.05). CONCLUSIONS: Our results therefore suggest that CFL phenotype is most likely associated with epigenetic effects induced by the captive environment and colony management strategy of extensive crossing of family lines to promote heterozygosity.

Simian Varicella Virus Infects Enteric Neurons and α4ß7 Integrin-Expressing Gut-Tropic T-Cells in Nonhuman Primates.

The pathogenesis of enteric zoster, a rare debilitating complication of reactivation of latent varicella-zoster virus (VZV) in the enteric nervous system (ENS), is largely unknown. Infection of monkeys with the closely related Varicellovirus simian varicella virus (SVV) mimics VZV disease in humans. In this study, we determined the applicability of the SVV nonhuman primate model to study Varicellovirus infection of the ENS. We confirmed VZV infection of the gut in latently infected adults and demonstrated that SVV DNA was similarly present in gut of monkeys latently infected with SVV using quantitative real-time PCR. In situ analyses showed that enteric neurons expressed SVV open reading frame (ORF) 63 RNA, but not viral nucleocapsid proteins, suggestive of latent ENS infection. During primary infection, SVV-infected T-cells were detected in gut-draining mesenteric lymph nodes and located in close vicinity to enteric nerves in the gut. Furthermore, flow cytometric analysis of blood from acutely SVV-infected monkeys demonstrated that virus-infected T-cells expressed the gut-homing receptor α4ß7 integrin. Collectively, the data demonstrate that SVV infects ENS neurons during primary infection and supports the role of T-cells in virus dissemination to the gut. Because SVV reactivation can be experimentally induced, the SVV nonhuman primate model holds great potential to study the pathogenesis of enteric zoster.

RNA-seq analysis of the transcriptome of the liver of cynomolgus monkeys with type 2 diabetes.

Genetic and environmental factors such as high-fat diet are involved in the development of type 2 diabetes mellitus (T2DM). Cynomolgus monkey shares similar genetic makeup, tissue structures, physiology and metabolic function to human. This study aimed to establish T2DM model in cynomolgus monkey and compare expression profiles of hepatic genes and their associated pathways in normal cynomolgus monkeys and those with T2DM. We employed RNA-seq technique and identified 1451 differentially expressed genes (DEGs) with a false discovery rate (FDR) of 0.1% between normal and T2DM animals. KEGG pathway analysis revealed that DEGs were associated with 12 KEGG pathways (P < 0.05). Two of these pathways were associated with metabolism and five were related to immunity. Unexpected, we found ECM-receptor interaction pathway. In conclusion, our data suggest that three major pathways may be implicated in the development of T2DM, including steroid biosynthesis, immune response and ECM. Further characterization of these pathways may provide new targets for the prevention and therapy of T2DM.

Neurodegenerative disease biomarkers Aß1-40, Aß1-42, tau, and p-tau181 in the vervet monkey cerebrospinal fluid: Relation to normal aging, genetic influences, and cerebral amyloid angiopathy.

Background: The Caribbean vervet monkey (Chlorocebus aethiops sabaeus) is a potentially valuable animal model of neurodegenerative disease. However, the trajectory of aging in vervets and its relationship to human disease is incompletely understood. Methods: To characterize biomarkers associated with neurodegeneration, we measured cerebrospinal fluid (CSF) concentrations of Aß1-40, Aß1-42, total tau, and p-tau181 in 329 members of a multigenerational pedigree. Linkage and genome-wide association were used to elucidate a genetic contribution to these traits. Results: Aß1-40 concentrations were significantly correlated with age, brain total surface area, and gray matter thickness. Levels of p-tau181 were associated with cerebral volume and brain total surface area. Among the measured analytes, only CSF Aß1-40 was heritable. No significant linkage (LOD > 3.3) was found, though suggestive linkage was highlighted on chromosomes 4 and 12. Genome-wide association identified a suggestive locus near the chromosome 4 linkage peak. Conclusions: Overall, these results support the vervet as a non-human primate model of amyloid-related neurodegeneration, such as Alzheimer's disease and cerebral amyloid angiopathy, and highlight Aß1-40 and p-tau181 as potentially valuable biomarkers of these processes.

Autosomal recessive congenital cataract in captive-bred vervet monkeys (Chlorocebus aethiops).

BACKGROUND: The aim of the study was to evaluate the genetic predisposition of congenital cataract in a colony of captive-bred vervet monkeys. METHODS: Four congenital cataract genes: glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat shock transcription factor 4 (HSF4), crystallin alpha A (CRYAA) and lens intrinsic membrane protein-2 (LIM2) were screened, sequenced and analysed for possible genetic variants in 36 monkeys. Gene expression was also evaluated in these genes. RESULTS: Fifteen sequence variants were identified in the coding regions of three genes (GCNT2, HSF4 and CRYAA). Of these variations, only three were missense mutations (M258V, V16I and S24N) and identified in the GCNT2 transcripts A, B and C, respectively, which resulted in a downregulated gene expression. CONCLUSION: Although the three missense mutations in GCNT2 have a benign effect, a possibility exists that the candidate genes (GCNT2, HSF4 and CRYAA) might harbour mutations that are responsible for total congenital cataract.

Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques.

Simian varicella virus (SVV), the primate counterpart of varicella-zoster virus, causes varicella (chickenpox), establishes latency in ganglia, and reactivates to produce zoster. We previously demonstrated that a recombinant SVV expressing enhanced green fluorescent protein (rSVV.eGFP) is slightly attenuated both in culture and in infected monkeys. Here, we generated two additional recombinant SVVs to visualize infected cells in vitro and in vivo One harbors eGFP fused to the N terminus of open reading frame 9 (ORF9) (rSVV.eGFP-2a-ORF9), and another harbors eGFP fused to the C terminus of ORF66 (rSVV.eGFP-ORF66). Both recombinant viruses efficiently expressed eGFP in cultured cells. Both recombinant SVV infections in culture were comparable to that of wild-type SVV (SVV.wt). Unlike SVV.wt, eGFP-tagged SVV did not replicate in rhesus cells in culture. Intratracheal (i.t.) or i.t. plus intravenous (i.v.) inoculation of rhesus macaques with these new eGFP-tagged viruses resulted in low viremia without varicella rash, although SVV DNA was abundant in bronchoalveolar lavage (BAL) fluid at 10 days postinoculation (dpi). SVV DNA was also found in trigeminal ganglia of one monkey inoculated with rSVV.eGFP-ORF66. Intriguingly, a humoral response to both SVV and eGFP was observed. In addition, monkeys inoculated with the eGFP-expressing viruses were protected from superinfection with SVV.wt, suggesting that the monkeys had mounted an efficient immune response. Together, our results show that eGFP expression could be responsible for their reduced pathogenesis.IMPORTANCE SVV infection in nonhuman primates has served as an extremely useful animal model to study varicella-zoster virus (VZV) pathogenesis. eGFP-tagged viruses are a great tool to investigate their pathogenesis. We constructed and tested two new recombinant SVVs with eGFP inserted into two different locations in the SVV genome. Both recombinant SVVs showed robust replication in culture but reduced viremia compared to that with SVV.wt during primary infection in rhesus macaques. Our results indicate that conclusions on eGFP-tagged viruses based on in vitro results should be handled with care, since eGFP expression could result in attenuation of the virus.