Identification of novel mammalian viruses in tree shrews ( Tupaia belangeri chinensis).

The Chinese tree shrew ( Tupaia belangeri chinensis), a member of the mammalian order Scandentia, exhibits considerable similarities with primates, including humans, in aspects of its nervous, immune, and metabolic systems. These similarities have established the tree shrew as a promising experimental model for biomedical research on cancer, infectious diseases, metabolic disorders, and mental health conditions. Herein, we used meta-transcriptomic sequencing to analyze plasma, as well as oral and anal swab samples, from 105 healthy asymptomatic tree shrews to identify the presence of potential zoonotic viruses. In total, eight mammalian viruses with complete genomes were identified, belonging to six viral families, including Flaviviridae, Hepeviridae, Parvovirinae, Picornaviridae, Sedoreoviridae, and Spinareoviridae. Notably, the presence of rotavirus was recorded in tree shrews for the first time. Three viruses - hepacivirus 1, parvovirus, and picornavirus - exhibited low genetic similarity (<70%) with previously reported viruses at the whole-genome scale, indicating novelty. Conversely, three other viruses - hepacivirus 2, hepatovirus A and hepevirus - exhibited high similarity (>94%) to known viral strains. Phylogenetic analyses also revealed that the rotavirus and mammalian orthoreovirus identified in this study may be novel reassortants. These findings provide insights into the diverse viral spectrum present in captive Chinese tree shrews, highlighting the necessity for further research into their potential for cross-species transmission.

Cognitive Deficits and Alzheimer's Disease-Like Pathologies in the Aged Chinese Tree Shrew.

Alzheimer's disease (AD) is the most common chronic progressive neurodegenerative disease in the elderly. It has an increasing prevalence and a growing health burden. One of the limitations in studying AD is the lack of animal models that show features of Alzheimer's pathogenesis. The tree shrew has a much closer genetic affinity to primates than to rodents and has great potential to be used for research into aging and AD. In this study, we aimed to investigate whether tree shrews naturally develop cognitive impairment and major AD-like pathologies with increasing age. Pole-board and novel object recognition tests were used to assess the cognitive performance of adult (about 1 year old) and aged (6 years old or older) tree shrews. The main AD-like pathologies were assessed by Western blotting, immunohistochemical staining, immunofluorescence staining, and Nissl staining. Our results showed that the aged tree shrews developed an impaired cognitive performance compared to the adult tree shrews. Moreover, the aged tree shrews exhibited several age-related phenotypes that are associated with AD, including increased levels of amyloid-ß (Aß) accumulation and phosphorylated tau protein, synaptic and neuronal loss, and reactive gliosis in the cortex and the hippocampal tissues. Our study provides further evidence that the tree shrew is a promising model for the study of aging and AD.

Yersiniabactin-Producing E. coli Induces the Pyroptosis of Intestinal Epithelial Cells via the NLRP3 Pathway and Promotes Gut Inflammation.

The high-pathogenicity island (HPI) was initially identified in Yersinia and can be horizontally transferred to Escherichia coli to produce yersiniabactin (Ybt), which enhances the pathogenicity of E. coli by competing with the host for Fe3+. Pyroptosis is gasdermin-induced necrotic cell death. It involves the permeabilization of the cell membrane and is accompanied by an inflammatory response. It is still unclear whether Ybt HPI can cause intestinal epithelial cells to undergo pyroptosis and contribute to gut inflammation during E. coli infection. In this study, we infected intestinal epithelial cells of mice with E. coli ZB-1 and the Ybt-deficient strain ZB-1Δirp2. Our findings demonstrate that Ybt-producing E. coli is more toxic and exacerbates gut inflammation during systemic infection. Mechanistically, our results suggest the involvement of the NLRP3/caspase-1/GSDMD pathway in E. coli infection. Ybt promotes the assembly and activation of the NLRP3 inflammasome, leading to GSDMD cleavage into GSDMD-N and promoting the pyroptosis of intestinal epithelial cells, ultimately aggravating gut inflammation. Notably, NLRP3 knockdown alleviated these phenomena, and the binding of free Ybt to NLRP3 may be the trigger. Overall, our results show that Ybt HPI enhances the pathogenicity of E. coli and induces pyroptosis via the NLRP3 pathway, which is a new mechanism through which E. coli promotes gut inflammation. Furthermore, we screened drugs targeting NLRP3 from an existing drug library, providing a list of potential drug candidates for the treatment of gut injury caused by E. coli.

Hemolysin Co-Regulatory Protein 1 Enhances the Virulence of Clinically Isolated Escherichia coli in KM Mice by Increasing Inflammation and Inducing Pyroptosis.

Hemolysin-coregulated protein 1 (Hcp1) is an effector released by the type VI secretion system (T6SS) in certain pathogenic strains of Escherichia coli (E. coli) that causes apoptosis and contributes to the development of meningitis. The exact toxic consequences of Hcp1 and whether it intensifies the inflammatory response by triggering pyroptosis are yet unknown. Here, utilizing the CRISPR/Cas9 genome editing method, we removed the gene expressing Hcp1 from wild-type E. coli W24 and examined the impact of Hcp1 on E. coli virulence in Kunming (KM) mice. It was found that Hcp1-sufficient E. coli was more lethal, exacerbating acute liver injury (ALI) and acute kidney injury (AKI) or even systemic infections, structural organ damage, and inflammatory factor infiltration. These symptoms were alleviated in mice infected with W24Δhcp1. Additionally, we investigated the molecular mechanism by which Hcp1 worsens AKI and found that pyroptosis is involved, manifested as DNA breaks in many renal tubular epithelial cells. Genes or proteins closely related to pyroptosis are abundantly expressed in the kidney. Most importantly, Hcp1 promotes the activation of the NLRP3 inflammasome and the expression of active caspase-1, thereby cleaving GSDMD-N and accelerating the release of active IL-1ß and ultimately leading to pyroptosis. In conclusion, Hcp1 enhances the virulence of E. coli, aggravates ALI and AKI, and promotes the inflammatory response; moreover, Hcp1-induced pyroptosis is one of the molecular mechanisms of AKI.

Mechanism of Thymus Rejuvenation in Elderly Macaques.

Senile thymus atrophy is an important factor leading to decreased immune function. Repairing the atrophic thymus tissue structure, rebuilding immune function, and replenishing the number of exogenous stem cells may be ideal methods. In this study, bone marrow mesenchymal stem cells were intravenously infused into elderly macaques. We found that thymus volume was substantially increased, some thymus tissue regeneration was observed, the degree of thymus tissue fibrosis decreased, collagen fiber deposition decreased, cortical and medulla structures emerged gradually, the number of apoptotic cells decreased significantly, and the expression of apoptosis-related proteins decreased. For the effects of stem cell therapy on aging-related genes, we performed transcriptomic analysis of thymus tissue. The results show the expression pattern of the tissue transcriptome tended to be similar to the thymus expression pattern in young macaques compared with the elderly group, reverse aging-related proteins. Based on the results, it is suggested that stem cell therapy is an ideal method to prevent or reverse the aging of the thymus.

Aging induces severe SIV infection accompanied by an increase in follicular CD8+ T cells with overactive STAT3 signaling.

The number of elderly people living with HIV is increasing globally, and the condition of this population is relatively complicated due to the dual effects of aging and HIV infection. However, the impact of HIV infection combined with aging on the immune homeostasis of secondary lymphoid organs remains unclear. Here, we used the simian immunodeficiency virus mac239 (SIVmac239) strain to infect six young and six old Chinese rhesus macaques (ChRMs) and compared the infection characteristics of the two groups in the chronic stage through multiplex immunofluorescence staining of lymph nodes. The results showed that the SIV production and CD4/CD8 ratio inversion in old ChRMs were more severe than those in young ChRMs in both the peripheral blood and the lymph nodes, especially when a large number of CD8+ T cells infiltrated the follicles and germinal centers. STAT3 in these follicular CXCR5+CD8+ T cells was highly activated, with high expression of granzyme B, which might be caused by the severe inflammatory milieu in the follicles of old ChRMs. This study indicates that aging may be a cofactor involved in SIV-induced immune disorders in secondary lymphoid tissues, affecting the effective antiviral activity of highly enriched follicular CXCR5+CD8+ cells.

Optimization of Milk Substitutes for the Artificial Rearing of Chinese Tree Shrews (Tupaia belangeri chinensis).

The Chinese tree shrew (Tupaia belangeri chinensis) has the potential to replace the use of non-human primates in biomedical research. To increase the availability of this species, we have undertaken the ambitious task of establishing inbred lines of the Chinese tree shrew; however, we have been hindered by a low survival rate of inbred pups. Here, we report our artificial rearing (AR) of Chinese tree shrew pups using four different milk substitutes: the formula described by Tsang and Collins (milk TC) and three commercially available milk substitutes intended for possums (milk A and milk C) and for guinea pigs (milk B). We compared the effects of these milk substitutes and maternal milk on the daily milk consumption, growth performance, and survival of the pups. We also assessed the life span and reproductive performance of the F1 individuals given the best milk substitute as compared to the maternally reared (MR) pups. Milk B was found to be appropriate for AR. Pups fed with milk B had a high survival rate at the weaning age compared to those fed with the other milk substitutes. The AR pups fed with milk B had a life span similar to that of MR pups. AR females fed with milk B had an earlier age of the first reproduction, a larger number of litters, and a higher rate of survival of the offspring at the weaning age compared with the MR females. The successful optimization of a milk substitute for AR of Chinese tree shrew pups will undoubtedly facilitate the wide usage of this experimental animal.

Characterizing the role of Tupaia DNA damage inducible transcript 3 (DDIT3) gene in viral infections.

DNA damage inducible transcript 3 (DDIT3, also known as CHOP) belongs to the CCAAT/enhancer-binding protein (C/EBP) family and plays an essential role in endoplasmic reticulum stress. Here, we characterized the potential role of the Chinese tree shrew (Tupaia belangeri chinensis) DDIT3 (tDDIT3) in viral infections. The tDDIT3 protein is highly conserved and has a species-specific insertion of the SQSS repeat upstream of the C-terminal basic-leucine zipper (bZIP) domain. Phylogenetic analysis of DDIT3 protein sequences of tree shrew and related mammals indicated a closer genetic affinity between tree shrew and primates than between tree shrew and rodents. Three positively selected sites (PSSs: Glu83, Pro93, and Ser172) were identified in tDDIT3 based on the branch-site model. Expression analysis of tDDIT3 showed a constitutively expressed level in different tissues and a significantly increased level in tree shrew cells upon herpes simplex virus type 1 (HSV-1) and Newcastle disease virus (NDV) infections. Overexpression of tDDIT3 significantly increased the production of HSV-1 and vesicular stomatitis virus (VSV) in tree shrew primary renal cells (TSPRCs), whereas tDDIT3 knockout in tree shrew stable cell line (TSR6 cells) had an inhibitory effect on virus production. The enhanced effect on viral infection by tDDIT3 was not associated with the three PSSs. Mechanistically, tDDIT3 overexpression inhibited type I IFN signaling. tDDIT3 interacted with tMAVS through CARD and PRR domains, but not with other immune-related factors such as tMDA5, tSTING and tTBK1. Collectively, our results revealed tDDIT3 as a negative regulator for virus infection.

Comprehensive annotation of the Chinese tree shrew genome by large-scale RNA sequencing and long-read isoform sequencing.

The Chinese tree shrew (Tupaia belangeri chinensis) is emerging as an important experimental animal in multiple fields of biomedical research. Comprehensive reference genome annotation for both mRNA and long non-coding RNA (lncRNA) is crucial for developing animal models using this species. In the current study, we collected a total of 234 high-quality RNA sequencing (RNA-seq) datasets and two long-read isoform sequencing (ISO-seq) datasets and improved the annotation of our previously assembled high-quality chromosome-level tree shrew genome. We obtained a total of 3 514 newly annotated coding genes and 50 576 lncRNA genes. We also characterized the tissue-specific expression patterns and alternative splicing patterns of mRNAs and lncRNAs and mapped the orthologous relationships among 11 mammalian species using the current annotated genome. We identified 144 tree shrew-specific gene families, including interleukin 6 (IL6) and STT3 oligosaccharyltransferase complex catalytic subunit B (STT3B), which underwent significant changes in size. Comparison of the overall expression patterns in tissues and pathways across four species (human, rhesus monkey, tree shrew, and mouse) indicated that tree shrews are more similar to primates than to mice at the tissue-transcriptome level. Notably, the newly annotated purine rich element binding protein A (PURA) gene and the STT3B gene family showed dysregulation upon viral infection. The updated version of the tree shrew genome annotation (KIZ version 3: TS_3.0) is available at http://www.treeshrewdb.org and provides an essential reference for basic and biomedical studies using tree shrew animal models.

Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys.

Although CRISPR/Cas9-mediated gene editing is widely applied to mimic human disorders, whether acute manipulation of disease-causing genes in the brain leads to behavioral abnormalities in non-human primates remains to be determined. Here we induced genetic mutations in MECP2, a critical gene linked to Rett syndrome (RTT) and autism spectrum disorders (ASD), in the hippocampus (DG and CA1-4) of adolescent rhesus monkeys (Macaca mulatta) in vivo via adeno-associated virus (AAV)-delivered Staphylococcus aureus Cas9 with small guide RNAs (sgRNAs) targeting MECP2. In comparison to monkeys injected with AAV-SaCas9 alone (n = 4), numerous autistic-like behavioral abnormalities were identified in the AAV-SaCas9-sgMECP2-injected monkeys (n = 7), including social interaction deficits, abnormal sleep patterns, insensitivity to aversive stimuli, abnormal hand motions, and defective social reward behaviors. Furthermore, some aspects of ASD and RTT, such as stereotypic behaviors, did not appear in the MECP2 gene-edited monkeys, suggesting that different brain areas likely contribute to distinct ASD symptoms. This study showed that acute manipulation of disease-causing genes via in vivo gene editing directly led to behavioral changes in adolescent primates, paving the way for the rapid generation of genetically engineered non-human primate models for neurobiological studies and therapeutic development.

Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in Rhesus macaques.

The 2019 novel coronavirus (SARS-CoV-2) outbreak is a major challenge for public health. SARS-CoV-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~6.4% worldwide (based on World Health Organization daily situation report). However, the dynamics of viral infection, replication and shedding are poorly understood. Here, we show that Rhesus macaques are susceptible to the infection by SARS-CoV-2. After intratracheal inoculation, the first peak of viral RNA was observed in oropharyngeal swabs one day post infection (1 d.p.i.), mainly from the input of the inoculation, while the second peak occurred at 5 d.p.i., which reflected on-site replication in the respiratory tract. Histopathological observation shows that SARS-CoV-2 infection can cause interstitial pneumonia in animals, characterized by hyperemia and edema, and infiltration of monocytes and lymphocytes in alveoli. We also identified SARS-CoV-2 RNA in respiratory tract tissues, including trachea, bronchus and lung; and viruses were also re-isolated from oropharyngeal swabs, bronchus and lung, respectively. Furthermore, we demonstrated that neutralizing antibodies generated from the primary infection could protect the Rhesus macaques from a second-round challenge by SARS-CoV-2. The non-human primate model that we established here provides a valuable platform to study SARS-CoV-2 pathogenesis and to evaluate candidate vaccines and therapeutics.

The anatomy of the skin of the Chinese tree shrew is very similar to that of human skin.

The Chinese tree shrew (Tupaia belangeri chinensis) is a small mammal closely related to primates. It has a small body size, low maintenance cost, and a relatively short reproductive cycle, all of which has made it the ideal model for the study of a variety of human diseases. In this study, we compared the anatomy of the skin of the Chinese tree shrew with that of the rhesus macaque, mouse and human, with the intention of providing the basic data required for the creation of skin disease models using this animal. Paraffin sections, hematoxylin-eosin (H&E) staining, masson staining and immunohistochemical techniques were used to examine the dorsal skin structure of the Chinese tree shrew. The epidermis was shown to be composed of 1-2 layers of cells. There were hair follicles, sebaceous glands and sweat glands in the dermis and the subcutaneous tissue, with apocrine glands being more common than eccrine glands. Both Keratin5 (KRT5) and Keratin10 (KRT10) were expressed in the skin of the Chinese tree shrew, with a localization in the cytoplasm. Overall, the skin morphology and histology of the Chinese tree shrew was basically the same as that of the human. We propose that the Chinese tree shrew has a strong potential to be used for creating animal models to help elucidate the molecular mechanisms underlying a variety of skin diseases.

Chromosomal level assembly and population sequencing of the Chinese tree shrew genome.

Chinese tree shrews (Tupaia belangeri chinensis) have become an increasingly important experimental animal in biomedical research due to their close relationship to primates. An accurately sequenced and assembled genome is essential for understanding the genetic features and biology of this animal. In this study, we used long-read single-molecule sequencing and high-throughput chromosome conformation capture (Hi-C) technology to obtain a high-qualitychromosome-scale scaffolding of the Chinese tree shrew genome. The new reference genome (KIZ version 2: TS_2.0) resolved problems in presently available tree shrew genomes and enabled accurate identification of large and complex repeat regions, gene structures, and species-specific genomic structural variants. In addition, by sequencing the genomes of six Chinese tree shrew individuals, we produced a comprehensive map of 12.8 M single nucleotide polymorphisms and confirmed that the major histocompatibility complex (MHC) loci and immunoglobulin gene family exhibited high nucleotide diversity in the tree shrew genome. We updated the tree shrew genome database (TreeshrewDB v2.0: http://www.treeshrewdb.org) to include the genome annotation information and genetic variations. The new high-quality reference genome of the Chinese tree shrew and the updated TreeshrewDB will facilitate the use of this animal in many different fields of research.

Molecular characterization of the 2',5'-oligoadenylate synthetase family in the Chinese tree shrew (Tupaia belangeri chinensis).

Virus infection induces type I interferons (IFNs) that in turn exert their pleiotropic effects through inducing a large number of interferon-stimulated genes (ISGs). The IFN-induced 2',5'-oligoadenylate synthetases (OASs) have been identified as a member of the ISGs family characterized by the ability to synthesize 2',5'-oligoadenylate (2-5A), which can induce the degradation of viral RNA by activating RNase L within the infected cells to block viral replications. In this study, we characterized the OASs of the Chinese tree shrew (Tupaia belangeri chinensis), a small mammal genetically close to primates and has the potential as animal model for viral infections. We identified 4 putative tree shrew OASs (tOASs, including tOAS1, tOAS2, tOASL1, and tOASL2) and characterized their roles in antiviral responses. Tree shrew lost tOAS3 that was presented in human and mouse. Phylogenetic analyses based on the protein sequences showed a close relationship of tOASs with those of mammals. Constitutive mRNA expression of tOASs was found in seven tissues (heart, liver, spleen, lung, kidney, small intestine and brain). Moreover, tOASs were significantly up-regulated upon various virus infections. Overexpression of tOASs significantly inhibited DNA virus and RNA virus replications in tree shrew primary renal cells. tOAS1 and tOAS2, but not tOASL1 and tOASL2, exerted their anti-HSV activity in an RNase L-dependent pathway. Collectively, our results revealed the evolutionary conservation of tOASs in tree shrew and might offer helpful information for creating viral infection models using the Chinese tree shrew.

Effects of a bone graft substitute consisting of porous gradient HA/ZrO2 and gelatin/chitosan slow-release hydrogel containing BMP-2 and BMSCs on lumbar vertebral defect repair in rhesus monkey.

Dense biomaterial plays an important role in bone replacement. However, it fails to induce bone cell migration into graft material. In the present study, a novel bone graft substitute (BGS) consisting of porous gradient hydroxyapatite/zirconia composite (PGHC) and gelatin/chitosan slow-release hydrogel containing bone morphogenetic protein 2 and bone mesenchymal stem cells was designed and prepared to repair lumbar vertebral defects. The morphological characteristics of the BGS evaluated by a scanning electron microscope showed that it had a three-dimensional network structure with uniformly distributed chitosan microspheres on the surfaces of the graft material and the interior of the pores. Then, BGS (Group A), PGHC (Group B), or autologous bone (Group C) was implanted into lumbar vertebral body defects in a total of 24 healthy rhesus monkeys. After 8 and 16 weeks, anteroposterior and lateral radiographs of the lumbar spine, microcomputed tomography, histomorphometry, biomechanical testing, and biochemical testing for bone matrix markers, including Type I collagen, osteocalcin, osteopontin, basic fibroblast growth factor, alkaline phosphatase, and vascular endothelial growth factor, were performed to examine the reparative efficacy of the BGS and PGHC. The BGS displayed excellent ability to repair the lumbar vertebral defect in rhesus monkeys. Radiography, microcomputed tomography scanning, and histomorphological characterization showed that the newly formed bone volume in the interior of the pores in the BGS was significantly higher than in the PGHC. The results of biomechanical testing indicated that the vertebral body compression strength of the PGHC implant was lower than the other implants. Reverse-transcription polymerase chain reaction and western blot analyses showed that the expression of bone-related proteins in the BGS implant was significantly higher than in the PGHC implant. The BGS displayed reparative effects similar to autologous bone. Therefore, BGS use in vertebral bone defect repair appears promising.

Long-term propagation of tree shrew spermatogonial stem cells in culture and successful generation of transgenic offspring.

Tree shrews have a close relationship to primates and have many advantages over rodents in biomedical research. However, the lack of gene manipulation methods has hindered the wider use of this animal. Spermatogonial stem cells (SSCs) have been successfully expanded in culture to permit sophisticated gene editing in the mouse and rat. Here, we describe a culture system for the long-term expansion of tree shrew SSCs without the loss of stem cell properties. In our study, thymus cell antigen 1 was used to enrich tree shrew SSCs. RNA-sequencing analysis revealed that the Wnt/ß-catenin signaling pathway was active in undifferentiated SSCs, but was downregulated upon the initiation of SSC differentiation. Exposure of tree shrew primary SSCs to recombinant Wnt3a protein during the initial passages of culture enhanced the survival of SSCs. Use of tree shrew Sertoli cells, but not mouse embryonic fibroblasts, as feeder was found to be necessary for tree shrew SSC proliferation, leading to a robust cell expansion and long-term culture. The expanded tree shrew SSCs were transfected with enhanced green fluorescent protein (EGFP)-expressing lentiviral vectors. After transplantation into sterilized adult male tree shrew's testes, the EGFP-tagged SSCs were able to restore spermatogenesis and successfully generate transgenic offspring. Moreover, these SSCs were suitable for the CRISPR/Cas9-mediated gene modification. The development of a culture system to expand tree shrew SSCs in combination with a gene editing approach paves the way for precise genome manipulation using the tree shrew.