Molecular Characterization and Function of the Nogo-66 Receptor (NgR1) Gene in the Chinese Tree Shrew.

BACKGROUND: Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. METHODS: In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. RESULTS: Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. CONCLUSIONS: Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.

miR-204-3p downregulates KRT16 and promotes corneal repair in tree shrew fungal keratitis model.

OBJECTIVES: Corneal repair is critical for the treatment and recovery of corneal injuries. However, the molecular mechanism underlying corneal repair remains unclear. METHODS: A tree shrew model of corneal fungal infection was established by injecting Fusarium solani into the corneal stroma to study the role of miR-204-3p in repairing corneal injury induced by fungal keratitis and to explore the potential mechanisms underlying the repair process. RESULTS: miR-204-3p expression was significantly downregulated, while KRT16 expression was significantly upregulated after F. solani infection in the cornea of tree shrews. Moreover, miR-204-3p injection promoted corneal injury repair post-infection, potentially by downregulating KRT16 expression. Results of a luciferase reporter gene assay showed that miR-204-3p had a targeted relationship with KRT16. KRT16 protein expression levels decreased after miR-204-3p injection into the cornea with fungal keratitis, reducing the degree of corneal injury. CONCLUSIONS: In this study, we report for the first time that miR-204-3p and KRT16 influence the repair of corneal injury. In addition, their effects on the repair of corneal injury were studied in a tree shrew model, providing an experimental basis for the study of pathogenesis of human fungal keratitis.

Identification of SEC14 like lipid binding 2(SEC14L2) sequence and expression profiles in the Chinese tree shrew (Tupaia belangeri chinensis).

BACKGROUND: The product of the SEC14L2 (SEC14 Like Lipid Binding 2) gene belongs to a family of lipid-binding proteins including Sec14p, alpha-tocopherol transfer protein, and cellular retinol-binding protein. SEC14L2 expression enables replication of clinical hepatitis C virus (HCV) isolates in several hepatoma cell lines, and mutations in SEC14L2 may enhance HCV replication in vitro. The Chinese tree shrew (Tupaia belangeri chinensis) is a potential animal model for studying HCV replication, however, the cDNA sequence, protein structure, and expression of the Chinese tree shrew SEC14L2 gene have yet to be characterized. METHODS AND RESULTS: In the present study, we cloned the full-length cDNA sequence of the SEC14L2 in the Chinese tree shrew by using rapid amplification of cDNA ends technology. This led us to determine that, this is 2539 base pairs (bp) in length, the open reading frame sequence is 1212 bp, and encodes 403 amino acids. Following this, we constructed a phylogenetic tree based on SEC14L2 molecules from various species and compared SEC14L2 amino acid sequence with other species. This analysis indicated that the Chinese tree shrew SEC14L2 protein (tsSEC14L2) has 96.28% amino acid similarity to the human protein, and is more closely related to the human protein than either mouse or rat protein. The Chinese tree shrew SEC14L2 mRNA was detected in all tissues, and showed highest expression levels in the pancreas, small intestine and trachea, however the tsSEC14L2 protein abundance was highest in the liver and small intestine. CONCLUSION: The Chinese tree shrew SEC14L2 gene was closer in evolutionary relation to humans and non-human primates and expression of the tsSEC14L2 protein was highest in the liver and small intestine. These results may provide useful information for tsSEC14L2 function in HCV infection.

Molecular cloning and characterization of NPC1L1 in the Chinese tree shrew (Tupaia belangeri chinensis).

BACKGROUND: The Niemann-Pick C1-Like 1 protein, a multi-transmembrane domain molecule, is critical for intestinal cholesterol absorption, and is the entry factor for hepatitis C virus (HCV). The Chinese tree shrew (Tupaia belangeri chinensis) is closer to primates in terms of genetic evolution than rodents. Previous studies indicated that the tree shrew was suitable for HCV research; however, little is known about tree shrew NPC1L1. METHODS AND RESULTS: TsNPC1L1 cDNA was amplified by rapid amplification of cDNA ends (RACE) technology. The cDNA sequence, its encoded protein structure, and expression profile were analyzed. Results indicated that the tsNPC1L1 mRNA is 4948 bp in length and encodes a 1326 amino acid protein. TsNPC1L1 possesses 84.97% identity in homology to human NPC1L1 which is higher than both mouse (80.37%) and rat (81.80%). The protein structure was also similar to human with 13 conserved transmembrane helices, and a sterol-sensing domain (SSD). Like human NPC1L1, the tsNPC1L1 mRNA transcript is highly expressed in small intestine, but it was also well-expressed in the lung and pancreas of the tree shrew. CONCLUSION: The homology of tree shrew NPC1L1 was closer to human than that of rodent NPC1L1. The expression of tsNPC1L1 was the highest in small intestine, and was detectable in lung and pancreas. These results may be useful in the study of tsNPC1L1 function in cholesterol absorption and HCV infection.

Characteristics of Angiotensin I-converting enzyme 2, type II transmembrane serine protease 2 and 4 in tree shrew indicate it as a potential animal model for SARS-CoV-2 infection.

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.

Recapitulating Zika Virus Infection in Vagina of Tree Shrew (Tupaia belangeri).

Sexual transmission of Zika Virus (ZIKV) elevates the risk of its dissemination in the female reproductive tract and causes a serious threat to the fetus. However, the available animal models are not appropriate to investigate sexual transmission, dynamics of ZIKV infection, replication, and shedding. The use of tree shrew as a small animal model of ZIKV vaginal infection was assessed in this study. A total of 23 sexually mature female tree shrews were infected with ZIKV GZ01 via the intravaginal route. There was no significant difference in change of body weight, and the temperature between ZIKV infected and control animals. Viral RNA loads were detected in blood, saliva, urine, and vaginal douching. ZIKV RNA was readily detected in vaginal lavage of 22 animals (95.65%, 22/23) at 1 dpi, and viral load ranged from 104.46 to 107.35 copies/ml, and the peak of viral load appeared at 1 dpi. The expression of key inflammatory genes, such as IL6, 8, CCL5, TNF-a, and CXCL9, was increased in the spleen of ZIKV infected animals. In the current study, female tree shrews have been successfully infected with ZIKV through the vaginal route for the first time. Interestingly, at first, ZIKV replicates at the local site of infection and then spreads throughout the host body to develop a robust systemic infection and mounted a protective immune response. This small animal model is not only valuable for exploring ZIKV sexual transmission and may also help to explain the cause of debilitating manifestations of the fetus in vivo.

Transcriptome Analysis of Choroid and Retina From Tree Shrew With Choroidal Neovascularization Reveals Key Signaling Moieties.

Pathological neovascularization in choroid, a leading cause of blindness, is a characteristic of many fundus diseases, such as diabetic retinopathy and age-related macular degeneration. The present study aimed to elucidate the key signaling pathways in choroidal neovascularization (CNV) by analyzing the mRNA profiles of choroid and retina in tree shrews with CNV. We induced choroidal angiogenesis by laser photocoagulation in 15 tree shrews and obtained mRNA profiles of their choroids and retinas by high-throughput transcriptome sequencing. Hierarchical cluster analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, hematoxylin and eosin (HE) staining, CD31 immunohistochemistry (IHC), and reverse transcription quantitative PCR (RT-qPCR) were performed. After laser photocoagulation, we obtained a total of 350 differentially expressed genes (DEGs) in the choroid, including 59 genes in Module-FASN ("ME-FASN") module and 28 genes in Module-RPL ("ME-RPL") module. A total of 69 DEGs in retina, including 20 genes in Module-SLC ("ME-SLC") module. Bioinformatics analysis demonstrated that DEGs in choroid were mainly involved in membrane transport; DEGs in "ME-RPL" were prominent in pathways associated with IgA production, antigen presentation, and cell adhesion molecules (CAMs) signaling. DEGs in "ME-FASN" were involved in fatty acid metabolism and PPAR signaling pathway, while DEGs in "ME-SLC" were involved in GABAergic synapse, neuroactive life receptor interaction, cholinergic synapse, and retrograde endocannabinoid signaling pathway. PPI network analysis demonstrated that the ribosomal protein family genes (RPL31, RPL7, RPL26L1, and RPL19) are key factors of "ME-RPL," acyl-CoA superfamily genes (ACACA, ACAT1, ACAA2, and ACACB) and FASN are key factors of "ME-FASN" and superfamily of solid carrier genes (SLC17A6, SLC32A1, SLC12A5, and SLC6A1) and complement genes (C4A, C3, and C2) are key factors of "ME-SLC." In conclusion, the present study discovered the important signal transductions (fatty acid metabolic pathway and CAMs signaling) and genes (ribosomal protein family and the complement system) in tree shrew CNV. We consider that our findings hold implications in unraveling molecular mechanisms that underlie occurrence and development of CNV.

Genome-wide DNA methylome and whole-transcriptome landscapes of spontaneous intraductal papilloma in tree shrews.

BACKGROUND: Breast intraductal papilloma (IP) is mainly caused by the abnormal proliferation of ductal epithelial cells. Tree shrews have potential as an animal model for the study of breast tumours; however, little is known regarding the transcriptome and DNA methylome landscapes of breast IP in tree shrews. In this research, we conducted whole-genome DNA methylation and transcriptome analyses of breast IP and normal mammary glands in tree shrews. METHODS: DNA methylation profiles were generated from the whole-genome bisulfite sequencing and whole-transcriptome landscapes of IP and control groups of tree shrews through strand-specific library construction and RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses and gene set enrichment analysis were performed. Spearman's correlation analysis was used to identify statistical relationships between gene expression and DNA methylation. RESULTS: A genome-wide perspective of the epigenetic regulation of protein-coding genes in breast IP in tree shrews was obtained. The methylation levels at CG sites were considerably higher than those at CHG or CHH sites, and were highest in gene body regions. In total, 3,486, 82 and 361 differentially methylated regions (DMRs) were identified in the context of CG, CHG, and CHH, respectively, and 701 differentially methylated genes (DMGs) were found. Further, through transcriptomic analysis, 62 differentially expressed genes, 50 long noncoding RNAs, and 32 circular RNAs were identified in breast IP compared to normal mammary glands. Correlation analysis between the DNA methylation and transcriptome data revealed that 25 DMGs were also differentially expressed genes, among which the expression levels of 9 genes were negatively correlated with methylation levels in gene body regions. Importantly, integrated analysis identified 3 genes (PDZ domain-containing 1, ATPase plasma membrane Ca2+ transporting 4 and Lymphocyte cytosolic protein 1) that could serve as candidates for further study of breast IP in tree shrews. CONCLUSIONS: This research has unearthed the comprehensive landscape of the transcriptome and DNA methylome of spontaneous IP in tree shrews, as well as candidate tumorigenesis related genes in IP. These results will contribute to the use of tree shrews in animal models of breast tumours.

New ribotype Clostridioides difficile from ST11 group revealed higher pathogenic ability than RT078.

Clostridioides difficile is the predominant antibiotic-associated enteropathogen associated with diarrhoea or pseudomembranous colitis in patients worldwide. Previously, we identified C. difficile RT078 isolates (CD21062) from elderly patients in China, including two new ribotype strains (CD10010 and CD12038) belonging to the ST11 group, and their genomic features were also investigated. This study compared sporulation, spore germination, toxin expression, flagellar characteristics, and adhesion among these strains in vitro and analysed their pathogenic ability in vivo using animal models. The results showed sporulation and spore germination did not significantly differ among the three C. difficile strains. CD10010 and CD12038 showed higher transcriptional levels of toxins until 48 h; thereafter, the transcriptional levels of toxins remained constant among RT078, CD10010, and CD12038. RT078 showed a loss of flagellum and its related genes, whereas CD12038 showed the highest motility in vitro. Both CD10010 and CD12038 initially showed flg phase OFF, and the flagellar switch reversed to phase ON after 48 h in swim agar. Flagellar proteins and toxins were both upregulated when flg phase OFF changed to flg phase ON status, enhancing their pathogenic ability. CD12038 showed the highest adhesion to Hep-2 cells. Histopathology and inflammation scores demonstrated that CD12038 caused the most severe tissue damage and infection in vivo. The new ribotype strains, particularly CD12038, exhibit higher pathogenic ability than the typical RT078 strain, both in vitro and in vivo. Therefore, more attention should be paid to this new C. difficile strain in epidemiological research; further studies are warranted.

First report of Schistosoma sinensium infecting Tupaia belangeri and Tricula sp. LF.

Schistosoma sinensium belongs to the Asian Schistosoma and is transmitted by freshwater snails of the genus Tricula. Rodents are known definitive hosts of S. sinensium. In 2016, suspected schistosome eggs were found in the feces of the northern tree shrew (Tupaia belangeri) in a field in Lufeng County (latitude, 25°04'50″ N; longitude, 102°19'30″ E; altitude 1820 m), Yunnan Province, China. Morphological analysis suggested that the schistosome was S. sinensium. 18S, 12S and CO1 genes sequencing and phylogenetic analysis showed that this species had the highest similarity to and occupied the same evolutionary branch as S. sinensium from Mianzhu, Sichuan, China. Meanwhile, based on 16S and 28S rDNA sequencing and morphological identification, the snail intermediate host was identified as a species of Tricula, and was found in irrigation channels. Phylogeny indicated that Tricula sp. LF was a sister taxon to T. bambooensis, T. ludongbini. The S. sinensium was able to experimentally infect the captive-bred Tupaia belangeri, and Schistosoma eggs were recovered from all Tupaia belangeri exposed. In this study, we report the infection of Tupaia belangeri and Tricula sp. LF with S. sinensium in Lufeng, Yunnan, southwest China. These findings may improve our understanding of the host range, evolution, distribution, and phylogenetic position of S. sinensium.

Comparing the hippocampal miRNA expression profiles of wild and domesticated Chinese tree shrews (Tupaia belangeri chinensis).

BACKGROUND: The domestication of tree shrews represents an important advance in the development of standardized laboratory animals. Little is known regarding the miRNA changes that accompany the transformation of wild tree shrews into domestic tree shrews. RESULTS: By performing miRNA-seq analysis on wild and domestic tree shrews, we identified 2410 miRNAs and 30 differentially expressed miRNAs in the hippocampus during tree shrew domestication. A KEGG analysis of the differentially expressed genes showed that the differentially expressed miRNAs were associated with ECM-receptor interaction, the phosphatidylinositol signaling system, protein digestion and absorption, inositol phosphate metabolism, lysine degradation, fatty acid degradation and focal adhesion. Most of these pathways could be classified under environmental information processing, organismal systems and metabolism. The miRNAs exclusively expressed in wild and tame tree shrews GO enriched in terms of divergent functions. The miRNA-mRNA networks suggested that novel-m1388-5p and novel-m0746-5p might play regulatory roles in domestication of tree shrews. Real-time RT-PCR analysis was employed to verify the presence of these miRNAs. CONCLUSION: We identified a number of candidate miRNA-regulated domestication genes that may represent targets for selection during the domestication of tree shrews.

Characteristics of the tree shrew humoral immune system.

Preclinical studies require an immune response similar to that of humans in a small animal model that is convenient to operate. Based on genome alignment, tree shrews are small animals considered to be more similar to primates than are rodents, and many human disease models have been established with tree shrews. However, the characteristics of the humoral immune response of tree shrews remain to be elucidated. In this study, the genetic sequence of the heavy chain constant region of tree shrew immunoglobulin (Ig) was complemented, and the results of immunoglobulin domain homology and transcriptome analysis showed that the tree shrew genome encodes only four classes of antibodies and does not encode IgD. The oldest IgM antibody has the highest homology with primates. After the complete sequence of each type of antibody was obtained, the tree shrew antibody protein was further expressed and purified by in vitro recombination, and an IgG quantitative evaluation system was established. The highly effective immuno protective effect induced by HSV-1 infection and the significant bactericidal effect induced by Neisseria meningitidis group C polysaccharide immunization showed that tree shrews exhibited immune responses more similar to humans than to mice. This may provide better predictive value for vaccine preclinical research.

A tree shrew model for steroid-associated osteonecrosis.

Osteonecrosis is a common human disease in orthopedics. It is difficult to treat, and half of patients may need artificial joint replacement, resulting in a considerable economic burden and a reduction in quality of life. Hormones are one of the major causes of osteonecrosis and high doses of corticosteroids are considered the most dangerous factor. Because of the complexity of treatment, we still need a better animal model that can be widely used in drug development and testing. Tree shrews are more closely related to primates than rodents. As such, we constructed a successful tree shrew model to establish and evaluate steroid-associated osteonecrosis (SAON). We found that low-dose lipopolysaccharide (LPS) combined with high-dose methylprednisolone (MPS) over 12 weeks could be used to establish a tree shrew model with femoral head necrosis. Serum biochemical and histological analyses showed that an ideal model was obtained. Thus, this work provides a useful animal model for the study of SAON and for the optimization of treatment methods.

Response of the gut microbiota during the Clostridioides difficile infection in tree shrews mimics those in humans.

BACKGROUND: Clostridioides difficile is a major cause of antibiotic associated diarrhea. Several animal models are used to study C. difficile infection (CDI). The tree shrew has recently been developed as a model of primate processes. C. difficile infection has not been examined in tree shrews. We infected tree shrews with hyper-virulent C. difficile strains and examined the alterations in gut microbiota using 16S rRNA gene sequencing. RESULTS: C. difficile colonized the gastrointestinal tract of tree shrew and caused diarrhea and weight loss. Histopathologic examination indicated structures and mucosal cell destruction in ileal and colonic tissues. The gut microbial community was highly diversity before infection and was dominated by Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria. Antibiotic administration decreased the diversity of the gut microbiota and led to an outgrowth of Lactobacillus. The relative abundance of Proteobacteria, Gammaproteobacteria, Enterobacteriales, Lachnospiraceae, Enterobacteriaceae, Escherichia, Blautia, and Tyzzerella increased following C. difficile infection. These taxa could be biomarkers for C. difficile colonization. CONCLUSIONS: In general, the disease symptoms, histopathology, and gut microbiota changes following C. difficile infection in tree shrews were similar to those observed in humans.

Isolation and identification of two new strains of mammalian orthoreovirus from Chinese tree shrews.

Chinese tree shrews have been used extensively in studies of different types of cancer and for the modeling of viral infections. In the present study, we report the isolation and characterization of two strains of mammalian orthoreovirus (MRV), MRV1/TS/2011 and MRV3/TS/2012, which were isolated from the feces of tree shrews in Yunnan, China. These two strains of MRV were isolated and cultured in both primary tree shrew intestinal epithelial cells (pTIECs) and primary tree shrew alveolar epithelial cells (pTAECs). A neutralization test using immunofluorescence was employed to determine the subtype of each isolate. Viral RNA was extracted and analyzed by polyacrylamide gel electrophoresis (PAGE), and the sequence was determined by next-generation sequencing for construction of a phylogenetic tree and analysis of gene polymorphism. Electron microscopy examination revealed the presence of virus particles with the typical morphological characteristics of MRV. Serotype analysis showed that strain MRV1/TS/2011 was of type I and strain MRV3/TS/2012 was of type III. A sequence comparison showed that the isolates were 25.4% identical in the S1 gene.

WGCNA analysis of the subcutaneous fat transcriptome in a novel tree shrew model.

IMPACT STATEMENT: We constructed the transcriptomic network in adipose tissue in lean, moderate obesity and severe obesity groups of tree shrew for the first time. Compared to other laboratory animal models, the tree shrew is a prospective laboratory animal that has a closer genetic association with primates than with rodents. It is widely used in biomedical researches. Enrichment analyses revealed several molecular biological processes were involved in the ribosome, lysosome, and ubiquitin-mediated proteolysis process. These results provided insights into new targets for the prevention and therapy of obesity and a novel research model for obesity.

Comparative genomic analysis of Proteus spp. isolated from tree shrews indicated unexpectedly high genetic diversity.

Proteus spp. are commensal gastrointestinal bacteria in many hosts, but information regarding the mutual relationships between these bacteria and their hosts is limited. The tree shrew is an alternative laboratory animal widely used for human disease research. However, little is known about the relationship between Proteus spp. and tree shrews. In this study, the complete genome sequencing method was used to analyse the characteristics of Proteus spp. isolated from tree shrews, and comparative genomic analysis was performed to reveal their relationships. The results showed that 36 Proteus spp. bacteria were isolated, including 34 Proteus mirabilis strains and two Proteus vulgaris strains. The effective rate of sequencing was 93.53%±2.73%, with an average GC content of 39.94%±0.25%. Briefly, 3682.89±90.37, 2771.36±36.01 and 2832.06±42.49 genes were annotated in the NCBI non-redundant nucleotide database (NR), SwissProt database and KEGG database, respectively. The high proportions of macrolide-, vancomycin-, bacitracin-, and tetracycline-resistance profiles of the strains were annotated in the Antibiotic Resistance Genes Database (ARDB). Flagella, lipooligosaccharides, type 1 fimbriae and P fimbriae were the most abundantly annotated virulence factors in the Virulence Factor Database (VFDB). SNP variants indicated high proportions of base transitions (Ts), homozygous mutations (Hom) and non-synonymous mutations (Non-Syn) in Proteus spp. (P<0.05). Phylogenetic analysis of Proteus spp. and other references revealed high genetic diversity for strains isolated from tree shrews, and host specificity of Proteus spp. bacteria was not found. Overall, this study provided important information on characteristics of genome for Proteus spp. isolated from tree shrews.

Tree shrew bone marrow-derived mesenchymal stem cells express CD81, OCLN, and miR-122, facilitating the entire hepatitis C virus life cycle.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated cirrhosis, and hepatocellular carcinoma worldwide. At present, there is no prophylactic vaccine against HCV due to the lack of in vivo and in vitro model systems. Although most recombinants of all major HCV genotypes replicate in Huh-7 cell line and derivatives, these cells are human hepatoma-derived cell line. Therefore, the development of un-tumor-derived cell systems facilitating the entire HCV life cycle is urgently needed. In this study, we aimed to establish a novel tree shrew-derived bone marrow-derived mesenchymal stem cell (BM-MSC) system to reconstruct the HCV life cycle. We transduction cluster of differentiation 81 (CD81), occludin (OCLN), and microRNA-122 (miR-122) into BM-MSCs, then used a well-established HCV, produced from the J6/JFH1-Huh7.5.1 culture system, to infect the cells. We observed that BM-MSCs transduction with CD81/OCLN or CD81/OCLN/miR-122 support HCV RNA replication and infectious virus production. We also found that the addition of exogenous vascular endothelial growth factor (VEGF) can enhance HCV infectivity in BM-MSCs, with HCV virus load up to 105 copies/mL. In conclusion, we identified the minimum essential factors required for HCV replication in tree shrew-derived nonhuman nonhepatic BM-MSCs. Further, we identified that exogenous addition of VEGF, and exogenous expression of CD81, OCLN, and miR-122, facilitates efficient viral replication and production of infectious particles. Our results describe a novel cell system capable of supporting the entire HCV life cycle, which may provide an essential tool for anti-HCV drug discovery, vaccine development, and study of pathogenesis.

mRNA profiling reveals response regulators of decreased fungal keratitis symptoms in a tree shrew model.

Fungal keratitis is a corneal disease with a high blindness rate caused by pathogenic fungal infections. The pathogenesis of fungal keratitis and the immune response after fungal infection are still unclear. Notably, the pathological features of fungal keratitis in tree shrews are similar to those in humans. In the present study, mRNA profiling of tree shrew corneas with fungal keratitis was performed. GO and KEGG enrichment analyses were performed on the differentially expressed mRNAs, and the GO biological process ontology was used to analyze functional trends in the differentially expressed mRNAs. In total, 151 downregulated and 71 upregulated mRNAs were shared among the 7-day, 14-day and 30-day infection groups. These differentially expressed mRNAs were significantly enriched in the GO category immune response (GO: 0002376) and the KEGG pathways cytokine receptor binding (KEGG ID: tup04060) and cell adhesion (KEGG ID: tup04514). The downregulated mRNAs were significantly enriched in the corneal epithelial cell adhesion function. Fifty-eight initially upregulated mRNAs gradually decreased in expression, and these mRNAs were significantly enriched in the functions lipopolysaccharide (LPS) and antibacterial polypeptide recognition, cell differentiation, and cell rearrangement. Zeta chain of T-cell receptor associated protein kinase 70 (ZAP70), lymphocyte cytosolic protein 2 (LCP2), C-C motif chemokine and its receptor showed high degrees of connectivity in the protein-protein interaction (PPI) network. We speculate that the decrease in symptoms of tree shrew fungal keratitis may be related to the upregulation of genes involved in immune regulation and macrophage colony stimulation. This study showed that the C-C motif chemokine and its receptor may play a key role in regulating tree shrew fungal keratitis, providing a theoretical basis for studying the pathogenesis of human fungal keratitis.

The characteristics of gut microbiota and commensal Enterobacteriaceae isolates in tree shrew (Tupaia belangeri).

BACKGROUND: Tree shrew is a novel laboratory animal with specific characters for human disease researches in recent years. However, little is known about its characteristics of gut microbial community and intestinal commensal bacteria. In this study, 16S rRNA sequencing method was used to illustrate the gut microbiota structure and commensal Enterobacteriaceae bacteria were isolated to demonstrate their features. RESULTS: The results showed Epsilonbacteraeota (30%), Proteobacteria (25%), Firmicutes (19%), Fusobacteria (13%), and Bacteroidetes (8%) were the most abundant phyla in the gut of tree shrew. Campylobacteria, Campylobacterales, Helicobacteraceae and Helicobacter were the predominant abundance for class, order, family and genus levels respectively. The alpha diversity analysis showed statistical significance (P < 0.05) for operational taxonomic units (OTUs), the richness estimates, and diversity indices for age groups of tree shrew. Beta diversity revealed the significant difference (P < 0.05) between age groups, which showed high abundance of Epsilonbacteraeota and Spirochaetes in infant group, Proteobacteria in young group, Fusobacteria in middle group, and Firmicutes in senile group. The diversity of microbial community was increased followed by the aging process of this animal. 16S rRNA gene functional prediction indicated that highly hot spots for infectious diseases, and neurodegenerative diseases in low age group of tree shrew (infant and young). The most isolated commensal Enterobacteriaceae bacteria from tree shrew were Proteus spp. (67%) and Escherichia coli (25%). Among these strains, the antibiotic resistant isolates were commonly found, and pulsed-field gel electrophoresis (PFGE) results of Proteus spp. indicated a high degree of similarity between isolates in the same age group, which was not observed for other bacteria. CONCLUSIONS: In general, this study made understandings of the gut community structure and diversity of tree shrew.