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Simple Summary: The Chinese tree shrew, a squirrel-like mammal, has been widely used as a laboratory animal in biological research. However, the low survival rate of the pups has seriously hindered the establishment of inbred lines of this species and further limited its wider use. We found a milk substitute appropriate for artificial rearing of Chinese tree shrew pups independent of any obvious adverse effects on their survival, health, and reproductive performance compared to those of the maternally reared pups. The successful optimization of a milk substitute for the artificial rearing of Chinese tree shrew pups may increase the availability of this experimental animal. 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. [ABSTRACT FROM AUTHOR]

Abstract Following viral detection and interferons (IFNs) production, several hundreds of IFN-stimulated genes (ISGs) are subsequently induced to act as direct antiviral effectors or regulators of the IFN signaling. The guanylate-binding protein (GBP) family belongs to IFN-inducible GTPases defending the host against a diverse group of invading pathogens such as parasites, bacteria and viruses. The Chinese tree shrew (Tupaia belangeri chinese) has been increasingly used as an alternative experimental animal to primates in studying viral infectious diseases. Hitherto, the tree shrew GBP family has not been characterized. In this study, we identified five tree shrew GBP genes (tGBP1 , tGBP2 , tGBP4 , tGBP5 and tGBP7) and characterized their antiviral activities. All these tGBPs were ubiquitously expressed in heart, spleen, intestines, kidney, liver, lung and brain tissues of the tree shrew. IFN-γ treatment of tree shrew primary renal cells (TSPRCs) significantly induced the mRNA expression of tGBPs. Infections with Newcastle disease virus (NDV), encephalomyocarditis virus (EMCV) and type 1 herpes simplex virus (HSV-1) enhanced tGBPs mRNA expression in TSPRCs, but had no effect on the localization of tGBP proteins in the cytoplasm. tGBP1, but not the other four tGBPs, showed antiviral activity against vesicular stomatitis virus (VSV) and HSV-1 infections. Taken together, this study provided the first-hand information of the GBP family members in the Chinese tree shrew, which might assist the development of tree shrew animal model for infectious diseases. Highlights • The Chinese tree shrew has five GBP genes (tGBP1 , tGBP2 , tGBP4 , tGBP5 and tGBP7). • The tGBPs are ubiquitously expressed in seven tissues of the tree shrew. • IFN-γ treatment and viral infections can induce mRNA expression of tGBPs. • Overexpression of tGBP1 presents antiviral activity against VSV and HSV-1. • Understanding the tGBPs will help with creating animal models of infectious diseases. [ABSTRACT FROM AUTHOR]

In mammals, the toll-like receptors (TLRs) play a major role in initiating innate immune responses against pathogens. Comparison of the TLRs in different mammals may help in understanding the TLR-mediated responses and developing of animal models and efficient therapeutic measures for infectious diseases. The Chinese tree shrew ( Tupaia belangeri chinensis ), a small mammal with a close relationship to primates, is a viable experimental animal for studying viral and bacterial infections. In this study, we characterized the TLRs genes ( tTLRs ) in the Chinese tree shrew and identified 13 putative TLRs , which are orthologs of mammalian TLR1 - TLR9 and TLR11 - TLR13 , and TLR10 was a pseudogene in tree shrew. Positive selection analyses using the Maximum likelihood (ML) method showed that tTLR8 and tTLR9 were under positive selection, which might be associated with the adaptation to the pathogen challenge. The mRNA expression levels of tTLRs presented an overall low and tissue-specific pattern, and were significantly upregulated upon Hepatitis C virus (HCV) infection. tTLR4 and tTLR9 underwent alternative splicing, which leads to different transcripts. Phylogenetic analysis and TLR structure prediction indicated that tTLRs were evolutionarily conserved, which might reflect an ancient mechanism and structure in the innate immune response system. Taken together, TLRs had both conserved and unique features in the Chinese tree shrew. [ABSTRACT FROM AUTHOR]

Human mitochondrial antiviral signaling protein (hMAVS, also known as IPS-1, VISA, or Cardif) is essential for antiviral innate immunity. The Chinese tree shrew ( Tupaia belangeri chinenses ), a close relative of primates, is emerging as a potential animal model for investigating viral infection. However, there is a lack of biological knowledge about the antiviral innate immunity of the tree shrew. In this study, we identified and characterized the function of the Chinese tree shrew MAVS gene ( tMAVS ). The cDNA of tMAVS was 2771 bp in length and encoded a polypeptide of 501 amino acids. Phylogenetic analyses based on the amino acid sequences revealed a closer affinity of tMAVS with those of primates. Quantitative real-time PCR analysis indicated that tMAVS mRNA was constitutively expressed in all seven tissues analyzed in this study. The tMAVS mRNA expression was rapidly and significantly increased after RNA virus infections. Ectopic-expression of tMAVS significantly potentiated the virus-triggered activation of IRF3, NF-κB and interferon-β (IFN-β), whereas knockdown of tMAVS displayed the opposite effect. Furthermore, tMAVS mutants lacking the caspase activation and recruitment (CARD) domains or the transmembrane (TM) domain were unable to induce IFN-β. Similar with hMAVS, mitochondrial localization of tMAVS was dependent on its domain. Collectively, this study revealed evolutionary conservation of the MAVS antiviral signaling pathway in the Chinese tree shrew. [ABSTRACT FROM AUTHOR]

• tGBP1 exhibits antiviral activity against VSV infection. • GTPase activity and isoprenylation of tGBP1 are dispensable for anti-VSV activity. • tGBP1 does not up-regulate interferon expression or autophagy upon VSV infection. • tGBP1 represses VSV genome transcription. • tGBP1 competes with VSV-N in binding to VSV-P. Chinese tree shrews (Tupaia belangeri chinensis) are increasingly used as an alternative experimental animal to non-human primates in studying viral infections. Guanylate-binding proteins (GBP) belong to interferon (IFN)-inducible GTPases and defend the mammalian cell interior against diverse invasive pathogens. Previously, we identified five tree shrew GBP genes (tGBP1 , tGBP2 , tGBP4 , tGBP5 , and tGBP7) and found that tGBP1 showed antiviral activity against vesicular stomatitis virus (VSV) and type 1 herpes simplex virus (HSV-1) infections. Here, we showed that the anti-VSV activity of tGBP1 was independent of its GTPase activity and isoprenylation. In response to VSV infection, instead of regulating IFN expression and autophagy, tGBP1 competed with the VSV nucleocapsid (N) protein in binding to the VSV phosphoprotein (VSV-P), leading to the repression of the primary transcription of the VSV genome. These observations constitute the first report of the potential mechanism underlying the inhibition of VSV by GBP1. [ABSTRACT FROM AUTHOR]

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. • Tupaia DDIT3 (tDDIT3) is highly conserved and underwent positive selection. • tDDIT3 is expressed in different tissues and induced by viral infection. • tDDIT3 overexpression enhances virus production of HSV-1 and VSV. • tDDIT3 overexpression inhibits type I IFN signaling. • tDDIT3 interacts with tMAVS and affects the ubiquitination of tMAVS. [ABSTRACT FROM AUTHOR]