To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β at 31 wpi, which might have contributed to the establishment of chronic HBV infection.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. To address the molecular basis of HCV pathogenesis using tupaias (Tupaia belangeri), we characterized host responses upon HCV infection. Adult tupaias were infected with HCV genotypes 1a, 1b, 2a, or 4a. Viral RNA, alanine aminotransferase, anti-HCV core and anti-nonstructural protein NS3 antibody titres, reactive oxygen species (ROS), and anti-3β-hydroxysterol-Δ24reductase (DHCR24) antibody levels were measured at 2-week intervals from 0 to 41 weeks postinfection. All HCV genotypes established infections and showed intermittent HCV propagation. Moreover, all tupaias produced anti-core and anti-NS3 antibodies. ROS levels in sera and livers were significantly increased, resulting in induction of DHCR24 antibody production. Similarly, lymphocytic infiltration, disturbance of hepatic cords, and initiation of fibrosis were observed in livers from HCV-infected tupaias. Intrahepatic levels of Toll-like receptors 3, 7, and 8 were significantly increased in all HCV-infected tupaias. However, interferon-β was only significantly upregulated in HCV1a- and HCV2a-infected tupaias, accompanied by downregulation of sodium taurocholate cotransporting polypeptide. Thus, our findings showed that humoral and innate immune responses to HCV infection, ROS induction, and subsequent increases in DHCR24 auto-antibody production occurred in our tupaia model, providing novel insights into understanding HCV pathogenesis.

Previous studies have shown that intermittent cold stress (ICS) induces depression-like behaviors in mammals. Tupaia belangeri (the tree shrew) is the only experimental animal other than the chimpanzee that has been shown to be susceptible to infection by hepatitis B and C viruses. Moreover, full genome sequence analysis has revealed strong homology between host proteins in Tupaia and in humans and other primates. Tupaia neuromodulator receptor proteins are also known to have a high degree of homology with their corresponding primate proteins. Based on these similarities, we hypothesized that induction of ICS in Tupaia would provide a useful animal model of stress responses. We exposed young adult Tupaia to ICS and observed decreases in body temperature and body weight in both female and male Tupaia, suggesting that Tupaia are an appropriate animal model for ICS studies. We further examined the efficacy of a new small-molecule compound, C737, against the effects of ICS. C737 mimics the helical structure of neuron-restrictive silencer factor (NRSF/REST), which regulates a wide range of target genes involved in neuronal function and pain modulation. Treatment with C737 significantly reduced stress-induced weight loss in female Tupaia; these effects were stronger than those elicited by the antidepressant agomelatine. These results suggest that Tupaia represents a useful non-rodent ICS model. Our data also provide new insights into the function of NRSF/REST in stress-induced depression and other disorders with epigenetic influences or those with high prevalence in women.

α-1,3-Galactosyltransferase (α-GalT), an enzyme creating Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference-based suppression of endogenous α-GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi-quantitative RT-PCR (qRT-PCR) and the cytochemical method using a fluorescence-labeled Bandeiraea simplicifolia Isolectin B(4) (BS-I-B(4) ). Staining with BS-I-B(4) demonstrated that α-Gal epitope expression was first recognized at the 8-cell stage, and increased up to the hatched blastocyst stage. Single embryo-based qRT-PCR also confirmed this pattern. These results indicate that creation of α-Gal epitope is proceeded by de novo synthesis of α-GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.

Abstract. RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B 4 , a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of doublestranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation. Key words: Blastocyst, α-1,3-Galactosyltransferase, Pig, RNAi, Somatic cell nuclear transfer (J. Reprod. Dev. 58: [69][70][71][72][73][74][75][76] 2012) S uppression of endogenous target gene expression by gene targeting has been proven as a powerful tool for exploration of the biological function of a gene of interest. Recently, attempts to use antisense technology, now termed RNA interference (RNAi), as a convenient and valuable tool for understanding the in vivo function of a gene of interest have been made [1][2][3][4][5][6][7][8]. RNAi is a multiple-step process that involves the generation of 21-25 nt small interfering (si) RNA and results in degradation of the homologous RNA [2]. Since siRNA can be easily synthesized chemically, it is easy to study the role of endogenous genes by injecting double-stranded (ds) RNA or siRNA into early embryos. However, in the former cases, the effectiveness of dsRNA introduced into cultured cells is known to be retained for a relatively short time (less than one week) [5][6][7][8][9]. In contrast, use of siRNA should be more beneficial for conferring long-term expression of siRNA in transfected cells. In fact, this technology (which is termed "transgenic RNAi") was found to be effective in knocking down ta...