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 metatranscriptomic 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 crossspecies transmission.

清醒活动树鼩Tupaia(Tree Shrew)神经元单位放电的记录

鉴于原猿类的树鼩在进化中的特殊地位,有关它的神经生物学研究十分活跃。树鼩的单位放电研究已有报告(J. E. Abano et al. 1978;A. L. Humphrey et al. 1977、1980),但是采用慢性微电极技术记录行为状态的树鼩的单位放电方法迄今未见报道,本文报告一种记录清醒活动状态树鼩的单位放电技术考虑到树鼩形体甚小、体重仅100多克。要分离单位放电,必须拥有一种特殊规格的微型微推进器,以适应特殊实验之需。本研究设计一种采用差动结构原理实现微推进的装置,整个装置由微推进器与基座组成。微推进器的参数如下:重量8.8克;微调范围:2.0毫米;微调读数5微米;微调可控范围:小于2微米;外形尺寸:13×13×51毫米。微推进器相对于基座有直径为1.8毫米的径向偏心移动范围。基座重0.65克,实验在局麻下,在立体定向仪控制下埋植基座。术后第二天即可实验观察。

树鼩(Tupaia glis)在恒定光照条件下的近似昼夜运动性活动节律

根据“Aschoff rule”,昼行性动物近似昼夜节律周期的长度和光照强度呈负相关,活动时间与休息时间的比率以及活动量均与光照强度呈正相关。而夜行性动物的情况则相反(比宁,1964;Aschoff,1959,1960;Sollberger,1965)。这在爬行动物、鱼、鸟以及夜间活动的哺乳类已得到了肯定的证实,但对一些白天活动的哺乳类还不清楚。到目前为止,检查了7种白天活动的灵长类动物均不符合这条规律(Tokura et al.,1978,1979,1980)。

树鼩(Tupaia belangeri chinensis)消化系统解剖和组织学观察

本文对云南产中国树鼩的消化系统作了大体解剖和组织学观察,在此基础上描述了树鼩消化系统的主要特征,并与其他有关种类作了比较和讨论。

Tree shrew (Tupaia belangeri)as a novel laboratory disease animal model

The tree shrew （Tupaia belangeri） is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents. In addition, advantages such as small size, easy breeding, and rapid reproduction make the tree shrew an ideal subject for the study of human disease. Numerous tree shrew disease models have been generated in biological and medical studies in recent years. Here we summarize current tree shrew disease models, including models of infectious diseases, cancers, depressive disorders, drug addiction, myopia, metabolic diseases, and immune-related diseases. With the success of tree shrew transgenic technology, this species will be increasingly used in biological and medical studies in the future.

中缅树鼩(Tupaia belangeri)头骨特征的研究

头骨形态与动物的栖息环境密切相关,生境的变化可引起其头骨形态上的变化,因此动物头骨形态的研究具有十分重要的意义。主要研究了中缅树鼩的头骨特征。结果表明:中缅树鼩头骨特征与其它树鼩显著不同,尤其是中缅树鼩的头骨与马来树鼩存在显著差异,表现在颅全长、基长、颧宽、齿长、吻长、最大颅宽和最大腭宽。结合聚类分析和形态系统树表明头骨形态的差异说明中缅树鼩可能是一个独立的分类单位,而不是马来树鼩的亚种。中缅树鼩头骨特征与其它树鼩的差异可能与其食性、栖息方式、生活环境、所处的海拔和纬度有关。

The characterization of auditory brainstem response(ABR) waveforms: A study in tree shrews(Tupaia belangeri)

To characterize the patterns of ABR waves in tree shrews, we must understand the hearing sensitivity and auditory function of healthy adult tree shrews. Fifteen tree shrews(30 ears) were stimulated with clicks and tone-pips at 11 different frequencies from 1 to 60 kHz. The ABR waves were recorded and analyzed. The ABR consisted of five to seven positive waves in the first 10 ms after a click stimulus, and the average hearing threshold of component III was 27.86 ± 3.78 dB SPL. Wave III was the largest and most clear. The ABR threshold was related to the tone-pip sitmulus by a "U" shaped curve. The sensitive frequency was approximately 8 kHz in tree shrews. The latencies systematically decreased with increasing stimulus frequencies. The ABR amplitudes of wave III increased as the sound pressure level increased. All of these results provide an empirical basis for future studies of hearing diseases in tree shrews.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

模拟空间节律(L:D=0.75h:0.75h)对树鼩(Tupaia belangeri chinensis)“24h 昼夜节律”的影响

模拟地球外层空间400—500km 处的空间节律(L:D=0.75h:0.75h)显著打乱灵长类原宗树鼩(Tupaia belangeri chinensis)固有的昼夜节律。其表现为晨间活动量的最高峰(0600—1200显著跌落,而在晚间(1800—2400)出现活动量显著增高的新峰,24h 活动总量(A)的显著降低和恢复缓慢。本研究又表明给予上述空间节律前两天和给予空间节律期间的每天清晨(0600)或傍晚(1800)注射纯 α 型内源性促眠神经肽 Asp^5-α-DSIP(30μg/kg,i.p.),虽固有节律被打乱,但未见 A 的显著降低,同时恢复也较快。

Ectoparasite communities on Tupaia belangeri (Scandentia:Tupaiidae) in surrounding areas of Erhai Lake in Yunnan,China

Objective:To investigate the ectoparasite communities on Tupaia belangeri (T. belangeri) in the surrounding areas of Erhai Lake and their potential importance in medical and veterinary science. Methods: Different locations in surrounding areas of Erhai Lake in Yunnan were selected as the investigation spots. T. belangeri were randomly captured with baited mouse cages and then brought to the laboratory for identification according to their body shape, size and color, and measurements such as the lengths of their body, ear and hind feet. Ectoparasites on them were collected and identified to determine the species under microscope after specimens were mounted on slides, with the clearing and drying process completed. The constituent ratio (C), prevalence (P) and average ectoparasite abundance (A) were used to test the prevalence and density of ectoparasites; Non-parametric Mann-Whitney U-tests were used to test the differences in the abundance and richness of ectoparasites between female and male hosts; Spearman correlation analysis was used to analyze the relationship between ectoparasites and the body parameters of hosts. Results: There was very high species diversity of ectoparasites found on T. belangeri and some species of ectoparasites were reported associated with human diseases. A high proportion (88%) of 107 T. belangeri was found to be infested with ectoparasites. A total of 75 species of ectoparasites on them were collected, including 58 species of chigger mite, 12 species of gamasid mite, 4 species of flea and 1 species of sucking louse. Within this ectoparasite complex, 13 species were previously reported to be vectors of human disease agents. Non-parametric Mann-Whitney U-tests showed significant differences between male and female T. belangeri in terms of the abundance and richness of total ectoparasites. However, the species richness and abundance of chigger mites, sucking lice, fleas or gamasid mites presented no significant differences between male and female hosts. Spearman correlation analysis showed that none of correlations were observed between host body parameters with abundance and richness of total ectoparasites, chigger mites, suckinglice, fleas or gamatid mites. Conclusion: The results suggest that there is very high species diversity of ectoparasites on T. belangeri, consisting mainly of communities of sucking lice, fleas, chiggers and gamasid mites.

树鼩(Tupaia belangeri chinensis)耳蜗毛细胞铺片的观察

我们用树鼩14只,取耳蜗铺片后进行毛细胞计数和长度测量。发现树鼩毛细胞数及缺失率与人、猴、豚鼠不同,树鼩毛细胞失数少且稳定,蜗尖弃用部少,不同种群间的个体差异小,利于用作生物学实验动物,可为研究耳的生理、病理及临床服务。

Population genomics provides insights into the evolution and adaptation of tree shrews(Tupaia belangeri)in China

Physiological adaptation of tree shrews(Tupaia belangeri)to changing environmental temperature has been re-ported in detail.However,the T.belangeri origin(mainland or island),population history,and adaptation to histor-ical climate change remain largely unknown or controversial.Here,for thefirst time,we sequenced the simplified genome of 134 T.belangeri individuals from 12 populations in China and further resequenced one individual from each population.Using population genomic approaches,wefirst observed considerable genetic variation in T.be-langeri.Moreover,T.belangeri populations formed obvious genetic structure and reflected different demographic histories;they generally exhibited high genetic diversity,although the isolated populations had relatively low ge-netic diversity.The results presented in this study indicate that T.b.modesta and T.b.tonquinia were separated recently and with a similar population dynamics.Second,physical barriers rather than distance were the driving factors of divergence,and environmental heterogeneity may play an important role in genetic differentiation in T.belangeri.Moreover,our analyses highlight the role of historical global climates in the T.belangeri population dynamics and indicate that the decrease of the T.belangeri population size may be due to the low temperature.Finally,we identified the olfaction-associated adaptive genes between different altitude populations and found that olfactory-related genes of high-altitude populations were selectively eliminated.Our study provides demographic history knowledge of T.belangeri;their adaption history offers new insights into their evolution and adaptation,and provides valuable baseline information for conservation measures.

树鼩精子特异性钙通道CatSper生化特性及功能研究

【目的】探究树鼩精子中的特异性钙通道CatSper生化特性及其在树鼩精子中的亚细胞定位,并阐明CatSper对树鼩精子运动的调控作用。【方法】选择性成熟、1~2岁龄雄性树鼩,用CO_2处死后采集树鼩精液,统计精子活力并提取蛋白质。利用Western blotting和免疫组化检测精子中CatSper家族存在情况及其亚细胞定位。通过显微镜观察不同钙离子通道抑制剂(CatSper抑制剂HC-056456,瞬时受体电位香草酸亚型1(TRPV1)抑制剂Capsazepine)与孕酮(P_4)共孵育后树鼩精子的运动度。使用钙离子探针法检测不同抑制剂和P_4共孵育对树鼩精子钙离子浓度的影响。【结果】树鼩精子中存在CatSper及TRPV1通道蛋白,CatSper定位于精子头部及鞭毛。与对照组相比,当HC-056456浓度为100、200、400、1 000 nmol/L时,树鼩精子运动度和钙离子浓度均显著降低(P<0.05),Capsazepine浓度为10、20、40μmol/L时,树鼩精子运动度和钙离子浓度均显著降低(P<0.05)。与Capsazepine组相比,HC-056456与P_4共孵育时,树鼩精子钙离子浓度明显升高,且当P_4浓度为10、20、30μmol/L时两组间差异显著(P<0.05)。Capsazepine、HC-056456与P_4共孵育时,两组间树鼩精子运动度无显著差异(P>0.05)。【结论】CatSper通道对树鼩精子运动和钙离子转运调控至关重要,通过P_4激活从而提高精子内钙离子浓度,本研究结果对于深入研究精子功能及精子受精能力调控机制具有重要意义。

树鼩源碳青霉烯耐药性铜绿假单胞菌的分离鉴定及生物学特性研究

【目的】确定养殖过程中死亡树鼩的致病原因并分析病原的生物学特性、致病性和耐药情况,制定防治方法。【方法】对死亡树鼩进行病理剖检,记录肺脏、肝脏、脾脏、肠道等主要脏器的病变情况,通过平板划线法从主要病变脏器中分离单个菌落,观察菌落形态并进行革兰染色镜检,通过生化试验、药敏试验、致病性试验及16S rRNA基因测序和全基因组测序等手段对分离株进行进一步分析。【结果】从死亡滇西树鼩的脾脏、肠道中分离获得1株碳青霉烯耐药性铜绿假单胞菌,分离菌株在LB琼脂培养基中呈现产绿色水溶色素的灰白色菌落,镜检为革兰阴性杆菌;生化试验进一步证实分离菌为非发酵菌;通过序列进化分析可知,分离菌与GenBank收录的土壤来源铜绿假单胞菌F4相似性高达99%。药敏试验结果显示,分离菌对亚胺培南和美罗培南等碳青霉烯类及左氧氟沙星等喹诺酮类抗菌药均具有一定的耐药性。致病性试验结果显示,该分离株对成年昆明小鼠的半数致死量(LD 50)为3.382×108 CFU,对死亡小鼠脏器组织进行病理学观察,可见肺脏、肝脏、脾脏、肠道等的炎性损伤和坏死。对各脏器再次进行细菌分离,基于16S rRNA序列分析发现铜绿假单胞菌可在死亡小鼠的脾脏、肠道、血液中存在,具有较高的致病性。全基因组测序结果证实,分离菌具有碳青霉烯耐药性相关基因,并与铜绿假单胞菌高致病参考菌株具有相似的毒力因子基因。【结论】本研究从病死滇西树鼩体内分离出碳青霉烯耐药性铜绿假单胞菌,为树鼩微生物致病研究和驯化养殖提供了新的理论依据。

短期大量摄入酒精对树鼩行为学及运动机能的影响

目的:探讨短期大量摄入酒精对树鼩行为学及运动机能的影响,为后续神经科学研究奠定基础。方法:用不同体积的50%乙醇溶液构建树鼩酒精暴饮模型。将20只雄性滇西树鼩随机分为对照组、低剂量组、中剂量组与高剂量组,每组5只。采用旷场试验、步态分析试验综合评价树鼩饮酒前后的行为学基本特征,比较3个剂量组的运动总距离、平均速度、最高速度、穿越中央区次数以及步态线性度Δα、交替系数β、步长规律系数σ。结果:旷场试验显示,与对照组相比,中、高剂量组的运动总距离、平均速度、最高速度均显著降低(均P<0.01),3个剂量组树鼩穿越中央区次数均显著减少(均P<0.01),且高剂量组的变化最为明显。步态分析显示,与对照组相比,中、高剂量组树鼩的步态线性度Δα均显著升高(均P<0.01),3个剂量组的交替系数β、步长规律系数σ均显著升高(均P<0.01),且高剂量组升高最为明显。结论:树鼩短期大量摄入酒精对其身体产生明显的镇静作用,表现为探索欲望降低,步态紊乱,甚至共济失调。

树鼩（Tupaia belangeri）ISG15分子全长克隆及分子生物学功能分析

目的对丙型病毒性肝炎（hepatitis C virus，HCV）小动物模型树嗣的干扰素刺激基因15（interferon stimulated gene15，ISG15）分子的全长eDNA序列进行克隆及分子生物学功能分析，为树晌模型在HCV感染天然免疫中的研究提供分子生物学基础。方法根据Genbank中灵长类及其他哺乳类动物ISG15分子序列保守区设计引物，使用SmarterRace方法扩增树购ISG15全长序列。在序列两端设计特异性引物，引入酶切位点，进行全长片段扩增。全长PCR产物纯化回收后连接至pMD18-T载体，构建重组质粒pMD18-T-tbISG15。对重组质粒进行酶切鉴定、测序。对序列进行同源性及种系进化，同时使用SWISSMODEL同源建模方法进行蛋白二级、三级结构预测及功能分析。结果获得树购ISG15全长序列共687bp，编码157个氨基酸。树晌ISG15与其他哺乳动物ISG15高度同源，与人的核苷酸及氨基酸序列同源性分别高达72．99％及71．34％，核苷酸及氨基酸序列进化树分析均显示树嗣种系最为接近灵长类动物。结构域分析提示：树嗣ISG15主要由两个类泛素样结构域构成。软件预测所得树购ISG15三维结构与人ISG15三维结构高度相似，且具有类泛素样三维结构，动力学检测提示本试验预测的树嗣ISG15三维结构稳定，可信度高。结论对树胸ISG15的克隆进一步完善了对树购模型的认识。为进一步在体内研究HCV感染的天然免疫机制奠定了分子生物学基础。

Liver chimeric mice with tupaia hepatocyte transplantation as an animal model for hepatitis B virus infection and antiviral therapy

The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several weeks and to evaluate antiviral drugs.However,the drawbacks of PHHs include lack of available donors,poor expansion in vitro and ethical issues that limit the application of human liver chimeric mice,necessitating the search for alternatives.Here,we transplanted primary tupaia hepatocytes(PTHs)into the livers of immunodeficient mice and achieved high liver chimerism within six weeks.These tupaia liver chimeric mice are adequate to support chronic infection of the four common HBV genotypes A,B,C and D for 36 weeks,as well as evaluate of antiviral drugs,including hepatitis B immune globulin(HBIG),monoclonal antibody and nucleoside analogues(NAs),for preventative therapy and treatment post infection.In conclusion,the tupaia liver chimeric mouse model provides a convenient,efficient and stable animal model for chronic HBV infection and long-term drug evaluation.

中国中缅树鼩生理生态学研究30年

自20世纪90年代,开始了中国中缅树鼩(Tupaia belangeri)的生理生态学研究。回顾30年来中国中缅树鼩生理生态学的研究与发展:起源和分类、能量代谢和产热特征、脂肪组织、形态学、组学、化学通讯和行为学等。在科学发展的新时期,除进一步加强有关中缅树鼩对极端环境的生理适应研究外,还需要借助多组学、遥感与红外技术等,加强对动物生理适应的分子水平的机理性探究。结合全球温室效应的环境背景,从地理分布北线、食性、肠道微生物多样性以及实验动物建模等方面对中缅树鼩未来的研究方向进行了展望。

滇西树鼩源沙门氏菌的分离鉴定及生物学特性研究

【目的】确定试验滇西树鼩患病致死的原因,分析分离菌株的致病性与耐药情况。【方法】本研究按常规平板划线法对发病死亡的滇西树鼩心脏血、肝脏、脾脏、肺脏和肾脏进行病原菌分离,进一步对分离菌进行形态学观察、生化试验及16S rRNA基因鉴定,并进行药敏试验和致病性试验。【结果】从发病死亡的滇西树鼩肝脏和肺脏组织分离得到1株细菌。分离菌在麦康凯培养基上呈浅橘黄色单菌落,镜检为革兰氏阴性短杆菌。生化试验结果显示,分离菌能分解葡萄糖,不发酵乳糖,产气,产硫化氢等,符合沙门氏菌生化特性。遗传进化分析显示,分离株与肉用仔鸡源沙门氏菌CP082916菌株亲缘关系最近,16S rRNA基因核苷酸相似性为99%。药敏试验结果显示,分离菌对氯霉素、庆大霉素、新霉素、诺氟沙星等药物敏感;对克林霉素、万古霉素不敏感。致病性试验结果显示,该菌对树鼩及成年小鼠具有较强致病性,对小鼠的半数致死量(LD 50)为1.6×106 CFU。病理组织学观察显示,感染组树鼩和小鼠肺脏肺泡间隔增宽、间隔内可见炎性细胞浸润;肝脏细胞排列紊乱,见大量坏死细胞崩解,窦状隙变形或减小,中央静脉淤血,周围有炎性细胞浸润;脾脏部分细胞结构紊乱。【结论】沙门氏菌是引起这次试验滇西树鼩发病的致死原因,分离菌对氯霉素、庆大霉素、新霉素敏感,具有较强的致病性。

一例瑶山亚种树鼩原发多形性脂肪肉瘤的诊断

在常规饲养5年的老龄雌性瑶山亚种树鼩中发现1例背腹部近髋关节处自发一大肿块。大体观察发现,该瘤体巨大,表面皮毛完整,未发生溃烂;该患病树鼩行动不便,精神状态尚可。异氟烷(2%~4%)吸入麻醉后,采血法安乐死,解剖剥离瘤体,肿瘤边界不清,向周围组织浸润。HE染色结果显示,肿瘤组织可见小灶多形性及大片脂肪母细胞,多形性肉瘤区域主要由异型的上皮样细胞组成,核分裂易见;脂肪母细胞大小显著不同,可见较多多形性脂肪母细胞,细胞体积大,核深染、畸形,边缘见压迹,胞质见多泡状脂滴。免疫组织化学染色结果显示,肿瘤细胞vimentin阳性,小灶多形性脂肪母细胞S-100阳性,Ki-67较高比例阳性。结合HE染色及免疫组织化学结果,综合诊断该例树鼩的自发肿瘤为多形性脂肪肉瘤。