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.

Progress in biological research on Tupaia belangeri

Tupaia belangeri,as a typical tropical species,has high value in biological research.It has the characteristics of small body size,high brain to body mass ratio,short reproductive cycle,short lifespan,and low maintenance cost.In biomedical research,experimental animals considered as substitutes for primates.This review discusses the systematic classification,distribution,and research progress of Scandentia.Emphasis was focus on the research progress,developmental status,and basic biological research in T.belangeri.Believing that the T.belangeri,as the closest species to humans,has many advantages as an experimental animal,and it can be further widely applied in human disease research.Therefore,how to create a clear and stable strain of T.belangeri with a clear genetic background is crucial for the development of the national economy and scientific research.

THE RELATION BETWEEN EVOLUTION OF SPATIAL WORKING MEMORY FUNCTION AND OF MORPHOLOGY OF THE DORSOLATERAL PREFRONTAL CORTEX AMONG THE RHESUS MONKEY, SLOW LORIS AND TREE SHREW

The relation between evolution of spatial working memory function and of morphology of the dorsolateral prefrontal cortex among the rhesus monkey (Macaca mulatta), the slow loris (Nycticebus coucang) and the tree shrew (Tupaia belangen chinensis) were reported in present paper. The results read as follows: In the DR performance with training, the rhesus monkeys and slow lorises could reach a criterion of 90% correct response at 1.1 ± 3.2 seconds, and 3.8±0.4 seconds delay interval, respectively, by 1000 training trails. The tree shrews failed to reach the criterion of 90% correct response even at 0 seconds delay interval by 1000 training trails. If a delay interval was tested in one session (30 trails) only, doing the DR performamce without training, the rhesus monkeys reached a correct of 80% or higher in each session at 0, 1, 2, 3, 4, and 5 seconds delay, respectively. The percent correct in each session of the slow lorises showed no differences from the rhesus monkeys at 0, 1, 2, 3, and 4 seconds delay. However, when the delay interval was increased to 5 seconds, the percent correct of the DR performance declined to 70% or lower in the slow lorises. In the tree shrews the percent correct in each session reached to 70% or lower at 0, 1, 2, 3, 4, and 5 seconds delay interval, respectively. The morphological studies revealed that the size of the prefrontal cortex increased, and the structure got complex in the course of the evolution in primates. It is suggested that the relation of evolution between the spatial working memory function and anatomy in the prefrontal cortex might be significant among the three species, both the development of morphology and that of the spatial working memory function in the dorsolateral prefrontal cortex are later than other regions of cerebral cortex in phylogenetic evolution course.

Inhibitory effects of antisense oligodeoxynucleotide on replication and expression of HDV genome in vitro and in vivo

Objective:To studytheinhibitoryeffectsof antisenseoligodeoxynucleotide(ASODN)anditsthiophosphate(S-ASODN)on thereplicationandexpressionof hepatitisD virus(HDV)in H1δ9cellsandin tupaiabody.Methods:After15mer-ASODNandS-ASODNweresynthesized,differentconcentrationsof ASODNandS-ASODNwereaddedto theculturemediumof H1δ9celllineandthenHDAgin thesupernatantof theculturewas examinedwithELISAand HDV-RNAinthecellsdeterminedwithdotblothybridization.SixteentupaiaeweresuccessfullyinfectedwithHDVand thenequallyrandomizedinto2groups.In thetreatedgroup,theanimalsreceivedintravenousinjectionsof S-ASODN3mg everyotherdayfor7timesandthecontrolgroupwereinjected withsamevolumeof normalsaline.On the5th,10th,15thand20thdayaftertheadministration,samplesof bloodandlivertissueswereexaminedwithimmunohistochemical methodforHDAganddotblothybridizationandin situ hybridizationforHDV-RNA.Results:Twenty-fourhoursafter theadditionof a finalconcentrationof6μmol/Lof S-ASODN,thereplicationof HDV-RNAandthereleaseof HDAgin theH1δ9cellsweresuppressedby84.5%and76.14%respectively.Theinhibitionwas dose-dependentwhenthefinal concentrationsof2,4and6μmol/Lof S-ASODNweregiven.WhenASODNandS-ASODNwereadministeredinthe samedosage,theinhibitionshowedno significantlydifferencebetweenthe2agents.On thelastdayof theadministration of S-ASODN,7outof the8tupaiaeof thetreatedgroupshowednegativeHDAgandHDV-RNAinthelivertissuewhile only1outof the8tupaiaeof thecontrolgroupwasnegative.Tendaysafterthecessationof drugadministration,3tupaiae of thetreatedgroupand7of controlwerepositiveof HDAgandHDV-RNA.Conclusion:OurfindingsshowthatS-ASODNefficientlyinhibitesthereplicationandexpressionof HDVgeneinH1δ9cellsandin thebodyof tupaia,which providesanexperimentalbasisfortheanti-HDVapplicationof antisenseoligonucleotides.

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)。

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.

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.

Viral and cellular determinants involved in hepadnaviral entry

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus （HBV） constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses （e.g. HBV） and avihepadnaviruses （e.g. duck hepatitis B virus [DHBV]）. Two important discoveries have been made recently regarding HBV： （1） primary hepatoo/tes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and （2） a human hepatoma cell line （HepaRG） was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D （CPD） has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes （PDH）, investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to： （1） functionally dissect the hepadnaviral entry pathways, （2） perform reverse genetics （e.g. test the fitness of escape mutants）, （3） titrate and map neutralizing antibodies, （4） improve current vaccines to combat acute and chronic infections of hepatitis B, and （5） develop entry inhibitors for future clinical applications.

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.

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.

A bright future for the tree shrew in neuroscience research:Summary from the inaugural Tree Shrew Users Meeting

Tree shrews(Tupaia spp.)have been used in neuroscience research since the 1960s due to their evolutionary proximity to primates.The use of and interest in this animal model have recently increased,in part due to the adaptation of modern neuroscience tools in this species.These tools include quantitative behavioral assays,calcium imaging,optogenetics and transgenics.To facilitate the exchange and development of these new technologies and associated research findings,we organized the inaugural“Tree Shrew Users Meeting”which was held online due to the COVID-19 pandemic.Here,we review this meeting and discuss the history of tree shrews as an animal model in neuroscience research and summarize the current themes being investigated using this animal,as well as future directions.

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.

Age-related changes of corneal endothelial cell in healthy Chinese tree shrew measured by non-contact specular microscope

AIM：To determine the impact of age on the morphology of endothelial cells and central corneal thickness（CCT） in Chinese tree shrew.METHODS：One-hundred and twenty eyes of 60 healthy Chinese tree shrews were studied.Based on age,the tree shrews were divided into four groups.After general anesthesia,the images of endothelium were acquired using non-contact specular microscope Topcon 3000 P.Eight parameters of corneal endothelial cells were measured by built-in software,including CCT,endothelial cell density（ECD）,percent hexagonality（HG%）,coeffi cient of variability（CV）,size of minimal cell（Smin）,size of maximal cell（Smax）,average cells size（Savg） and size standard deviation（Ssd）.Data were analyzed using STATA software.The differences of eight parameters among groups and correlations with age were analyzed.RESULTS：In all studied animals,the average CCT was 2 4 9.6 ± 2 .29 μ m（202-301 μm）,ECD was 308 0.72 ± 460.76 cells/mm^2（1239.6-4047.6 cells/mm^2） and CV was 29.10±7.60（13.6-54.6）.CV was significantly different among different groups（P〈0.001）.Strong correlation with age was found in ECD,Smax,Savg,Ssd and CV.CONCLUSION：Cornea of Chinese tree shrews had half CCT of human cornea and similar ECD,CV and size of corneal endothelial cells.Young adult tree shrews had higher ECD,HG% and low CV.ECD,Smax,Savg,Ssd and CV correlated with age signifi cantly.

Advances in Animal Models of Hepatitis B Virus Infection

Hepatitis B virus (HBV) infection seriously affects human health. Stable and reliable animal models of HBV infection bear significance in studying pathogenesis of this health condition and development of intervention measures. HBV exhibits high specificity for hosts, and chimpanzee is long used as sole animal model of HBV infection. However, use of chimpanzees is strictly constrained because of ethical reasons. Many methods were used to establish small-animal models of HBV infection. Tupaia is the only nonprimate animalthat can be infected by HBV. Use of HBV-related duck hepatitis virus and marmot hepatitis virus infection model contributed to evaluation of mechanism of HBV replication and HBV treatment methods. In recent years, development of human–mouse chimeric model provided possibility of using common experimental animals to carry out HBV research.These models feature their own advantages and disadvantages and can be complementary in some ways. This

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.

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.