Xenozoonoses in xenotransplantation

Pig to human xenotransplantation is one of the possible ways to solve the problem of organ shortage, but the potential risk of xenozoonoses hinders the progress of xenotransplantation. Among pathogens that might cause xenozoonoses, porcine endogenous retrovirus (PERV) is undoubtedly the most noticeable. Current researches show that PERV exists in pigs' genomes and is able to express in multiple tissues and organs in pigs. Other experiments also show that PERV could successfully infect several human cell lines in vitro and could infect SCID mice through pig to mouse islet transplantation. The discussion and research on the risk of PERV is one of the hottest topics in current xenotransplantation, but the question of whether PERV will transmit to human through xenotransplantation has not been answered due to the lack of suitable animal models. More work should be done to evaluate the risk of xenozoonoses caused by PERV after pig to human xenotransplantation.     

临床异种移植百年进展(述评)

美国最近连续报道4例基因工程猪供给人的异种器官移植，这些进展打破了异种移植临床研究领域近30年的沉静。尤其是实验性的猪心脏移植取得初步成功，引发了广泛的关注。异种移植面向未来的临床应用迈出了关键性一步。这4个案例，无论是创新精神、技术进步、审批流程，还是伦理学的同步跟进，都值得我国同行学习和借鉴。30年来，基因工程（GE）动物器官已经能在非人类灵长类动物（NHPs）模型中长时间存活。这些实验中，预存抗体、补体激活、凝血状态等机制参与的超急性排异反应基本上得到有效控制。异种移植已经达到临床应用的起跑点。然而，未来仍然有四大问题需要解决：免疫排斥、生物安全、跨种适配和伦理心理。本文简要概括临床异种移植百年来的主要进展，其困难程度可见一斑，但希望和探索从未终止。     

猪作为异种器官移植供体的研究进展

异种器官移植(Xenotransplantation,XT)是未来生物学科中的重要研究领域之一.猪作为为人类提供移植器官的供体源具有得天独厚的条件.文章就器官移植(OrganTransplantation,OT)的历史、原因、移植后的免疫排斥反应和猪内源性逆转录病毒(porcine endogenous retrovirous,PERV)感染问题及可能的解决途径进行了综述.     

The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity

Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration.     

Epstein-Barr virus infection and replication in a human epithelial cell system.

Epstein-Barr virus, a human herpesvirus with oncogenic potential, infects two target tissues in vivo: B lymphocytes, where the infection is largely non-productive, and stratified squamous epithelium in which virus replication occurs. The interaction with B cells, initiated through virus binding to the B-cell surface molecule CR2 (ref. 4), has been studied in vitro and the virus 'latent' genes associated with B-cell growth transformation defined. By comparison, viral infection of epithelium remains poorly understood, reflecting the lack of an appropriate cell-culture model. Here we describe the development of such a model using as targets CR2-expressing transfected cells of two independent human epithelial lines. A high proportion of these cells bind virus and become actively infected, expressing the small EBER RNAs (small non-polyadenylated virus-coded RNAs) and the Epstein-Barr nuclear antigen 1 but not other latent proteins; thereafter, under conditions favouring epithelial differentiation, up to 30% of the cells can be induced to enter virus productive cycle with some progressing to full virus replication. We find significant differences between laboratory virus strains in their ability to infect epithelium that do not correlate with their B-cell growth-transforming activity.     

Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, slw3 and slw6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after activation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.     

草原毛虫核型多角体病毒的安全性研究 Ⅰ.草原毛虫核型多角体病毒的致病性研究

用GrNPV对鱼、蛙、小白鼠、大白鼠、豚鼠、兔、鹌鹑、鸽、鸡、鹅、狗、猪、山羊、绵羊、牦牛、猴等16种366只脊椎动物,按国际病毒制剂安全性试验标准作了急性和亚急性感染试验,检测了长期接触GrNPV人员血清中对应抗体.结果表明,GrNPV对人体和脊椎动物无感染性、致病性和其它毒性.     

Green fluorescent protein （GFP） transsenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm animals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfecUon system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe- tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h resulted in the highest transfecUon rate （3.6%）. The percentage of GFP reconstructed embryos that de- veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Reconstructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 delivered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.     

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.     

非洲猪瘟舍内传播元胞自动机仿真研究

为了研究非洲猪瘟在封闭式猪舍内传播的规律,本文通过引入SI模型,并在此基础上进行改进,结合猪舍内猪只的移动规律及个体体质的差异,分析猪舍内猪只感染非洲猪瘟的概率;设计猪只个体接触后的感染规则,构建元胞自动机模型进行仿真,模拟非洲猪瘟在猪舍的传播情况;通过将元胞自动机模拟的非洲猪瘟的传播仿真结果与传统SI模型的传播数据进行对比分析。结果表明,元胞自动机仿真非洲猪瘟舍内传播能够与传统的SI模型传播数据可以实现较好的拟合,拟合度为0.96,同时使用元胞自动机作为非洲猪瘟的传播模型在预测方面具有优势,可以通过迭代对传播过程可视化,进而实时预测非洲猪瘟在密闭猪舍的传播轨迹,为相关传染病的仿真研究提供了理论指导。     

Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection

It has recently been established that both acute human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are accompanied by a dramatic and selective loss of memory CD4+ T cells predominantly from the mucosal surfaces. The mechanism underlying this depletion of memory CD4+ T cells (that is, T-helper cells specific to previously encountered pathogens) has not been defined. Using highly sensitive, quantitative polymerase chain reaction together with precise sorting of different subsets of CD4+ T cells in various tissues, we show that this loss is explained by a massive infection of memory CD4+ T cells by the virus. Specifically, 30-60% of CD4+ memory T cells throughout the body are infected by SIV at the peak of infection, and most of these infected cells disappear within four days. Furthermore, our data demonstrate that the depletion of memory CD4+ T cells occurs to a similar extent in all tissues. As a consequence, over one-half of all memory CD4+ T cells in SIV-infected macaques are destroyed directly by viral infection during the acute phase-an insult that certainly heralds subsequent immunodeficiency. Our findings point to the importance of reducing the cell-associated viral load during acute infection through therapeutic or vaccination strategies.     

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.     

Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system

The mechanism(s) by which infectious or malignant material is cleared by the host has long been an area of intensive study. We have used the murine model of infection with lymphocytic choriomeningitis virus (LCMV) to look at immune clearance during persistent infection. LCMV was selected because the mouse is its natural host, it easily induces acute or persistent infection in vivo, and the mechanism by which it is cleared in vivo during acute infection is now well understood. Clearance, although associated with several antiviral immune effector mechanisms, is primarily dependent on the activity of virus-specific cytotoxic T lymphocytes (CTL) restricted by H-2 molecules of the mouse major histocompatibility complex (MHC). If these cells fail to generate or are depleted, progression from acute to persistent infection occurs. Here, using molecular probes, we show that viral nucleic acid sequences, viral proteins and infectious materials can be efficiently and effectively cleared by adoptive transfer of antiviral H-2-restricted lymphocytes bearing the Lyt 2+ phenotype. Viral materials are cleared from a wide variety of tissues and organs where they normally lodge during persistent infection. Unexpectedly, the mode by which viral materials are removed from the central nervous system (CNS) differed markedly from the mechanism of clearance occurring at other sites. These observations indicate the possible use of adoptive lymphocyte therapy for treatment of persistent infections and suggest that immune clearance of products from the CNS probably occurs by a process distinct from those in other organs.     

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.     

Transferrin receptor 1 is a cellular receptor for New World haemorrhagic fever arenaviruses

At least five arenaviruses cause viral haemorrhagic fevers in humans. Lassa virus, an Old World arenavirus, uses the cellular receptor alpha-dystroglycan to infect cells. Machupo, Guanarito, Junin and Sabia viruses are New World haemorrhagic fever viruses that do not use alpha-dystroglycan. Here we show a specific, high-affinity association between transferrin receptor 1 (TfR1) and the entry glycoprotein (GP) of Machupo virus. Expression of human TfR1, but not human transferrin receptor 2, in hamster cell lines markedly enhanced the infection of viruses pseudotyped with the GP of Machupo, Guanarito and Junin viruses, but not with those of Lassa or lymphocytic choriomeningitis viruses. An anti-TfR1 antibody efficiently inhibited the replication of Machupo, Guanarito, Junin and Sabia viruses, but not that of Lassa virus. Iron depletion of culture medium enhanced, and iron supplementation decreased, the efficiency of infection by Junin and Machupo but not Lassa pseudoviruses. These data indicate that TfR1 is a cellular receptor for New World haemorrhagic fever arenaviruses.     

Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

Great progress have been made in animal cloning in China, as evidenced by the live births of cloned cat- tle[1,2], goats[3,4], and sheep[5]. In contrast, pig cloning is still in its infancy though limited fundamental studieshave been conducted[6]. It is g…     

Use of a cDNA clone to identify a supposed precursor protein containing valosin

Valosin, a novel 25-amino-acid peptide isolated recently from pig intestine, has several effects on the digestive system of dogs. We report here that the valosin-specific complementary DNA clone from pigs codes for a polypeptide unlike most precursors of biologically active peptides. The predicted protein lacks a characteristic amino-terminal hydrophobic signal sequence and contains no processing signals of the type acted upon by endopeptidases to generate other active peptides from precursors. Antibodies to synthetic valosin have been used to show that nearly all valosin immunoreactivity is in the cytoplasm and that the protein detected (valosin-containing protein, VCP), although smaller than the predicted product of the cDNA sequence, is much larger than valosin. Valosin-specific messenger RNA is found in extracts from many pig tissues, which contrasts with the restricted occurrence expected of a biologically active peptide. We conclude that valosin is an artefact of the purification procedure and does not occur in vivo.     

HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

The worldwide shortage in the supply of human do-nor organs is becoming more and more pronounced. Xenotransplantation may probably give the hope to over-come the problem ultimately. Because it has a great pros-pect of clinical application, xenotransplantation has drawn great attention[1]. The pig appears to be an ideal source for human transplantation. But the xenograft has to face the challenge of three severe rejections (the hyperacute rejec-tion, the delayed xenograft rejection and acute ce…