美培育出不会将病毒传染给人体的猪

美国马萨诸塞州生物移植公司的科学家说,他们新培育出一种小猪,其细胞中所携带的猪生反转酶病毒不会传染给人体细胞.这可能为异种器官移植研究带来新的希望. 据悉,猪生反转酶病毒(PERV)普遍存在于猪体内,这种病毒已经与猪本身的基因结合,无法去除,并具有传染性,能够感染人体细胞.PERV病毒对猪无害,对人体是否有害还不得而知. 生物移植公司的科学家认为,这批新品种猪是为医学研究而培育的.试验表明,这种猪细胞中的PERV病毒不会传染给人体细胞.科学家尚不清楚其中的原因.如果进一步的试验表明这种猪体内的PERV病毒也不会感染活体动物,…     

聚合酶链反应检测4种猪源细胞系中的内源性反转录病毒

为了建立检测猪内源性反转录病毒（porcine endogenous retrovirus,PERV)的特异性方法，根据巳发表的PERV的序列，设计并合成了针对PERV核心蛋白（gag）、多聚酶（pol)、囊膜蛋白（env）基因的3对引物，预期扩增片段分别为361、150、265bp。应用PCR技术检测了PERV在4株猪源细胞中的整合情况。结果表明，在所有被检细胞的基因组中均存在有PERV的前病毒序列，应用RT-PCR检测上述4株猪源细胞中PERV特异性MRNA的表达，结果均为阳性。试验还对建立的上述2种方法的特异性进行了探讨，结果表明试验建立的PERV检测法具有较高的特异性。该方法的建立为进一步研究PERV奠定了基础，还可对异种移植动物模型及异种移植受体进行病原安全性监测。     

猪内源性反转录病毒囊膜蛋白基因的克隆及其蛋白二级结构和B细胞表位预测

猪内源性反转录病毒（PERV）是与猪-人异种移植病原安全性密切相关的一类病毒。env基因编码病毒的囊膜蛋白,它与病毒的亚型分类、宿主感染范围、细胞的嗜性以及对宿主细胞的感染机制、诱导宿主产生中和抗体等密切相关。本研究利用RT-PCR的方法,从五指山小型猪外周血淋巴细胞中扩增PERV的囊膜蛋白基因并进行测序,随后用生物信息学相关软件和方法,对PERV-Env蛋白二级结构及B细胞表位进行预测。经综合分析评价,结果发现PERV-Env蛋白有18个可能的B细胞优势抗原表位区域,7个可能的糖基化位点。该分析预测结果不但有利于PERV疫苗的设计、单抗及诊断试剂研制,而且将有助于分析Env蛋白的功能及PERV对人源细胞的感染机制。     

中国小型猪内源性反转录病毒研究进展与对策

作者介绍了国内外猪内源性反转录病毒（PERV）研究进展,简述了中国不同品种小型猪PERV存在情况、PERV前病毒全基因克隆与序列分析、细胞水平鉴定方法、感染HEK293细胞研究平台的创建、猪A3F对PERV的抑制作用研究,以及从分子遗传学上揭示不同品系PERV特异性等创新性研究成果,分析了五指山小型猪近交系具有PERV基因拷贝少且没有PERV-C等特异性,以及展望培育中国PERV阴性猪新品系方法等研究,以期为人类异种器官移植和生物医用材料产品安全性研发,解决小型猪PERV疾病传播危险性提供科学对策和新的方向。     

巴马小型猪内源性反转录病毒感染对HEK293细胞周期调控相关基因mRNA表达的影响

以巴马小型猪内源性反转录病毒(porcine endogenous retrovirus,PERV)感染HEK293细胞模型(PERV-BMHEK293)为研究对象,分析PERV整合对HEK293细胞活性的影响,同时建立7种与人细胞增殖和周期调控密切相关基因的实时荧光定量PCR(q-PCR)检测方法,并对细胞感染模型中7个基因的mRNA表达情况进行分析。细胞活力分析结果显示,随着培养代次及培养时间增加,相对于母源HEK293细胞PERV感染模型的细胞活力下降;qPCR检测方法建立结果显示,所构建的cyclinD1、CDK1、CDK4、k-ras、c-myc、p53、p16基因检测方法检测各基因在1.0×10~1~1.0×10~9 copies/μL反应范围内有很好的线性关系,扩增产物熔解曲线只出现特异性单峰,各组内变异系数在0.31%~2.01%之间,组间变异系数在0.46%~2.19%之间,重复性好,可稳定地用于相关基因mRNA的定量检测。用建立的q-PCR检测方法对PERV感染模型进行细胞周期调控相关基因mRNA表达分析,结果显示,与母源细胞相比,模型细胞cyclinD1、CDK1、CDK4、k-ras、p53和p16基因的mRNA表达无明显变化,原癌基因c-myc基因表达下调,提示PERV感染可能抑制c-myc基因表达。该研究结果可为评价巴马小型猪来源PERV在人-猪异种移植中生物安全性提供参考。     

一种检测猪基因组中内源性反转录病毒C方法的建立

猪内源性反转录病毒使异种移植猪细胞、组织和器官存在风险,猪内源性反转录病毒A和猪内源性反转录病毒B都出现在猪基因组中,并能在体外感染人.猪内源性反转录病毒C只感染猪细胞,并可整合到大多数猪基因组中,但不是全部.猪内源性反转录病毒A和C的重组能感染人细胞,并大量复制.为了避免这种重组,猪内源性反转录病毒C阳性动物不能用于异种移植.为检测猪内源性反转录病毒C阳性猪,建立了多种不同的方法,如用不同引物建立的特异性PCR技术、异种高灵敏度的巢氏PCR技术和可定量前病毒拷贝数的实时定量PCR技术.实时定量PCR技术可用于区分污染和真正的前病毒分子.这些PCR方法经过优化,具有稳定的检测灵敏性.首先进行PCR1,如果检测结果为阴性,则进行PCR2或PCR5或巢氏PCR；如果检测结果是阳性,则进行实时定量PCR来排除污染.使用这些方法可评估猪内源性反转录病毒C的流行程度和识别未感染猪内源性反转录病毒C的动物.由于可能从其它动物细胞带来污染,不是使用耳活体检测,而是采用血细胞检测.     

猪内源性逆转录病毒(PERV)γ1长末端重复序列(LTRs)的分子特性及鉴定

猪基因组中的猪内源性逆转录病毒(PERV)γ1可能是异种移植(从猪到人)中的有害因子。众所周知,长末端重复序列(LTRs)是强启动元件,能够控制PERV元件和邻近功能基因片段的转录活性。本试验通过生物信息学和试验分析对猪组织中PERVγ1 LTRs的转录活性进行了研究。经RT-PCR扩增及测序鉴定出69个不同的LTR转录元件。并根据种内变异将69个LTR元件分为6个类型(15个亚型),包括串联重复序列,插入和缺失(INDEL)。更值得注意的是,所有的种内变异都发生在LTR元件的U3区域。本试验结果表明,不同PERV LTR转录体的分子特性及鉴定为进一步研究PERV及其转录奠定了基础。     

异种移植的现状与未来

应用猪的细胞、组织或器官移植治疗人类疾病的研究方兴未艾.本文综述了异种移植的最新进展,侧重阐明了异种移植存在的生理学和免疫学障碍及解决异种移植排斥的策略.讨论了应用猪作为供体进行异种移植可能存在的一些潜在危险性,特别是猪内源性逆转录病毒造成的一系列问题.     

异种移植的现状与未来

应用猪的细胞、组织或器官移植治疗人类疾病的研究方兴未艾。本文综述了异种移植的最新进展，侧重阐明了异种移植存在的生理学和免疫学障碍及解决异种移植排斥的策略。讨论了应用猪行为供体进行异种移植可能存在的一些潜在危险性，特别是猪内源性逆转录病毒造成的一系列问题。     

广西巴马小型猪内源性反转录病毒整合后传代次数对HEK293细胞中HERV-W表达和变异的影响

检测不同代次广西巴马小型猪内源性反转录病毒(PERV)感染HEK293细胞模型(HEK293-PERV-BM)中,HERV-W各基因mRNA和syncytin-1蛋白表达水平,以及HERV-W各基因变异情况,评价PERV的整合对HERV-W的影响。结果显示,与HEK293细胞相比,PERV整合后,不同代次HEK293-PERV-BM中HERV-W gag、pol、env和syncytin-1的mRNA相对表达量出现不同程度的改变,且改变趋势相似,即P10或P20后开始升高,至P30最高;选取相对表达量差别较大代次,检测其syncytin-1蛋白,各代次间syncytin-1蛋白表达量的改变较mRNA的差别小。此外,PERV整合后,在P1～P55中HERV-W结构基因仅发生个别位置的点突变,未见发生固定位置的点突变或基因片段的缺失、插入及重组。本试验为系统评价PERV-BM在异种器官移植中的安全性提供参考。     

Research Progress and Countermeasures of Porcine Endogenous Retrovirus in Chinese Miniature

The paper is a brief introduction of porcine endogenous retrovirus (PERV) innovative research results in the world and also expounds the passing PERV existence situation on different varieties of miniature pig, analyzes the PERV-virus gene cloning and sequence, appraises method on cell level, create the platform of infection HEK293 cells research, study on pigs A3F inhibition of PERV, and reveal the innovative research results on the specific molecular genetics in the different strain of PERV, analyzes the advantages of Wuzhishan miniature pig inbred line such as low gene copy of PERV,and there is no passing PERV-C specificity and as well as looking forward to cultivate the methods for new strain of PERV negative pigs. It will provide a scientific counter measure and new perspective to solve the spread of disease risk of miniature pig PERV and product safety for human xenotransplantation and biomedical materials of research and development.     

中国巴马小型猪内源性反转录病毒的检测

目的 :对我国特有巴马小型猪内源性反转录病毒 ( porcine endogenous retrovirus,PERV)的存在与 m RNA的表达情况进行检测 ,了解巴马小型猪内源性反转录病毒的携带情况。方法 :根据以往建立的 PCR、RT-PCR检测方法 ,对来自于巴马小型猪外周血淋巴细胞的 DNA和RNA样品进行 PERV核心蛋白基因 ( gag)、多聚酶基因 ( pol)及囊膜基因 ( env)的存在与表达进行检测 ;同时 ,根据目前通用的 env基因分型方法检测 PERV env-A、env-B、env-C的存在与表达。结果 :在 1 2个被检的 DNA样品中均检出了PERV特异性 DNA的存在 ;同样 ,在 1 2个被检的 RNA样品中均有 PERV特异性 RNA的表达 ,且所表达的 PERV均为 A型和 B型 ;其中有9个 DNA样品检测出 PERV-C型的存在 ,所有样品中均未检出 C型 PERV的表达。结论 :检测结果表明 1 2个被检巴马小型猪基因组中存在着内源性反转录病毒序列 ,且能以 m RNA的形式表达 ,这一结果为我国特有小型猪的开发、利用及其病毒安全性评价奠定了基础。     

Porcine endogenous retrovirus copy number in different pig breeds is not related to genetic diversity

The risk of zoonoses is a major obstacle to xenotransplantation. Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection, and its control is a prerequisite for the development of clinical xenotransplantation. The copy number of PERV varies among different breeds, and it has been suggested that the PERV integrations number is increased by inbreeding. The purpose of this study was (i) to examine the copy number of PERV in different Spanish pig breeds, Spanish wild boar and commercial cross-bred pigs from five different farms and genetic background (CCP1-CCP5) and (ii) to investigate the correlation between PERV copy number and the genetic background of the pigs in order to improve the selection of pigs for xenotransplantation. PERV copy number was determined by quantitative, real-time polymerase chain reactions. Thirty-four microsatellite markers were genotyped to describe the genetic diversity within populations (observed and expected heterozygosities, Ho and He, respectively) and the inbreeding coefficient (F). Pearson's correlation coefficient was used to determine the relationship between PERV copy number and Ho, He and F. The copy number of PERV among different pig breeds was estimated to range between three (CCP1) and 43 copies (Iberian Pig). Statistical differences were found among the studied populations concerning PERV copy number. No correlation was found between the PERV copy number and the heterozygosity (calculated at an individual level or at a population level) or the inbreeding coefficient of each population. Our data suggest that pigs inbreeding does not increase PERV copy number and support the idea that careful selection of pigs for organ donation with reduced PERV copy number will minimize the risk of retrovirus transmission to the human receptor.     

Porcine endogenous retroviruses (PERV): in vitro artifact or a big problem for xenotransplantation?]

The pig is the favorized donor species for clinical xenotransplantation. However, PATIENCE et al. could show, that porcine endogenous retroviruses (PERV), released by a porcine kidney cell line, are capable of infecting human cell lines in vitro. Based on this discovery there is an ongoing discussion concerning the risks of zoonosis combined with xenotranplantation, which culminated in the demand for a moratorium on clinical transplantation of porcine organs. Recent findings exclude the possibility of an artifact due to the use of an immortalized cell line: Release of infectious PERV was also shown for mitogenic stimulated primary porcine peripheral blood mononuclear cells and, even more important, for primary porcine endothelial cells. In contrast, none of the recent retrospective in vivo studies showed evidence for PERV transmission, neither in patients after transplantation of porcine pancreas islet cells or after extracorporal perfusion of porcine kidneys, nor in baboons after transplantation of porcine endothelial cells. Currently it is not known, whether impairments of the immunological responses against foreign pathogens, which are associated with different xenotransplantation strategies, could enable PERV in vivo infection. Only in vivo experiments, if possible in suitable subhuman primate models, offer the prospect for a final risk assessment.     

Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.     

Large-scale survey of porcine endogenous retrovirus in Chinese miniature pigs

We conducted a large-scale survey on the existence and expression status of porcine endogenous retrovirus (PERV) in seven breeds of Chinese miniature pigs. Genotyping of PERV was examined by PCR using type-specific primers according to the env genotyping method. The presence and expression status of viral gag, pol and env genes were further analyzed in Wuzhishan pigs (WZSP) and Bama minipigs (BMP). The results showed that PERV existed in all 348 genomic DNA samples. The genotype distribution was subtype A-74.43%, subtype B-95.40% and subtype C-30.46%. No expression of subtype C was found in WZSP and BMP. This research obtained an adequate level of information on the molecular epidemiology of PERV in China. The results indicated that it is possible to monitor pig herds for individuals with the lowest PERV prevalence, especially lacking PERV-C.