五指山小型猪不同器官内猪内源性反转录病毒的存在与表达

目的:检测五指山小型猪不同器官内猪内源性反转录病毒(PERV)的存在与表达情况。方法:提取心、肝、脾、肺、肾、胸腺等6种器官基因组DNA与总RNA,用PCR、反转录PCR对PERV结构基因gag、pol、env进行定性检测,用实时定量反转录PCR对pol基因进行相对定量检测。结果:6种器官中均能检测到PERV结构基因gag、pol、env的存在与表达;从m RNA水平上看,不同器官内pol基因的相对表达量不存在显著性差异。结论:五指山小型猪不同器官内PERV存在与表达的检测,对进一步阐明五指山小型猪来源的PERV分子生物学特性及深入评价猪-人异种移植病原安全性具有重要意义。     

广西巴马小型猪内源性反转录病毒类型群体检测

抽样检测广西巴马小型猪内源性反转录病毒(PERV),了解广西巴马小型猪携带PERV的情况。为培育无PERV广西巴马小型猪提供依据。对广西巴马小型猪PERV-A、PERV-B基因亚型以及陆川猪PERV-C基因亚型进行基因克隆分析,三种亚型基因克隆片段与NCBI上的目的片段一致,确保了PCR分型方法检测PERV的准确性。根据通用的env基因分型方法检测广西巴马小型猪群体内PERV-A、PERV-B、PERV-C基因的存在情况。在50份巴马小型猪样品中,96%的样品中存在PERV-A亚型,100%的样品存在PERV-B亚型,所有广西巴马小型猪样品中都不存在PERV-C亚型;其中,有96%的样品同时存在PERV-A、PERV-B两种亚型,而在24份陆川猪样品中有58.3%存在PERV-C型。广西巴马小型猪没有检测到PERV-C亚型,可以作为无PERV小型猪的培养对象,具有很好的应用前景。     

猪肝细胞和培养上清液中猪内源性逆转录病毒的检测

建立了猪肝细胞及其培养上清液中猪内源性逆转录病毒(PERV)的检测方法,探讨了其在猪肝细胞生物人工肝应用中的意义。以PERV gag基因为靶序列,选用特定的引物,PCR检测中国实验用小型猪肝细胞PERV前病毒DNA;RT-PCR检测猪、犬、大鼠以及HBV阳性病人血清和猪肝细胞培养6h、24h时的上清液PERV RNA,同时检测猪肝细胞猪线粒体DNA(mtDNA)。研究结果表明：检测5份中国实验用小型猪血清、肝细胞及培养猪肝细胞24h时的上清液PERV均为阳性,而5份培养猪肝细胞6h时的上清液、5份犬血清、5份大鼠血清和5份HBV阳性病人血清PERV检测结果均为阴性,猪肝细胞中均可检测到猪mtDNA。因此,中国实验用小型猪肝细胞携带PERV;PERV可释放到血清中;猪肝细胞培养24h后该病毒颗粒已释放到培养液中;PCR和RT-PCR方法检测PERV具有特异性强、简便的特点。     

湖南沙子岭猪内源性逆转录病毒的研究

为评价从猪到人异种移植的生物安全性提供依据,从湖南沙子岭猪的保种群内随机采集31头个体的耳样组织,应用PCR和RT-PCR技术分别检测这些组织中内源性逆转录病毒（porcine endogenous retrovirus,PERV）的前病毒DNA和mRNA,并对PCR扩增的灵敏性进行评估。多组织RT-PCR检测3头沙子岭猪肾、心、肝、肺、脾 等组织中PERV的表达情况,了解其在各组织中的分布情况;最后,扩增、测序该猪种的env基因,结果用NCBI中的BLAST软件进行分析。PCR和RT-PCR结果表明,所检测的31头沙子岭猪均带有PERV前病毒DNA,耳样组织中均有PERV mRNA表达,其中有2头个体携带 env-A、env-B、env-C 3种囊膜蛋白基因,而其余的29头个体只带有env-A、env-B 两种囊膜蛋白基因,未检测到env-C基因。多组织RT-PCR扩增结果表明,3头沙子岭猪的肾、心、肝、肺、脾等组织中,pol、gag、env-A、env-B 基因均有表达,未检测到env-C基因表达。测序沙子岭猪的env基因,结果发现,沙子岭猪env-B 和env-C基因与其他猪种序列比较分别存在2 和10个碱基的差异,而env-A基因序列没有差异,说明不同的猪种之间 env基因存在多态性。以上结果表明,沙子岭猪种群携带PERV,其亚型主要以PERV-A,B为主;PERV在该猪种肾、心、肝、肺、脾等多种组织中的分布没有明显组织特异性,且93.5 % (29/31)个体表现为 env-C 基因缺失,提示沙子岭猪作为候选猪种可能在异种移植中具有较好的应用前景。     

PERV在猪外周血白细胞DNA和组织mRNA中的表达及其差异性分析

为了解我国家猪猪内源性逆转录病毒(PERV)生物学的基本特征,为评价应用猪器官、组织、细胞进行猪-人间跨种移植的生物安全性提供理论基础.本文采用PCR方法调查12个家猪品系外周血白细胞DNA基因组PERV的生物学特征,并应用SS-SSCP、RFLP-PCR方法分析PERV基因片段的差异性及采用RT-PCR方法和半定量方法分析2个品系小型猪13种组织PERV表达的差异.结果表明12个品系猪外周血白细胞DNA基因组普遍存在PERV-A、-B基因序列,未发现单链构象多态性;部分品系猪PERV env基因序列片段存在限制性片段长度多态性.分析2个品系13种组织均表达PERV-A、-B、-C,肾、淋巴结、肝为高表达器官,胰腺和脑组织为低表达器官,PERV-C mRNA丰度明显低于PERV-A、-B mRNA.PERV env存在限制性片段长度多态性、PERVA存在碱基缺失和错配的现象,有可能在猪异种移植中构成PERV感染的潜在危险性,这是在猪异种移植过程中值得高度关注的问题.     

应用RNA干扰沉默猪内源性反转录病毒

目的:尝试应用RNA干扰(RNAi)沉默猪源PK-15细胞中的猪内源性反转录病毒(PERV),并通过反转录酶活性及pol基因相对荧光定量PCR检测沉默效果。方法:依据GenBank公布的PERV pol基因序列,采用Invitro-gen公司的BLOCK-iT RNAi Designer软件设计Stealth小干扰RNA(siRNA)序列;将合成的siRNA转染PK-15细胞,72 h后检测细胞上清PERV反转录酶活性及细胞内pol基因拷贝数并评价沉默效果。结果:反转录酶活性及pol基因拷贝数检测结果表明,设计的3条Stealth siRNA序列中,位于pol基因3272～3296 bp的序列能有效沉默PERV。结论:RNAi方法可有效使猪源PK-15细胞中的PERV沉默,为进一步研究天然抗病毒分子与PERV的相互作用提供了实验基础,同时也为猪源异种移植研究中去除PERV提供了一种可供尝试的方法。     

PERV在猪外周血白细胞DNA和组织mRNA中的表达及其差异性分析

为了解我国家猪猪内源性逆转录病毒(PERV)生物学的基本特征,为评价应用猪器官、组织、细胞进行猪一人间跨种移植的生物安全性提供理论基础。本文采用PCR方法调查12个家猪品系外周血白细胞DNA基因组PERV的生物学特征,并应用SS-SSCP、RFLP-PCR方法分析PERV基因片段的差异性及采用RT-PCR方法和半定量方法分析2个品系小型猪13种组织PERV表达的差异。结果表明12个品系猪外周血白细胞DNA基因组普遍存在PERV-A、-B基因序列,未发现单链构象多态性;部分品系猪PER Venv基因序列片段存在限制性片段长度多态性。分析2个品系13种组织均表达PERV-A、-B、-C,肾、淋巴结、肝为高表达器官,胰腺和脑组织为低表达器官,PERV-C mRNA丰度明显低于PERV-A、-B mRNA。PERV env存在限制性片段长度多态性、PERV-A存在碱基缺失和错配的现象,有可能在猪异种移植中构成PERV感染的潜在危险性,这是在猪异种移植过程中值得高度关注的问题。     

中国两头乌猪品种内源性逆转录病毒基因研究

目的对5个中国两头乌猪品种(通城猪、东山猪、沙子岭猪、赣西两头乌猪和金华猪)及3个国外品种(大白猪、长白猪和杜洛克猪)猪内源性逆转录病毒(PERV)的核心蛋白(gag)基因、多聚酶(pol)基因、囊膜(env)基因的3个亚型A、B、C,分别从DNA和RNA水平上进行研究,以发现中国两头乌猪品种在异种器官移植中的资源优势。方法利用PCR方法在DNA水平上对PERV基因的三个亚型进行鉴定,并通过半定量PCR方法在RNA水平上检测通城猪和大白猪PERV各亚型在心、肝、脾、肺、肾、肌肉、脂肪、淋巴和脑组织中的表达谱。结果4个华中两头乌猪种中env-AB型为主要PERV亚型,分别占被测总数的92%～100%。在这4个品种中均没有检测到C亚型,金华猪以及3个国外猪种中均检测到了C亚型,病毒亚型种类也更丰富。半定量PCR实验结果显示gag、pol基因在两个品种9个组织中广泛表达,env-A在通城猪的心、肝、肺、脂肪和淋巴组织中表达量较低,env-B在通城猪的心脏和淋巴组织中表达量较低,而env-B在大白猪的肾脏中表达很低,其他所测8个组织中表达量都较高。结论通城猪、东山猪、赣西两头乌猪和沙子岭猪可以做为较佳的异种移植候选供体,具有良好的应用前景。     

猪内源性反转录病毒囊膜基因env真核表达质粒的构建及鉴定

目的:构建猪内源性反转录病毒(PERV)囊膜基因env的真核表达质粒pHCMV-env并加以鉴定,为研究PERV的细胞嗜性和宿主范围奠定基础。方法:用RT-PCR方法扩增五指山猪来源PERV的env基因,将其插入pGEM-T easy载体中,构建重组质粒pGEM-T-env,酶切鉴定正确后,将pGEM-T-env与pHCMV-VSV-G表达质粒同时经EcoRⅠ酶切消化后连接,构建重组表达质粒pHCMV-env,并进行酶切、测序鉴定;将鉴定正确的质粒pHCMV-env转染HEK293T细胞,采用PCR、RT-PCR检测转染后env基因的整合和转录情况。结果:扩增得到五指山猪来源PERV的env基因,并构建了pHCMV-env真核表达质粒,转染HEK293T细胞系后,该细胞系中有目的基因的整合和转录。结论:构建了真核表达质粒pHCMV-env,并且在HEK293T细胞中能够整合并转录,为研究PERV的细胞嗜性和宿主范围奠定了基础。     

用RACE技术快速扩增五指山猪来源的猪内源性反转录病毒3'LTR

测定我国小型猪来源的猪内源性反转录病毒(PERV)3'LTR,以便于PERV全基因的克隆和分析.用cDNA末端快速扩增(RACE)技术,从五指山猪外周血淋巴细胞mRNA中扩增到PERV-3'LTR,并克隆入pGEM-T easy载体,将阳性克隆进行序列测定和同源性分析.测序结果显示该克隆3'端的尾部有一个由12个A组成的poly(A)信号;其R区与PERV-MSL的R区(约64 bp)基本一致,同源性分析表明其与PERV-MSL的3'LTR具有81%的序列同源性.说明成功扩增了我国五指山猪来源的PERV-3'LTR,将有利于PERV全基因的克隆.     

Absence of replication of porcine endogenous retrovirus and porcine lymphotropic herpesvirus type 1 with prolonged pig cell microchimerism after pig-to-baboon xenotransplantation

Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or alpha-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.     

Rapid Determination of Perv Copy Number From Porcine Genomic DNA by Real-Time Polymerase Chain Reaction

Here, we report the quantification of porcine endogenous retrovirus (PERV) copy numbers using real time PCR. After generating standard curves using plasmid DNA, copy numbers were determined for PERV pol and for a housekeeping gene, porcine estrogen receptor2 (ER2) with the same amount of genomic DNA. Using this method, we examined 6 pig breeds in Korea including two breeds of miniature pig, one domestic pig from Jeju, and imported pig breeds, Duroc, Landrace, and Yorkshire. All breeds showed PERV copy numbers ranging from 9 to 50. This method will be useful for monitoring of PERVs in a porcine xenograft.     

Rapid determination of perv copy number from porcine genomic DNA by real-time polymerase chain reaction

Here, we report the quantification of porcine endogenous retrovirus (PERV) copy numbers using real time PCR. After generating standard curves using plasmid DNA, copy numbers were determined for PERV pol and for a housekeeping gene, porcine estrogen receptor2 (ER2) with the same amount of genomic DNA. Using this method, we examined 6 pig breeds in Korea including two breeds of miniature pig, one domestic pig from Jeju, and imported pig breeds, Duroc, Landrace, and Yorkshire. All breeds showed PERV copy numbers ranging from 9 to 50. This method will be useful for monitoring of PERVs in a porcine xenograft.     

Quantitative Analysis of Porcine Endogenous Retroviruses in Different Organs of Transgenic Pigs Generated for Xenotransplantation

The pig appears to be the most promising animal donor of organs for use in human recipients. Among several types of pathogens found in pigs, one of the greatest problems is presented by porcine endogenous retroviruses (PERVs). Screening of the source pig herd for PERVs should include analysis of both PERV DNA and RNA. Therefore, the present study focuses on quantitative analysis of PERVs in different organs such as the skin, heart, muscle, and liver and blood of transgenic pigs generated for xenotransplantation. Transgenic pigs were developed to express the human α-galactosidase, the human α-1,2-fucosyltransferase gene, or both genetic modifications of the genome (Lipinski et al., Medycyna Wet 66:316–322, 2010; Lipinski et al., Ann Anim Sci 12:349–356, 2012; Wieczorek et al., Medycyna Wet 67:462–466, 2011). The copy numbers of PERV DNA and RNA were evaluated using real-time Q-PCR and QRT-PCR, respectively. Comparative analysis of all PERV subtypes revealed the following relationships: PERV A > PERV B > PERV C. PERV A and B were found in all samples, whereas PERV C was detected in 47 % of the tested animals. The lowest level of PERV DNA was shown in the muscles for PERV A and B and in blood samples for PERV C. The lowest level of PERV A RNA was found in the skin, whereas those of PERV B and C RNA were found in liver specimens. Quantitative analysis revealed differences in the copy number of PERV subtypes between various organs of transgenic pigs generated for xenotransplantation. Our data support the idea that careful pig selection for organ donation with low PERV copy number may limit the risk of retrovirus transmission to the human recipients.     

Characterization of two porcine endogenous retrovirus integration loci and variability in pigs

The pig (Sus scrofa) is a potential organ donor for man but porcine endogenous retroviruses (PERVs) represent an important concern for patients, and identification or engineering of PERV-free pigs suitable for xenotransplantation is a major undertaking. Consequently, studies of variability in pigs for the presence of PERVs at specific loci are a prerequisite. We identified genomic flanking sequences of two PERVs cloned in bacterial artificial chromosomes, a replication-competent PERV-A at locus 1q2.4 and a defective PERV-B at locus 7p1.1–2. PERV-A is embedded in the second repeat of a tandem of eight 190 bp repeats. A short duplicated 4 bp cellular motif, AGAC, was found at each flank of PERV-A and a degenerate 4 bp motif was found for PERV-B. At each locus, the PERV flanks matched expressed sequence tags available in public databases. Primer pairs were designed to amplify either genomic flanks or PERV-genomic junctions. Polymerase chain reaction screening was performed on pigs from 11 distinct Chinese breeds and from the European Large White breed. PERV-B at locus 7p1.1–2 was detected in all animals whereas the presence of PERV-A at locus 1q2.4 was variable. Our results suggest that a genetic selection can be designed to identify animals lacking a potentially active PERV at a specific locus and that Chinese and European pig breeds represent large biodiversity reservoirs to explore. Our results point also to the existence of PERVs that might be fixed in the pig genome, and that might not be eliminated by classical genetic selection.Accession numbers: Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under Accession numbers AY160111–AY160114