“如源一号”治疗猴免疫缺陷病毒感染恒河猴的药效观察

目的 评价“如源一号”治疗猴免疫缺陷病毒(SIV)感染中国恒河猴的效果.方法 选取经静脉注射感染SIVmac239的中国恒河猴15只,随机分为3组,分别为对照组和“如源一号”低剂量组、高剂量组.检测不同时期血常规、CD4细胞和CD8细胞亚群、SIV病毒载量的变化,并活检取淋巴结观察病理学的情况.结果 SIV感染后对照组...     

免疫缺陷病毒感染的中国恒河猴骨特异性碱性磷酸酶和甲状旁腺激素的动态变化

目的监测中国恒河猴在感染猴免疫缺陷病毒（SIV）前后骨特异性碱性磷酸酶（BAP）和甲状旁腺激素（PTH）水平的动态变化,探讨艾滋病毒对骨代谢的影响及可能机制。方法20只中国恒河猴感染SIVmac239,分别在感染前和感染后2、6、9、12、15和18个月空腹静脉采血,检测病毒载量、CD4＋细胞及血浆BAP和PrrH水平。结果中国恒河猴感染SIV后,BAP水平持续下降,在12—18个月时最为明显（P〈0．01）;胛H水平在感染早期（2个月）略下降,后期出现上升趋势。结论中国恒河猴免疫缺陷病毒感染可直接或间接累及骨代谢系统,表现为骨形成下降,甲状旁腺轴功能受损可能是其危险因素之一。     

编码弓形虫致密颗粒抗原-1（GRA1）质粒DNA免疫小鼠诱导抗弓形虫感染的保护性

目的 构建真核表达质粒pcGRA1，并观察其免疫小鼠的保护性。方法经PCR从cDNA克隆中扩增出GRAl编码基因，定向克隆入pcDNA3的EcoRI／XhoI位点，从而获得pcGRA1。用限制性内切酶消化、PCR及序列测定对该重组质粒进行鉴定。将100μg质粒于小鼠左后腿DNA肌注，于注射后2周和4周各加强1次；分别观察小鼠体内的特异性抗体滴度、GRA1基因在小鼠体内的分布、GRA1在肌细胞的表达以及免疫小鼠对弓形虫强毒株RH速殖子攻击后的保护性。结果DNA序列测定结果表明，将573bp的GRA1编码阅读框定向克隆到pcDNA3EcoRI／XhoI位点，成功构建了重组质粒pcGRA1；免疫小鼠血清中检测到特异性IgG；不同组织DNA为模板特异地扩增出GRA1编码基因；IFA检测pcGRA1免疫鼠肌细胞呈阳性反应；弓形虫RH株速殖子攻击感染pcGRA1免疫鼠，其存活时间长于对照组。结论重组质粒pcGRA1免疫小鼠可诱导特异性免疫反应，对弓形虫强毒株的攻击感染具有一定的保护性。     

HIV-1感染恒河猴外周血单个核细胞的生物学特性和C2-V3区基因序列变化

目的探讨艾滋病病毒1型(HIV-1)感染恒河猴外周血单个核细胞(PMBCs)，并在其中传代的可能性。方法用HIV-1感染原代猴PBMCs，采用酶联免疫吸附试验(ELISA)方法测定培养0、2、3、7天时上清液中的HIV-1P24抗原，以观察HIV-1在猴细胞中的复制情况，比较HIV-1分别在人和猴细胞感染的复制动力学。同时采用聚合酶链反应(PCR)方法扩增HIV-1的enV基因，并对其C2-V3区进行序列分析。结果HIV-1毒株SF33、89．6、ⅢB感染猴PBMCs后的第3天，P24抗原达到最高，随着培养时间的延长，P24抗原逐渐降低。将SF33、ⅢB、89．6、YNl9四个毒株分别感染猴PBMCs并盲传3代后，P24抗原测定结果均为阴性。提取盲传各代细胞脱氧核糖核酸(DNA)进行PCR扩增，结果显示，SF33、ⅢB、YN19、89．6感染猴细胞的第一代和89．6感染猴细胞的第二代，均可以检测到HIV-1前病毒核酸。比较原毒株enV基因C2-V3区的序列发现，传代后的基因序列SF33有5个碱基改变外，其它HIV-1只有0～2个核苷酸改变。与SF33感染猴PBMCs的表现相反，其感染人PBMCs时。HIV-1的复制一直维持在较高的水平。结论应用3个实验株、1个临床分离株HIV-1，虽都能够感染恒河猴外周淋巴细胞，但均难以继代，提示能否用恒河猴作为HIV-1研究的动物模型，尚待进一步的体内外研究。     

SIV感染恒河猴大脑神经毒性基因型的渐变选择

据医学空间网3月10日报道（原载AIDS 2006 Jan；20（2）：197-205），为了比较SIV基因型在感染了该病毒的恒河猴大脑和外周血单核细胞里RNA水平上的区别以及该病毒在急性，无症状和晚期病毒感染中DNA水平上的差异，科学家们做了以下试验：实验采集了SIV病毒感染急性期、无症状期和晚期恒河猴脑组织和外周血单核细胞（PBMC）中的RNA和脑组织中的DNA，比较感染不同时期病毒的基因型。     

新疫苗可大幅降低猿类艾滋病病毒感染率

据英国《自然》杂志网站1月5日(北京时间)报道,美国科学家开发出的一种实验性猿类免疫缺陷病毒疫苗,可大幅降低恒河猴感染猿类艾滋病病毒的风险,让接触一次SIV的恒河猴感染病毒的概率减少80%。SIV是导致人类感染艾滋病的HIV的"远亲",因此,最新研究为人类研制出有效的HIV疫苗指明了新方向。     

中国恒河猴SIV急性感染期模型CD4细胞和MBL的变化

目的研究中国恒河猴猴免疫缺陷病毒(SIV)mac239急性期感染模型外,周血CD4+T淋巴细胞(简称CD4细胞)、淋巴结CD4细胞和血清甘露糖结合凝集素(MBL)的变化情况,探索感染早期固有免疫与适应性免疫的病理变化。方法选用5只健康中国恒河猴,静脉接种SIVmac239病毒株,分别于感染前、感染后2周、4周和10周检测血常规、血浆病毒载量,流式细胞仪分析外周血淋巴细胞亚群,免疫组织化学染色法观察淋巴结CD4细胞,酶联免疫吸附试验法检测血清MBL。结果感染后CD4细胞比值较感染前降低27.3%(P0.05),CD8细胞比值较感染前升高44.3%(P0.05),外周血CD4细胞绝对数下降23.6%,淋巴结CD4细胞上升14.9%,但是差异无统计学意义。血清MBL在感染后10周内持续下降至感染前水平的24.5%(P0.01)。结论 SIV急性感染期固有免疫破坏显著,适应性免疫主要表现为体液免疫与细胞免疫的失衡。     

基于BALB/c裸小鼠构建SIV感染动物模型

目的构建可进行猴免疫缺陷病毒(SIV)复制的裸小鼠模型,为抗艾滋病病毒药物筛选的体内实验提供一种廉价、易于操作的艾滋病小动物模型。方法向BALB/c裸小鼠腹腔内接种已感染猴免疫缺陷病毒(SIVmac251)的TZM-b1细胞,构建SIV体内复制的裸小鼠动物模型,通过采集小鼠血浆,进行反转录实时荧光定量聚合酶链反应法(RT-PCR)检测血浆中SIVmac251病毒载量,并且通过HE染色法检测小鼠主要器官的组织学改变,通过高效联合抗反转录病毒疗法(HAART)对该模型进行药物干预,验证该模型的抗病毒药物评价效果。结果 1)BALB/c裸小鼠成功接种已感染SIVmac251的TZM-b1细胞。2)连续7周在BALB/c裸小鼠血浆里检测出SIVmac 251病毒载量。3)在小鼠主要器官出现了组织学改变。4)验证了HAART对小鼠体内SIVmac 251复制的抑制作用。结论构建了支持SIV复制的小动物模型,该模型能够支持SIVmac251的复制,并表现出组织学病理变化,为研究抗艾滋病药物体内抑制病毒复制提供了廉价、易于操作的小动物模型。     

日本血吸虫大陆株泛蛋白缀合酶E(SjUCEE)基因的A-T克隆

目的　日本血吸虫中国大陆株泛蛋白缀合酶 E (ubiquitin- conjugating enzyme E ,Sj UCEE)的基因克隆和鉴定。方法　特定寡核苷酸引物 PCR扩增目的基因 ;应用分子克隆常规操作方法将扩增产物克隆至载体 pu Cm - T中。结果　 PCR特异性扩增出 Sj UCEE编码区基因序列 ,其片段大小为 75 7bp;经酶切、PCR及测序鉴定表明所构建的质粒 pu Cm - T/Sj UCEE中含有所扩增的基因序列。结论　 PCR扩增的 Sj U CEE抗原编码区基因序列与预期长度相符合 ,成功构建了含目的基因的原核质粒 pu Cm- T/Sj U CEE     

巢式RT-PCR分析日本血吸虫组织蛋白酶L1(SjCL1)的基因5''末端序列

目的：测定日本血吸虫组织蛋白酶L1（SjCL1)基因的5'端序列。方法：从日本血吸虫成虫中提取总RNA，以该RNA为模板，进行巢式RT－PCR，扩增SjCL1基因5'端序列。将其与pMD18-T载体连接得到含SjCL1基因5' 端序列的重组质粒，并测定上述DNA插入片段的序列。结果：通过巢式RT－PCR扩增出332bp SjCL1基因5'端序列，测序后，与报道的SjCL1基因部分序列拼接后，可得到一个编码317个氨基酸的完整开放阅读框。结论：使用巢式RT－PCR技术，扩增得到SjCL1基因的5'端序列。为进一步对其进行功能研究奠定了基础。     

巢式PCR检测猴免疫缺陷病毒基因

位于ＳＩＶ的ｇａｇ保守基因区内的特异巢式ＰＣＲ引物用于体外扩增ＳＩＶ病毒基因，其检出敏感性达到０．１ｆｇ水平，较常规ＰＣＲ敏感性高１００倍。巢式ＰＣＲ检测粗提ＳＩＶｍａｃ感染猴后期或经过药物治疗后的猴外周血淋巴细胞ＤＮＡ，证明９６．５％（２８／２９）的猴体内仍有ＳＩＶ病毒感染存在。巢式ＰＣＲ是一种敏感性极高，特异性强，操作简便易行的ＳＩＶ基因检测技术，它对确证ＳＩＶ病毒感染和正确评价药物和疫苗在猴艾滋病模型中的体内治疗效果提供了良好依据。     

Genetic variation of simian immunodeficiency viruses in nonhuman primates.

The generation of biologically active proviral DNA clones of simian immunodeficiency virus (SIV) that give rise to infectious virions has allowed the detailed examination of genetic variation in experimentally inoculated monkeys. Studies of nucleotide sequences derived directly from circulating leukocytes of infected monkeys show that the SIV genome undergoes rapid and dramatic variation during the course of infection. The env gene is a major site for variation, and within the Env protein, hypervariable regions analogous to those previously defined for the human immunodeficiency virus type 1 (HIV-1) env gene are apparent. A major exception is the region corresponding to the V3 domain in HIV-1, which has been highly conserved in all SIV studies to date. These data notwithstanding, the role of SIV genetic variation in the pathogenesis of AIDS in monkeys remains unclear. Genetic variation within the env gene does not appear to be sufficient for the development of AIDS since significant variation is observed in both pathogenic and nonpathogenic SIV infections. Furthermore, although it generally is believed that env gene variation might allow HIV and SIV to avoid recognition and elimination by host immune responses, this premise has not been rigorously proven. The use of molecularly cloned SIV in monkey models has provided important quantitative and qualitative information on in vivo sequence variation, and these data, in turn, have laid the groundwork for addressing the undoubtedly complex functional significance of this variation.     

Isolation and characterization of simian immunodeficiency viruses from two subspecies of African green monkeys

Cercopithecus aethiops (African Green monkey), a nonhuman primate species distributed throughout subsaharan Africa, has been shown to have high seroprevalence rates of antibodies to simian immunodeficiency virus (SIV), and therefore, has been proposed as a natural reservoir for immunodeficiency viruses. Our laboratories have isolated SIV-like viruses from two East African subspecies of C. aethiops designated grivet and vervet monkeys. Analysis of the structural proteins based on the molecular weights and immunologic cross-reactivity to the prototypic SIV(MAC), HIV-1, and HIV-2 isolates suggests that these viruses are distinctly different. Heterogeneity was observed in the molecular weights of the gag, pol, and env gene products between SIV isolates from vervets [SIV(AGM(VER))] and grivets [SIV(AGM(GRI))]. Phenotypically, SIV(AGM(VER)) isolates were distinguishable from SIV(AGM(GRI)) isolates by the apparent size difference of the major core antigen p24. All SIV(AGM(GRI)) and SIV(AGM(VER)) isolates were found to encode a transmembrane protein of approximately 40 kD (gp40) in contrast to gp32 of SIV(MAC). Furthermore, the transmembrane protein was shown to be encoded by the entire env open reading frame, unlike gp32 of SIC(MAC) or gp36 of SIV(AGM(TYO-1)). These data indicate that viruses from C. aethiops share common features with SIV(MAC) and HIV-1, but represent diverse SIV-like viruses which may vary according to subspecies and geographic location.     

Pattern of SIVagm infection in patas monkeys suggests that host adaptation to simian immunodeficiency virus infection may result in resistance to infection and virus extinction

Patas monkeys were not reported to carry species‐specific simian immunodeficiency virus (SIV), but cross‐species transmission of SIVagm to patas monkeys occurred in the wild. We report that patas monkeys share immunophenotypic features with natural hosts of SIV; that is, low levels of CD4+ T cells and low CCR5 expression on CD4+ T cells. In 1 patas monkey with undetectable levels of CD4+ T cells, experimental exposure to SIVagm did not result in infection. The other experimentally infected patas monkeys showed an infection pattern similar to SIV infection in natural hosts. Thus, down‐regulation of CD4 and CCR5 expression on CD4+ T cells may effectively control human immunodeficiency virus acquisition and result in SIV extinction.     

Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys

We engineered recombinant derivatives of simian immunodeficiency virus (SIV) to express enhanced green fluorescent protein (EGFP). Replacement of vpr sequences with EGFP resulted in a genome that did not produce detectable levels of replication-competent virus. Replication-competent virus and bright fluorescence of infected cells were obtained with two other constructs, one in which SIV nef sequences were replaced by EGFP and another in which EGFP was inserted into the SIV nef locus and HIV-1 nef sequences were expressed by downstream placement of an internal ribosomal entry site. These strains were infectious in rhesus monkeys and green fluorescing cells were detected in the tissues of infected monkeys by FACS analysis and by direct microscopic visualization. EGFP sequences were absent from recovered virus by 8 weeks following infection. We conclude that recombinant SIV that is engineered to express EGFP can be used to directly detect productively infected cells and aid in the immunophenotypic characterization of these cells within the first 2 weeks of infection of rhesus monkeys.     

Prevention of HIV-2 and SIV infections in cynomolgus macaques by prophylactic treatment with 3'-fluorothymidine.

The aim of this study was to determine the usefulness of human immunodeficiency virus type 2 (HIV-2) for in vivo evaluation of antiviral drugs in monkeys and to study if prophylactic treatment with 3'-fluorothymidine (FLT) could prevent infection against a low challenge dose of HIV-2 or simian immunodeficiency virus (SIV). Protection against infection was assessed by virus isolation and polymerase chain reaction (PCR) on monkey peripheral blood mononuclear cells (PBMC) as well as by antibody and viral antigen assays. Prophylactic treatment with FLT 3 x 5 mg/kg/day, starting 8 h prior to virus inoculation, prevented HIV-2 infection in 3 of 8 monkeys. In another experiment 2 of 4 monkeys resisted 2-10 monkey infectious doses (MID50) of SIV with the same prophylactic treatment. All control animals (HIV-2 n = 8, SIV n = 4) became infected. Thus, FLT treatment prevented HIV-2 and SIV infection in 5 of 12 animals.     

Transmission of retroviral infection by transfusion of seronegative blood in nonhuman primates

Techniques such as polyclonal B cell activation with pokeweed mitogen (PWM) and polymerase chain reaction (PCR) analysis have documented the existence of simian immunodeficiency virus (SIV)-and human immunodeficiency virus type 1-seronegative but infected humans and nonhuman primates. To establish whether blood from such seronegative but PWM- and PCR-positive monkeys can transmit infection, naive macaques were transfused with whole blood (n = 2) or cultured cells and supernatant fluid (n = 2) from two seronegative but PWM- and PCR-positive sooty mangabeys. After transfusion, three of the four recipients seroconverted, and peripheral blood mononuclear cells from all four recipients secreted SIV-reactive antibodies upon polyclonal activation in vitro and were SIV-positive by PCR that used highly specific gag primer pairs and probe. In addition, CD8+ cells from all four recipients markedly inhibited replication of SIV in autologous cells in vitro. These data suggest caution in the sole use of serologic tests for the detection of retroviral infection and document the ability of such blood samples to transmit infection.     

Infection of cynomolgus monkeys with HIV-2 protects against pathogenic consequences of a subsequent simian immunodeficiency virus infection

Simian immunodeficiency virus (SIV) infection in cynomolgus macaques leads to severe immunodeficiency with a fatal outcome. In contrast, HIV-2 infects these primates without apparently causing any immunological abnormalities. In this study three cynomolgus monkeys were experimentally infected with HIV-2 strain SBL-K135 and 168 days later challenged with 10-100 animal infectious doses of the closely related SIV strain SM to study protective immunity. At the time of SIV challenge the HIV-2-infected monkeys had neutralizing antibodies against HIV-2, but virus could no longer be recovered from their peripheral blood mononuclear cells (PBMCs) and no clinical symptoms or decrease in CD4+ lymphocytes were observed. Follow-up for 9 months after challenge with SIV showed that the HIV-2-infected monkeys were protected against SIV-induced immunodeficiency (no decrease of CD4+ lymphocytes) and lymphadenopathy. However, they were not resistant to SIV infection since virus could be recovered from their PBMCs and they developed anamnestic antibody responses. Four naive control monkeys which were inoculated with the same dose of SIV became persistently infected and developed a decrease of the absolute numbers of CD4+ cells and showed a marked lymphadenopathy. Two out of four control animals died 58-265 days postinfection with an immunosuppressive disease. Immunohistochemical examination showed abundant viral antigen in lymph-node biopsies from the SIV-infected control monkeys but absence of SIV or HIV-2 antigens in the biopsies from the three HIV-2-preinfected and SIV-superinfected monkeys. The present study demonstrates possibilities for induction of immunity against immunodeficiency induced by a primate lentivirus, a concept with application also to HIV infection and AIDS in man.     

Identification of broadly reactive continuous antigenic determinants of simian immunodeficiency virus glycoproteins.

The env polyprotein sequences of several simian immunodeficiency virus (SIV) isolates were analyzed using computer algorithms designed to predict immunologically reactive protein segments. Peptides corresponding to predicted epitopes were synthesized and employed in peptide enzyme-linked immunosorbent assay (ELISA) screening of serum panels from experimentally infected macaques, as well as naturally infected, asymptomatic mangabeys and African green monkeys. Several of the peptides are recognized by a high percentage of antisera from each panel of monkeys indicating that they represent group-specific antigenic determinants of SIV. Several type-specific determinants also were identified. These peptides may be a useful tool for studying the kinetics of SIV glycoprotein-specific immune responses produced by infected and vaccine-protected monkeys at the epitope level.