作者要感谢的患者参加的HIV - 1的许多研究。 江铃汽车的FM柯比基金会的支持下与美国癌症协会的研究教授。

1。从大量的血浆提取的RNA 在图1和2提供了一个单拷贝检测（SCA）的概述和单基因组测序（SGS）的协议。 <ol><li>要获得7毫升血浆，旋转约14毫升的血液收集在15毫升EDTA，15分钟，在2600 XG（肝素）无制动收集管。移液器（一定要避免巴菲涂层）为15毫升管的血浆。 </li><li>如果RNA是单拷贝检测（SCA），秒杀Rous肉瘤病毒内部的病毒粒子控制（RCAS）30000份血浆中提取。 RCAS病毒前准备执行政制事务局局长如前所述（20）。简言之，通过RT活性量化RCAS病毒从细胞培养上清，稀释至终浓度的每200 UL 30000的病毒颗粒，含5％FBS的RPMI组织培养基稀释，并储存于-80 ° C在单一使用等份。 RCAS病毒不应该被冻结/解冻之前，在实验中使用。 RCAS添加到患者血浆RNA提取的效率和精度的PCR定量控制。在下面的链接： <a href="http://home.ncifcrf.gov/hivdrp/RCAS/">http://home.ncifcrf.gov/hivdrp/RCAS/</a>可以找到更多关于合会的信息。 </li><li> “预旋”等离子，15毫升锥形离心管中，在2600 XG 15分钟，分离出任何脂类和细胞碎片，它可以干扰RNA定量准确。 </li><li>标签西顿易于密封的永久性标记，以确定样本数和颗粒的位置将形成管离心管。前自旋后，转让等离子到西顿易于密封的离心管中，吹打的血浆，并避免在表面上的任何细胞碎片和/或血脂。记录血浆的输入量。 </li><li>添加的Tris缓冲液（TBS），以填补剩余量的西顿简易封管螺纹脖子底部。确保无气泡，目前在管或管会崩溃的超速离心机。确保管的标志是朝外管放入转子。 </li><li>密封可重复使用的瓶盖和一个预冷Sorval T1270转子和Sorval 170000 XG（50,000 RPM）在4 ° C为30分钟90SE超速离心机离心机样本。 </li><li>离心后除去上清液和添加90 UL分子级的水和10 UL蛋白酶K（20毫克/毫升）病毒颗粒（这将是不可见的的）。 </li><li>在55 ° C水浴30分钟孵化广场管。确保管一边是沉浸在蛋白酶K和水的混合物，使沉淀的角度。 </li><li>孵化后不久，在表离心机旋转管，收集所有液体的试管底部（约5秒），并添加315 UL 6M胍硫氰酸解决方案和10 UL 20毫克/毫升糖原（注：按照适当的这种有害物质的处置准则;不使用漂白剂或酸混合，丢弃在单独的容器 ） 。 </li><li>涡轻轻旋不久（约5秒）。在室温下孵育10分钟，然后各管的内容传送到一个​​新标记的1.5毫升核糖核酸酶免费离心管。 </li><li> 495 UL的分子级异丙醇添加到每个管，离心旋涡10秒，在室温为30分钟，在21000 XG。 </li><li>弃上清，和70％的乙醇添加500 UL。 </li><li> 21000 XG用于在室温下15分钟10秒和离心旋涡。取出用移液管的乙醇。再次旋转和删除任何残留的乙醇用移液器。空气干燥10分钟。 （SGS的协议溶于40 5毫米的Tris - HCl pH值8.0 UL的RNA，并继续与SGS协议）。 </li><li> 55 RNA缓冲UL溶解沉淀。 RNA缓冲是通过添加10 UL 100毫米的DTT和25 Rnasin 40 U / UL UL 965 UL的Tris - HCl（pH值8.0，5毫米）。置于冰上。 </li></ol> 2。单拷贝检测一个96孔板拥有8患者的样本，包括艾滋病毒和RCAS标准和控制。该协议将描述一个单盘的设置。 <ol><li>开始准备2 1毫升RNA缓冲区中的HIV - 1的RNA和RCAS RNA转录的RNA标准曲线将稀释分装。 RNA缓冲描述下1.13。 </li><li>准备在2毫升离心管冰稀释。加入的HIV - 1 RNA的股票（1 × 10 6份/ UL）的25到54 UL RNA 缓冲3x10 5份/ UL UL一半的HIV - 1 RNA的RNA缓冲记录日志稀释。 </li><li>继续通过连续的半对数的稀释（25 UL + 54 UL）下降到0.3份每10 UL（注：0.3，1，3稀释将不会在这些分析的标准曲线的一部分被用作推断值并应设置为未知数DURING的分析）。 RCAS成绩单的放养密度为1 × 10 6份/ UL。同样，准备在RNA缓冲RCAS笔录半日志稀释，但每10 UL下降到100份。 </li><li>准备如表1所列的cDNA合成的RT鸡尾酒。准备RT和牢记，以弥补一点额外的考虑了在传输过程中可能发生的任何损失，如表1所列的尼古丁替代疗法的反应。注：为尼古丁替代疗法的鸡尾酒，逆转录酶是由水所取代，以控制从DNA扩增。 注意：此步骤最近一直在钢板上Qiagen公司的机器人（原本科贝特）设立自动化。 </li><li>设置光学96孔板（如图3所示），每口井的RT反应混合物中加入20 UL。在标记的8口井的“尼古丁替代疗法”，添加没有逆转录酶（简称NRT反应混合物）的鸡尾酒。加入鸡尾酒板后，加水10 UL井标有“无模板对照（NTC）的”。 10艾滋病毒成绩单UL标记为“人类免疫力缺乏病毒（HIV）”，如图3所示的浓度井。 10 RCAS笔录UL标有“RCAS”根据浓度的水井。添加在板图上标有“样品”的3个相邻井的10个患者样本UL。洗脱样品，加入10 UL标记的井“尼古丁替代疗法”。新增5 UL同一样品和5水UL标记的井“内部人控制”。 </li><li>密封板和热循环仪上运行：25 ° C为15分钟，42 ° C时40分钟，85℃10分钟，25℃30分钟和5 ° C举行。 </li><li>准备PCR扩增混合，按表2。 </li><li>当cDNA合成完成后，移动到指定的区域，利用cDNA。在此指定的区域中，添加20 PCR扩增预混UL以及每个艾滋病毒和RCAS的cDNA板。重要的是要在指定的区域中执行此步骤，以避免任何可能污染。 </li><li>旋转盘，ABI或罗氏光学与光毯（毯只有必要的ABI 7700）盖盖和密封。罗氏480或ABI的7700上运行。 PCR条件：95℃10分钟，然后45个循环95 ° C，持续15秒，60 ° C为1分钟。 RCAS和艾滋病毒进行单独的分析是必需的。如图4所示的结果的例子。一个正常的低拷贝数笔录泊松分布的标准曲线的斜率会受到影响。为了确保最准确的斜坡，作标准曲线，这些低拷贝数的标准（0.3，1和3份）为“未知” 。17 </li></ol> 3。单基因组测序<ol><li> cDNA合成。加入UL的10毫米dNTPs和5 5 2毫米的基因特异性引物（POL或 env）良好的UL在一个96孔PCR板（BIORAD） 。新增40所提取的RNA UL。密封板。 </li><li>在热循环中的RNA混合物在65 ° C变性10分钟。 </li><li>混合cDNA合成，表3中所列的试剂。变性步骤后，放置在冰PCR板，每个样品中添加50 UL的cDNA混合物。 </li><li>运行PCR板热循环：45 ° C为50分钟，85 ° C为10分钟和4 ° C举行。 </li><li>第一次PCR，10 UL反应。准备PCR反应试剂托盘在使用P6 - RT或 env（表3中所列的试剂，在表5中列出的引物）的第一轮扩增引物。 </li><li>使用多道移液器免除8.0 PCR混合物73口井的UL 96孔PCR板（70个样本和3个阴性对照）。 </li><li> cDNA合成完成后，移动的cDNA和PCR板，PCR反应混合物中含有利用cDNA指定的区域。在指定的区域中，添加40的Tris - HCl pH值8.0的UL每个cDNA样本，使终体积为140 UL。添加2.0 cDNA的UL 70口井的水和2.0 UL，每个阴性对照。密封PCR板。热循环，计划在表4上运行。 </li><li>巢式PCR - 20ul反应。混合试剂托盘（表3中所列的试剂，在表5中列出的引物）的巢式PCR试剂。 </li><li> 18 UL的巢式PCR混合物添加一个96孔PCR板73口井，多道移液器</li><li>第一次PCR 1:5稀释，加2 UL第一轮PCR巢式PCR。运行PCR反应的热循环，表4中列出的条件下，。 </li><li> 1％琼脂糖凝胶上运行的样本。为了确保大多数的PCR产物是一个单分子的cDNA的结果，不超过30％的水井应是积极的。该工艺已使用贝克曼Biomek调频机器人自动加载的内容，以1％的PCR板，96孔E -凝胶（Invitrogen公司）。 E -凝胶是运行在E -基地为7分钟。使用表5中列出的引物的序列产品。 </li></ol> 4。代表性的成果： 政制事务局局长： 所有的控制应以正确考虑测量的HIV - 1 RNA传递。如果阴性对照positiVE，运行应该被忽视，因为可能的污染。为了控制损失的RNA提取步骤，至少有50％的添加血浆中的合会应收回。如果平均RCAS &lt;15000份，样品失败，并应被忽略，因为相当数量的RNA可以在提取或该协议的其他步骤丢失。这发生在约10％，所有患者的样本测试，可能是由于高血脂在血浆6。 RCAS病毒的复苏，是为了控制质量的提取和cDNA合成和PCR扩增效率。它不是用于艾滋病毒恢复正确的，因为艾滋病毒有可能略有不同的组织培养分离病毒的免疫球蛋白和其他人类蛋白的约束。在我们的实验室合会的恢复平均25,716 + / - 4057拷贝/反应混合物，或约95％+ / -15％的RCAS RNA添上17 。净吨（无逆转录酶）控制并行运行，以测试从DNA扩增。的NRT值是每三个HIV - 1 RNA值相减，然后平均计算，如果这个数字是小于零（DNA扩增超过RNA扩增），其结果必然失败，应该被忽视，因为而不是RNA的DNA扩增的风险。如果HIV - 1 RNA只有1 / 3口井的放大，重新测试样品的建议，以确保扩增是真正的实际样品被检测，而不是由于一个可能的污染物。 如果通过了​​所有的控制，HIV - 1的平均血浆中的RNA拷贝数可以计算的。 例1：如果平均HIV - 1病毒的拷贝数是零，检测限检测血浆量的基础上计算。作为一个例子，比方说，7毫升血浆检测。本来可以用这个实验中发现的RNA的量最小，本来1份，平均每口井0.33副本的其他两个井的井和0份之一。然后由RNA的洗脱总套数（有10 UL在每口井，但总的洗脱体积为55 UL）为5.5倍乘以平均拷贝数。然后检测下限：5.5 × 0.33除以7毫升= 1.83 / 7 = 0.3拷贝/ ml的副本。在这个例子中的HIV - 1 RNA的血药浓度&lt;0.3拷贝/ ml。 例2：HIV - 1病毒RNA被觉察。 RNA提取从7毫升血浆。 HIV - 1 RNA每平均拷​​贝数以及计算为2.0％，以及副本。然后，每毫升血浆的平均拷贝数为5.5毫升× 2.0份/ 7 = 1.6 HIV - 1的RNA /毫升血浆。 模板Excel工作表来计算拷贝数可以从网站上下载。 SGS： 如果一个阴性对照的是积极的运行应该被忽视，由于可能的污染。从每个运行的产品的数量将取决于每个样本中的病毒载量和样本的条件。根据我们的经验，很多血浆中的脂类或细胞会减少获得产品的机会。储存条件和以前的冻结和解冻的样品，还将大大影响RNA的复苏。在一般情况下，与病毒载量低于50拷贝/ ml，产品（凝胶波段）的样品时，预计将在约1 / 3 -1 / 2处理的样品。根据上述因素，每盘1-5条带应被视为一个很好的结果，由于在这些患者中的低的病毒载量。 <table border="1"><tbody><tr><td> 合成cDNA（RT鸡尾酒/ cDNA反应） </td><td> 1反应</td><td>无逆转录酶（NRT）的鸡尾酒/ cDNA的反应（反应） </td></tr><tr><td>分子级H 2 O </td><td> 8.1 UL </td><td> 8.2 UL </td></tr><tr><td> 25毫米氯化镁2 </td><td> 6 UL </td><td> 6 UL </td></tr><tr><td> 25毫米dNTPs </td><td> 0.6 UL </td><td> 0.6 UL </td></tr><tr><td> 100 mM的数码地面电视</td><td> 0.2 UL </td><td> 0.2 UL </td></tr><tr><td>随机六聚体（0.1微克/ UL） </td><td> 1.5 UL </td><td> 1.5 UL </td></tr><tr><td> 10X的TaqMan缓冲液A </td><td> 3.0 UL </td><td> 3.0 UL </td></tr><tr><td> Rnasin（40 U / UL） </td><td> 0.5 UL </td><td> 0.5 UL </td></tr><tr><td> Strategene RT（200 U / UL） </td><td> 0.1 UL </td><td>不添加</td></tr><tr><td></td><td></td><td></td></tr><tr><td>共有</td><td> 20 UL </td><td> 20 UL </td></tr><tr><td>样品的RNA </td><td> 10 UL </td><td> 10 UL </td></tr><tr><td>总成交量</td><td> 30 UL </td><td> 30 UL </td></tr></tbody></table> 表1反应mixtURES在单拷贝含量的cDNA合成。 <table border="1"><tbody><tr><td> PCR扩增预混 </td><td> 1反应 </td><td> 底漆 </td></tr><tr><td> H 2 O </td><td> 15.1 UL </td><td>艾滋病毒正向引物5&#39; - CATGTTTTCAGCATTATCAGAAGGA - 3&#39; </td></tr><tr><td> 10X PCR检测金缓冲区</td><td> 2.0 UL </td><td>艾滋病毒的反向引物5&#39; - TGCTTGATGTCCCCCCACT - 3&#39; </td></tr><tr><td> 25毫米氯化镁2 </td><td> 2.0 UL </td><td>艾滋病毒Probe5&#39;FAM CCACCCCACAAGATTTAAACACCATGCTAA TAMRA 3“ </td></tr><tr><td> *正向引物（100 UM） </td><td> 0.3 UL </td><td></td></tr><tr><td> *反向引物（100 UM） </td><td> 0.3 UL </td><td> RCAS正向Primer5&#39; - GTCAATAGAGAGAGGGATGGACAAA 3“ </td></tr><tr><td> *探头（100 UM） </td><td> 0.05 UL </td><td>合会反向Primer5&#39; - TCCACAAGTGTAGCAGAGCCC 3&#39; </td></tr><tr><td> TaqGold（5 U / UL） </td><td> 0.25 UL </td><td> RCAS探头5&#39;FAM TGGGTCGGGTGGTCGTGCC - TAMRA 3&#39; </td></tr><tr><td>共有</td><td> 20.0 UL </td><td></td></tr></tbody></table> 见表2。PCR扩增预混试剂和引物进行单拷贝含量。 <table border="1"><tbody><tr><td> cDNA的鸡尾酒/ cDNA的反应 </td><td> 1 RNA样品 </td><td> 第一次PCR扩增鸡尾酒</td><td> 1板（75反应） </td><td> 巢式PCR检测鸡尾酒</td><td> 1板（75反应） </td></tr><tr><td> 10X RT缓冲液（Invitrogen公司） </td><td> 10 UL </td><td> 10X PCR缓冲液（Invitrogen公司） </td><td> 75 UL </td><td> 10X PCR缓冲液</td><td> 150 UL </td></tr><tr><td> 25毫米氯化镁2 </td><td> 20 UL </td><td> 50毫米硫酸镁 </td><td> 30 UL </td><td> 50毫米硫酸镁 </td><td> 60 UL </td></tr><tr><td> 0.1M的数码地面电视</td><td> 1 UL </td><td> 10毫米dNTPs </td><td> 15 UL </td><td> 10毫米dNTPs </td><td> 30 UL </td></tr><tr><td>无RNase水</td><td> 17.5 UL </td><td> 50 UM引物（EA） </td><td> 3 UL </td><td> 50 UM引物（EA） </td><td> 6 UL </td></tr><tr><td> RNase的输出（Invitrogen公司） </td><td> 1 UL </td><td>白金Taq酶的Hi - Fi酶（Invitrogen公司） </td><td> 6 UL </td><td>白金Taq酶的Hi - Fi酶（Invitrogen公司） </td><td> 12 UL </td></tr><tr><td>三标（200 U / UL）（Invitrogen公司） </td><td> 0.5 UL </td><td>分子级水</td><td> 468 UL </td><td>分子级水</td><td> 1086 UL </td></tr></tbody></table> 表1的cDNA和单基因组测序的PCR混合物。 <table border="1"><tbody><tr><td> P6 - RT PCR程序 </td><td> 环境保护1 PCR程序 </td></tr><tr><td> 1。 94℃2分钟</td><td> 1。 94℃2分钟</td></tr><tr><td> 2。 94℃30秒</td><td> 2 0.94 ° C为30秒</td></tr><tr><td> 2 0.94 ° C为30秒</td><td> 3。 52℃30秒</td></tr><tr><td> 4。 72℃1分30秒</td><td> 4。 72℃1分钟</td></tr><tr><td> 5。 ＃2，44个循环</td><td> 5。 ＃2，44个循环</td></tr><tr><td> 6。 72℃3分钟</td><td> 6。 72℃3分钟</td></tr><tr><td> 7。 4℃举行</td><td> 7。 4℃举行</td></tr><tr><td> P6 - RT 2 PCR程序 </td><td> 环境保护2 PCR程序 </td></tr><tr><td> 1。 94℃2分钟</td><td> 1。 94℃2分钟</td></tr><tr><td> 2。 94℃30秒</td><td> 2。 94℃30秒</td></tr><tr><td> 3。 55℃30秒</td><td> 3。 56℃30秒</td></tr><tr><td> 4。 72℃1分钟</td><td> 4。 72℃45秒</td></tr><tr><td> 5。 ＃1，40（共41周期） </td><td> 5。 ＃1，40（共41周期） </td></tr><tr><td> 6。 72℃3分钟</td><td> 6。 72℃3分钟</td></tr><tr><td> 7。 4℃举行</td><td> 7。 4℃举行</td></tr></tbody></table> 表4热循环条件为单基因组测序分析。 <table border="1"><tbody><tr><td> 反应 </td><td> 底漆 </td><td> 序列 </td></tr><tr><td> P6 - RT基因cDNA </td><td> 3500 - </td><td> 5&#39;CTATTAAGTATTTTGATGGGTCATAA 3&#39; </td></tr><tr><td> ENV基因</td><td> E115 - </td><td> 5&#39;AGAAAAATTCCCCTCCACAATTAA 3“ </td></tr><tr><td> P6 - RT 1。 PCR扩增</td><td> 3500 - </td><td> 5&#39;CTATTAAGTATTTTGATGGGTCATAA 3&#39; </td></tr><tr><td> P6 - RT 1。 PCR扩增</td><td> 1849 + </td><td> 5&#39;GATGACAGCATGTCAGGGAG 3&#39; </td></tr><tr><td> P6 - RT2.PCR </td><td> 1870年+ </td><td> 5&#39;GAGTTTTGGCTGAGGCAATGAG 3&#39; </td></tr><tr><td> P6 - RT 2.PCR </td><td> 3410 - </td><td> 5&#39;CAGTTAGTGGTATTACTTCTGTTAGTGCTT 3&#39; </td></tr><tr><td> ENV 1。 PCR扩增</td><td> E115 - </td><td> 5&#39;AGAAAAATTCCCCTCCACAATTAA 3“ </td></tr><tr><td> ENV 1。 PCR扩增</td><td> E20 + </td><td> 5&#39;GGGCCACACATGCCTGTGTACCCACAG 3“ </td></tr><tr><td> ENV 2。 PCR扩增</td><td> E30 + </td><td> 5&#39;GTGTACCCACAGACCCCAGCCCACAAG3“ </td></tr><tr><td> ENV 2。 PCR扩增</td><td> E125 - </td><td> 5&#39;CAATTTCTGGGTCCCCTCCTGAGG 3“ </td></tr><tr><td> P6 - RT测序</td><td> 2030 + </td><td> 5&#39;TGTTGGAAATGTGGAAAGGAAGGAC 3“ </td></tr><tr><td> P6 - RT测序</td><td> 2600 +的</td><td> 5&#39;ATGGCCCAAAAGTTAAACAATGGC3“ </td></tr><tr><td> P6 - RT测序</td><td> 2610 - </td><td> 5&#39;TTCTTCTGTCAATGGCCATTGTTTAAC3“ </td></tr><tr><td> P6 - RT测序</td><td> 3330 - </td><td> 5&#39;TTGCCCAATTCAATTTTCCCACTAA 3“ </td></tr><tr><td> ENV测序</td><td> E30 + </td><td> 5&#39;GTGTACCCACAGACCCCAGCCCACAAG3“ </td></tr><tr><td> ENV测序</td><td> E125 - </td><td> 5&#39;CAATTTCTGGGTCCCCTCCTGAGG 3“ </td></tr></tbody></table> 表5。引物为单基因组测序分析。 <img src="/files/ftp_upload/2960/2960fig1.jpg" alt="图1" /> 图1。辛格基因组测序检测（SGS）的概述。 <img src="/files/ftp_upload/2960/2960fig2.jpg" alt="图2" /> 图2单拷贝含量（SCA）的概述。 <img src="/files/ftp_upload/2960/2960fig3.jpg" alt="图3" /> 图3。板设置为单拷贝检测。 <img src="/files/ftp_upload/2960/2960fig4.jpg" alt="图4" /> 图4：从ABI拍摄的画面7700。 A.显示病人样本和B. RCAS标准曲线呈现出飙升的患者样本的HIV - 1 RNA标准曲线。

<ol>
	<li>Amara, R. R., Villinger, F., Altman, J. D., Lydy, S. L., O'Neil, S. P., Staprans, S. I., Montefiori, D. C., Xu, Y., Herndon, J. G., Wyatt, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+mucosal+challenge+and+prevention+of+AIDS+by+a+multiprotein+DNA/MVA+vaccine.">Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.</a> Vaccine. 20, 1949-1955 (2002).</li><li>Dinoso, J., Kim, S., Blankson, J., Siliciano, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Dynamics+of+Elite+Suppressors+in+HIV-1+Infection.">Viral Dynamics of Elite Suppressors in HIV-1 Infection.</a> Conference on Retroviruses and Opportunistic Infections. , (2008).</li><li>Dinoso, J. B., Kim, S. Y., Wiegand, A. M., Palmer, S. E., Gange, S. J., Cranmer, L., O'Shea, A., Callender, M., Spivak, A., Brennan, T., Kearney, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+intensification+does+not+reduce+residual+HIV-1+viremia+in+patients+on+highly+active+antiretroviral+therapy.">Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 9403-9408 (2009).</li><li>Doria-Rose, N. A., Klein, R. M., Manion, M. M., O'Dell, S., Phogat, A., Chakrabarti, B., Hallahan, C. W., Migueles, S. A., Wrammert, J., Ahmed, R., Nason, M., Wyatt, R. T., Mascola, J. R., Connors, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+and+phenotype+of+human+immunodeficiency+virus+envelope-specific+B+cells+from+patients+with+broadly+cross-neutralizing+antibodies.">Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies.</a> J. Virol. 83, 188-199 (2009).</li><li>Dornadula, G., Zhang, H., Uitert, B. V. a. n., Stern, J., Livornese, L., Ingerman, M. J., Witek, J., Kedanis, R. J., Natkin, J., DeSimone, J., Pomerantz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+HIV-1+RNA+in+blood+plasma+of+patients+taking+suppressive+highly+active+antiretroviral+therapy.">Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy.</a> JAMA. 282, 1627-1632 (1999).</li><li>Gandhi, R. T., Zheng, L., Bosch, R. J., Chan, E. S., Margolis, D. M., Read, S., Kallungal, B., Palmer, S., Medvik, K., Lederman, M. M., Alatrakchi, N., Jacobson, J. M., Wiegand, A., Kearney, M., Coffin, J. M., Mellors, J. W., Eron, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+raltegravir+intensification+on+low-level+residual+viremia+in+HIV-infected+patients+on+antiretroviral+therapy:+a+randomized+controlled+trial.">The effect of raltegravir intensification on low-level residual viremia in HIV-infected patients on antiretroviral therapy: a randomized controlled trial.</a> PLoS medicine. 7, (2010).</li><li>Gay, C., Dibben, O., Anderson, J. A., Stacey, A., Mayo, A. J., Norris, P. J., Kuruc, J. D., Salazar-Gonzalez, J. F., Li, H., Keele, B. F., Hicks, C., Margolis, D., Ferrari, G., Haynes, B., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-sectional+detection+of+acute+HIV+infection:+timing+of+transmission,+inflammation+and+antiretroviral+therapy.">Cross-sectional detection of acute HIV infection: timing of transmission, inflammation and antiretroviral therapy.</a> PLoS One. 6, 19617-19617 (2011).</li><li>Hatano, H., Delwart, E. L., Norris, P. J., Lee, T. H., Dunn-Williams, J., Hunt, P. W., Hoh, R., Stramer, S. L., Linnen, J. M., McCune, J. M., Martin, J. N., Busch, M. P., Deeks, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+persistent+low-level+viremia+in+individuals+who+control+human+immunodeficiency+virus+in+the+absence+of+antiretroviral+therapy.">Evidence for persistent low-level viremia in individuals who control human immunodeficiency virus in the absence of antiretroviral therapy.</a> J. Virol. 83, 329-335 (2009).</li><li>Havlir, D. V., Bassett, R., Levitan, D., Gilbert, P., Tebas, P., Collier, A. C., Hirsch, M. S., Ignacio, C., Condra, J., Gunthard, H. F., Richman, D. D., Wong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+predictive+value+of+intermittent+viremia+with+combination+hiv+therapy.">Prevalence and predictive value of intermittent viremia with combination hiv therapy.</a> JAMA. 286, 171-179 (2001).</li><li>Jordan, M. R., Kearney, M., Palmer, S., Shao, W., Maldarelli, F., Coakley, E. P., Chappey, C., Wanke, C., Coffin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+standard+PCR/cloning+to+single+genome+sequencing+for+analysis+of+HIV-1+populations.">Comparison of standard PCR/cloning to single genome sequencing for analysis of HIV-1 populations.</a> J Virol Methods. 168, 114-120 (2010).</li><li>Kaufmann, D. E., Kavanagh, D. G., Pereyra, F., Zaunders, J. J., Mackey, E. W., Miura, T., Palmer, S., Brockman, M., Rathod, A., Piechocka-Trocha, A., Baker, B., Zhu, B., Gall, S. L. e., Waring, M. T., Ahern, R., Moss, K., Kelleher, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upregulation+of+CTLA-4+by+HIV-specific+CD4++T+cells+correlates+with+disease+progression+and+defines+a+reversible+immune+dysfunction.">Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction.</a> Nat Immunol. 8, 1246-1254 (2007).</li><li>Kearney, M., Maldarelli, F., Shao, W., Margolick, J. B., Daar, E. S., Mellors, J. W., Rao, V., Coffin, J. M., Palmer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Population+Genetics+and+Adaptation+in+Newly+Infected+Individuals.">HIV-1 Population Genetics and Adaptation in Newly Infected Individuals.</a> J. Virol. 83, 2715-2727 (2008).</li><li>Kearney, M., Palmer, S., Maldarelli, F., Shao, W., Polis, M. A., Mican, J., Rock-Kress, D., Margolick, J. B., Coffin, J. M., Mellors, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+polymorphism+at+drug+resistance+sites+in+HIV-1+protease+and+reverse+transcriptase.">Frequent polymorphism at drug resistance sites in HIV-1 protease and reverse transcriptase.</a> AIDS. 22, 497-501 (2008).</li><li>Kearney, M., Spindler, J., Shao, W., Maldarelli, F., Palmer, S., Hu, S. L., Lifson, J. D., Kewal Ramani, V. N., Mellors, J. W., Coffin, J. M., Ambrose, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+simian+immunodeficiency+virus+encoding+HIV-1+reverse+transcriptase+persists+in+macaques+despite+antiretroviral+therapy.">Genetic diversity of simian immunodeficiency virus encoding HIV-1 reverse transcriptase persists in macaques despite antiretroviral therapy.</a> Journal of Virology. 85, 1067-1076 (2011).</li><li>Keele, B. F., Giorgi, E. E., Salazar-Gonzalez, J. F., Decker, J. M., Pham, K. T., Salazar, M. G., Sun, C., Grayson, T., Wang, S., Li, H., Wei, X., Jiang, C., Kirchherr, J. L., Gao, F., Anderson, J. A., Ping, L. H., Swanstrom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+transmitted+and+early+founder+virus+envelopes+in+primary+HIV-1+infection.">Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.</a> Proc. Natl. Acad. Sci. U.S.A. , 105-7552 (2008).</li><li>Liu, S. L., Rodrigo, A. G., Shankarappa, R. G., Learn, H., Hsu, L., Davidov, O., Zhao, L. P., Mullins, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+quasispecies+and+resampling.">HIV quasispecies and resampling.</a> Science. 273, 415-416 (1996).</li><li>Mahalanabis, M., Jayaraman, P., Miura, T., Pereyra, F., Chester, E. M., Richardson, B., Walker, B., Haigwood, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+viral+escape+and+selection+by+autologous+neutralizing+antibodies+in+drug-naive+human+immunodeficiency+virus+controllers.">Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers.</a> J. Virol. 83, 662-672 (2009).</li><li>Migueles, S. A., Connors, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+CD4(+)+and+CD8(+)+T+Cells+in+Controlling+HIV+Infection.">The Role of CD4(+) and CD8(+) T Cells in Controlling HIV Infection.</a> Curr. Infect. Dis. Rep. 4, 461-467 (2002).</li><li>Palmer, S., Kearney, M., Maldarelli, F., Halvas, E. K., Bixby, C. J., Bazmi, H., Rock, D., Falloon, J., Davey, R. T., Dewar, R. L., Metcalf, J. A., Hammer, S., Mellors, J. W., Coffin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple,+linked+human+immunodeficiency+virus+type+1+drug+resistance+mutations+in+treatment-experienced+patients+are+missed+by+standard+genotype+analysis.">Multiple, linked human immunodeficiency virus type 1 drug resistance mutations in treatment-experienced patients are missed by standard genotype analysis.</a> J. Clin. Microbiol. 43, 406-413 (2005).</li><li>Palmer, S., Wiegand, A. P., Maldarelli, F., Bazmi, H., Mican, J. M., Polis, M., Dewar, R. L., Planta, A., Liu, S., Metcalf, J. A., Mellors, J. W., Coffin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+real-time+reverse+transcriptase-initiated+PCR+assay+with+single-copy+sensitivity+for+human+immunodeficiency+virus+type+1+RNA+in+plasma.">New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma.</a> J. Clin. Microbiol. 41, 4531-4536 (2003).</li></ol>

没有利益冲突的声明。

艾滋病毒1感染者抗逆转录病毒药物联合治疗（车）或自然控制感染病毒载量非常低，通常约1副本每毫升血浆4，11，12，17，18。自然控制患者的病毒载量，往往个人设置1，2，17点左右波动。检测的灵敏度，此处所描述的高度依赖的血浆输入量。一般来说，我们已与7毫升血浆，但取得了积极的成果，可以从少量的血浆，以及根据实际血浆病毒载量。此处所描述的RNA提取方法“，这?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>随机六聚体（500微克/毫升） </td><td> Promega公司</td><td> C118A </td><td></td></tr><tr><td> TaqMan探针缓冲液A </td><td>应用生物系统公司</td><td> 4304441 </td><td></td></tr><tr><td> RNAsin（40 U / UL） </td><td> Promega公司</td><td> N211B </td><td></td></tr><tr><td>逆转录酶（200 U / UL） </td><td> Strategene </td><td> 600107-51 </td><td></td></tr><tr><td> Amplitaq黄金DNA聚合酶</td><td>应用生物系统公司</td><td> 4311814 </td><td> 6包</td></tr><tr><td> Amplitaq 10XGold PCR缓冲液II </td><td>应用生物系统公司</td><td> 4311814 </td><td>带有的酶</td></tr><tr><td> Magnesiumcloride 25毫米</td><td>应用生物系统公司</td><td> 4311814 </td><td>带有的酶</td></tr><tr><td> 1M的Tris - HCl缓冲液，pH 8.0，5毫米</td><td> Invitrogen公司</td><td> AM9855G </td><td></td></tr><tr><td>标III逆转录酶200 U / UL </td><td> Invitrogen公司</td><td> 18080-044 </td><td></td></tr><tr><td>标三10X缓冲</td><td> Invitrogen公司</td><td></td><td>带有的酶</td></tr><tr><td>数码地面电视100毫米</td><td> Invitrogen公司</td><td></td><td>带有的酶</td></tr><tr><td>白金Taq DNA聚合酶高保真5 U / UL </td><td> Invitrogen公司</td><td> 11304-102 </td><td></td></tr><tr><td> 10X高保真PCR缓冲液</td><td> Invitrogen公司</td><td> 11304-102 </td><td>带有的酶</td></tr><tr><td>蛋白酶K 20毫克/毫升</td><td> Ambion公司</td><td> 2546 </td><td></td></tr><tr><td>胍硫氰酸解决方案6M </td><td> FlukaBiochemica </td><td> 50983 </td><td></td></tr><tr><td>糖原20​​毫克/毫升</td><td>罗氏公司</td><td> 10901393001 </td><td></td></tr><tr><td>异丙醇</td><td> Sigma - Aldrich公司</td><td> 19516 </td><td></td></tr><tr><td>乙醇70％ </td><td> Sigma - Aldrich公司</td><td> E702 - 3 </td><td></td></tr><tr><td>分子级水</td><td> GIBCO / Invitrogen公司</td><td> 10977-015 </td><td></td></tr><tr><td> dNTPs（各25毫摩尔） </td><td> Bioline </td><td> BIO - 39025 </td><td></td></tr></tbody></table><table border="1"><tbody><tr><td> 对Euipment名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>易密封离心管（16x73mm） </td><td>西顿科学</td><td> 6041 </td><td></td></tr><tr><td>白聚甲醛树脂冠</td><td>西顿科学</td><td> 4020 </td><td>简易的密封管的上限</td></tr><tr><td>试管架Optiseal钟顶管，8.9毫升</td><td>贝克曼</td><td> 361642 </td><td></td></tr><tr><td>六角螺丝刀4.5公司开放</td><td>西顿科学</td><td> 4013 </td><td></td></tr><tr><td>盖帽拆除tupe </td><td>西顿科学</td><td> 4014 </td><td></td></tr><tr><td> 5毫升的血清移液器</td><td>配角</td><td> 4051 </td><td></td></tr><tr><td> Pipetboy </td><td>任何</td><td></td><td></td></tr><tr><td> 15毫升转移移液器</td><td> ISC的Bioexpress </td><td> P - 5005 - 7 </td><td></td></tr><tr><td> 5.8毫升Dispossable转移移液器，精尖</td><td> VWR </td><td> 14670-329 </td><td></td></tr><tr><td> 15 mL离心管</td><td>鹘</td><td></td><td></td></tr><tr><td> 1 mL离心管</td><td>任何</td><td></td><td></td></tr><tr><td> Tris缓冲盐水片</td><td> Sigma - Aldrich公司</td><td> T5030 - 100TAB </td><td></td></tr><tr><td>超速离心机和转子</td><td> Sorval </td><td> 90SE和T - 1270 </td><td></td></tr><tr><td> 96孔PCR板</td><td> BIORAD </td><td> HSS - 9601 </td><td></td></tr><tr><td> 50毫升的试剂水库</td><td>配角</td><td> 4870 </td><td></td></tr><tr><td>微安光学96孔反应板</td><td>应用生物系统公司</td><td> N801 - 0560 </td><td></td></tr><tr><td>光板覆盖</td><td>应用生物系统公司</td><td> 4333183 </td><td></td></tr><tr><td>微安光学压缩垫</td><td>应用生物系统公司</td><td> 4312639 </td><td></td></tr><tr><td> MicrosealA片</td><td> BIORAD </td><td> MSA - 5001 </td><td></td></tr><tr><td> 2毫升离心管</td><td>任何</td><td></td><td></td></tr><tr><td> 1％琼脂糖凝胶（E -凝胶，Invitrogen公司） </td><td> Invitrogen公司</td><td> G - 7008 - 01 </td><td></td></tr><tr><td>电子基地（Invitrogen公司） </td><td> Invitrogen公司</td><td> EB - M03 </td><td></td></tr></tbody></table> EDTA的收集管任何品牌

amplifying and quantifying hiv-1 rna in hiv infected individuals with viral loads below the limit of detection by standard clinical assays

，获得患者的洞察力病毒动态和病毒宿主相互作用，病毒基因放大和量化，在HIV - 1病毒载量低于检测标准检测的限制（低于50-75拷贝/ ml）感染者HIV - 1 RNA是必要的日志自然控制的感染和抗逆转录病毒联合疗法（CART）的那些。 在这里，我们介绍了如何由单一的基因组测序（SGS协议）13，19和如何准确地量化在与病毒载量低（单拷贝检测（SCA）协议）12，20的患者的HIV - 1 RNA病毒的基因组扩增。 单拷贝检测的实时PCR检测的灵敏度取决于被检测血浆量。如果一个单一的病毒基因组是7毫升血浆中检测到，然后报道RNA拷贝数为0.3拷贝/ ml。该检测RNA提取效率的内部控制测试，并可能扩增DNA或污染控制。测量患者样本一式三份。 ，现在已经广泛使用，被视为非劳动密集的3，7，12，14，15的单基因组测序法（SGS）的，是一个限制的稀释检测，其中端点cDNA产物稀释96孔蔓延板。根据泊松分布，不到1 / 3的水井时给予的产品，有一个80％的机会的PCR产物被放大的结果，从一个单一的基因分子。 SGS已超过克隆没有受到重采样和PCR引入重组19偏颇的优势。然而，SCA和SGS的扩增成功取决于引物的设计。两种检测为HIV - 1 B亚型，但可以适应不断变化的引物，探针和标准的其他亚型和基因组的其他地区。

Watch this Scientific Journal Video about Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays at JoVE.com

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

量化血浆中HIV - 1 RNA水平和测序单一的HIV - 1病毒载量低于检测限（50-75拷贝/ ml）与个人的基因组是困难的。在这里，我们描述了如何提取和量化使用实时PCR检测方法能够可靠措施，HIV - 1 RNA下降到0.3拷贝/ ml，以及如何放大由单一的基因组测序的病毒基因组病毒载量非常低的样品，血浆病毒RNA。

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

amplifying-quantifying-hiv-1-rna-hiv-infected-individuals-with-viral

Research

JoVE Journal

Immunology and Infection

31.3K 次观看.  NCI-Frederick. 量化血浆中HIV - 1 RNA水平和测序单一的HIV - 1病毒载量低于检测限（50-75拷贝/ ml）与个人的基因组是困难的。在这里，我们描述了如何提取和量化使用实时PCR检测方法能够可靠措施，HIV - 1 RNA下降到0.3拷贝/ ml，以及如何放大由单一的基因组测序的病毒基因组病毒载量非常低的样品，血浆病毒RNA。

视频: Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.