These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

<ol>
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作者宣称，他们有没有竞争的财务权益。这里表达的观点是作者的，不应被解释为官员或代表美国国防部部或陆军部的意见。

提出的方案包括两个主要部分:施工重组HIV-1分子克隆和增长竞争分析。为了区分这两种病毒在一个双重感染的细胞培养，但重要的是相互竞争的分子克隆含有序列标签，这可以通过一个RT-qPCR的引物 - 探针测定法或通过Sanger测序来检测。这个协议利用了VIFA和VIFB标签，它们占据的HIV-1的NL4-3 的vif同一区域和编码相同氨基酸序列，但相差6同义突变。这些突变显示不影响 病毒复制健身20。在这个协议中使用的基于PCR的克隆方法在选择的克隆位点提供了更多的灵活性，相对于限制性位点克隆。然而，PCR克隆的效率降低为插入尺寸增大。插入大小的电流限制为5〜34 KB。用于基于PCR的克隆和位点定向mutagenesis时，使用高保真DNA聚合酶的关键是减少的额外的突变的可能性。使用得到的产品足够量的所需要的PCR循环的最小数目的还建议。根据我们的经验，我们没有发现这里所描述的基于PCR的克隆和位点定向诱变步骤后的任何额外突变。尽管如此，PCR产物应测序，以检查是否有任何不希望的突变。理想的情况下，整个的HIV编码区应当重新测序。 至少三个采样时间点应该在指数生长期9内进行检查。该病毒生长动力学必须首先使用每日取样，以确定适当的培养周期和采样时间点的成长竞争建立。影响病毒生长动力学的一个因素是感染复数（MOI）的多重性。该协议使用的0.005共MOI为monoinfection和增长的竞争，因为它被证明产生更多的RO胸围结果低于9的MOI。尽管如此，低的MOI可用于在必要时，得到较长的指数增长阶段，但在结果的一致性为代价的。这个协议建议的50:50的初始感染比率，假定的病毒适应度差异一般未知预先。然而，使用不相等的输入比率是合适的时，有初步数据表明在病毒复制动力学显著差异。在这些情况下，以70:30的感染比率，推荐以允许大型健身差的检测，其中所述不太适合病毒被放置在过量9。 该协议确定TCID 50，病毒生长动力学，以及用于执行使用HIV-1的B亚型的生长竞争试验进行了优化，NL4-3 COTB-p24和来自单一供体的外周血单个核细胞的PBMC已经证明一致易感性艾滋毒1感染在体外 。培养期并在这个协议中提出的采样时间点都可能是适合研究HIV-1的M组病毒在人PBMC。使用的PBMC来自单一来源，强烈建议，以获得一致的结果作为病毒复制可以在不同的供体细胞35而变化。尽管不太希望，PBMC中从多个供体汇集的可以用作一个替代条件是相同的池用于所有的实验。另一种替代方法是使用细胞系。这里介绍的协议被成功用于与T细胞系CEMx174 23,36。但是，重要的是，细胞接种的号码重新优化以实现一致的细胞生长。病毒生长动力学，还必须重新建立的，因为它很可能在不同的细胞系而异，在生长竞争步骤，以确定适当的采样时间点。 两种不同的方法来确定病毒比率用于计算健身都包含在protoc醇。第一种方法使用RT-qPCR的测量病毒cDNA拷贝数在每个采样时间点。病毒复制健身然后从病毒比计算，是根据cDNA的拷贝数。可替代地，病毒比率可以基于色谱峰高在VifAB标签位点的比值来确定。这两种方法产生了类似的结果（ 图4）。色谱峰高度的方法可以适用于其它HIV-1病毒株未经改造的序列标签。用于RT-qPCR的，使用引物或探针来区分病毒变异体必须首先仔细评估（见20）。然而，RT-qPCR的提供用少量的病毒RNA，如那些从指数生长期中的第一个时间点样品的灵敏度。病毒cDNA的直接测量还可以检测可能产生的RNA提取和cDNA合成技术问题。利用分子克隆与序列标签提供一个具有成本效益的解决方案牛逼澳RT-qPCR的方法，因为只需要两对引物和探针来研究多种病毒。这一策略也避免了高峰高度变化的顺序问题色谱图，由于邻近的基地37，测序完成在该研究中所有的病毒在同一网站。制备用于RT-qPCR的和Sanger测序PCR产物也可以是与其他方法使用，以确定病毒的比例如本体测序单个克隆12,13，HTA 4,7,17,19，或寡核苷酸连接测定法（OLA）38 。

病毒复制健身被定义为病毒，以产生感染性子代在给定的环境1的能力，并在确定病毒的变体在群体水平的患病率随着时间的推移2的一个重要因素。作为体内健身研究是不致病的人类病毒，如HIV-1，各种体外和可行体外复制健身测定已开发研究从耐药性和免疫逃避突变，上位和进化所产生的健身效果病毒的人群3-6。在不同的健身测定法，生长竞争分析，确认得到的健身差别更敏感和有效的措施，为两个或更多的病毒变体竞争恰恰相同的环境条件下相同的细胞群，如发生在体内 1,7,8。开始增长竞争实验，几个variab前莱斯需要确定，包括使用不同感染多重性（MOI），病毒输入比，以及分析用取样定时。我们已经研究了这些参数对病毒生长动力学和上的竞争实验的结果的影响，并在细胞培养9确定所需的HIV-1的健身鲁棒测量的关键因素。 除了测定变量，有各种各样的用于定量在生长竞争实验的病毒变体的方法。散装10,11或克隆测序12,13已被用于确定基于在感兴趣的位点（多个）核苷酸频率竞争病毒的比例。相对适合从改变这个比例随着时间的推移得到。此方法是方便的DNA测序服务广泛提供。并行等位基因特异性测序（PASS）方法能够测序在多个位点和重组体14的检测</sup&gt;，但它也需要专门开发的试剂和检测系统。本质上，开发了这些方法，研究病毒株具有少量的在感兴趣的区域的核苷酸差异。其他方法使用小报道基因15或同义突变4,16-18作为标记来区分竞争病毒通过测序，异源跟踪测定（HTA）4,7,17,19或逆转录定量实时聚合酶链反应（RT-qPCR的）15,16,18,20，所有这些可以由适用于研究竞争菌株无关的序列相似性。一个附加的步骤是必需的，以引入标记插入病毒基因组和RT-qPCR分析也需要特定的试剂和仪器。我们已发现，散装Sanger测序产量比较的结果9。 继增长的竞争，病毒复制健身是作为相对健身，或T之间的比值健身WO病毒变种。病毒的相对适合度可以被定义为一种病毒变体通过在接种最初的比例或以两种相互竞争的病毒之间的净生长速率差标准化的最终比例。我们发现，后一方法中，使用仅在指数生长期纵向的数据点，产生了最健壮的结果9,20。 在体外实验健身主要用于研究生物克隆6-8和HIV-1感染性克隆的分子。后者，是适合于遗传操作，通常用于研究对从特定的突变或感兴趣3-5,21,22特定序列的健身效果。以下协议描述的工作流程从构建新的全长感染HIV-1的分子克隆用HIV-1载体含有序列标签，引入利益的突变，使得病毒的股票，建立的病毒生长动力学的角度，在执行的生长竞争测定和计算相对适合度（ 图1）。 使用我们的程序优化，我们创建了三个重组HIV-1突变体和确定其复制健身。首先构建通过更换pNL4-3，含有HIV-1毒株实验室的NL4-3全长基因组感染性的质粒的HIV-1 的gag-P24基因区域，用合成COTB（中心最先进重组分子克隆树，B亚型）GAG-P24序列23创建原型株。单个氨基酸变化（T186M，T242N和I256V），然后介绍了创建三个突变克隆。每个突变体竞争对原型病毒，观察在给定的遗传背景各突变的适合度的影响。这三种突变体展示不同的复制fsitness水平从轻微到比原型病毒显著降低。的T242N突变先前报道具有适度fitneSS花费24-26，类似于在这项研究中所示的结果。其他两个突变的适合度以前没有报道。

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name of Material/ Equipment</td>
			<td style="width:484px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:196px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Construction of recombinant clones</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Chimeric primer/Mutagenic/Sequencing primers</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom DNA oligos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pNL4-3VifA and pNL4-3VifB plasmid</td>
			<td style="width:484px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-Fidelity DNA polymerase</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-fidelity buffer</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems&#174; GeneAmp&#174; PCR System 9700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA loading dye</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td style="width:137px;">R0631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1kb Plus DNA ladder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAquick PCR Purification Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick gel extraction kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">DpnI enzyme</td>
			<td style="width:484px;">New England Biolabs</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">TOP10 chemically competent Escherichia coli</td>
			<td style="width:484px;">Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Luria Broth base powder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">12795-084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Carbenicillin</td>
			<td style="width:484px;">Research Products International</td>
			<td style="width:137px;">C46000-25.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep kit&#160;</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">27104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">12362</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">X-tremeGENE 9 DNA transfection reagent</td>
			<td style="width:484px;">Roche</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HEK 293T-17</td>
			<td style="width:484px;">ATCC</td>
			<td style="width:137px;">CRL-11268</td>
			<td>http://www.atcc.org/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.22um filter top tube</td>
			<td style="width:484px;">VWR International</td>
			<td>89220-716&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#8242;s Modified Dulbecco&#8242;s Medium (IMDM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>31980-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RPMI-1640 media</td>
			<td style="width:484px;">Life Technologies</td>
			<td>61870-036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phytohemagglutinin (PHA)</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>R30852801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Human Interleukin-2</td>
			<td style="width:484px;">Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum</td>
			<td style="width:484px;">JR Scientific</td>
			<td style="width:137px;">43640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicillin and Streptomycin</td>
			<td style="width:484px;">Corning Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">73520-906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">62407-338</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat-bottomed plate</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td style="width:137px;">T-3015-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well round-bottomed plate</td>
			<td style="width:484px;">BD Falcon</td>
			<td style="width:137px;">353077</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 ml Microcentrifuge Tube</td>
			<td style="width:484px;">Mt. Baker Bio</td>
			<td style="width:137px;">MBD-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 mL tubes w/ O-ring</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">89004-290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">50 mL conical tube</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td>C-3317-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Triton-X 100</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phosphate buffer saline (PBS)</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum (FBS)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>F0392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">glycerol</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td style="width:137px;">G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tween 20</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-HIV-1 SF2 p24 antiserum</td>
			<td style="width:484px;">NIH AIDS Reagent Program</td>
			<td style="width:137px;">4250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit HRP</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">474-1516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMB Microwell Peroxidase Substrate</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">52-00-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">H2SO4</td>
			<td>Fisher Scientific</td>
			<td>A300-500</td>
			<td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 minutes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HIV p24 standard</td>
			<td style="width:484px;">AIDS and Cancer Virus program (NCI-Frederick)</td>
			<td style="width:137px;">N/A</td>
			<td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microplate reader</td>
			<td style="width:484px;">Molecular Devices</td>
			<td style="width:137px;">N/A</td>
			<td>VMax Kinetic ELISA Microplate Reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA isolation cDNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxtractor</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">N/A</td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAamp Viral RNA Mini Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">52906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">SuperscriptIII</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">First-strand buffer&#160;</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Rnase inhibitor</td>
			<td style="width:484px;">Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RnaseH</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18021-071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Real-time qPCR machine</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems 7300 Real-Time PCR&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan&#174; Gene Expression Master Mix</td>
			<td style="width:484px;">Life Technologies</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Forward, reverse primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Probe</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">optical 96 well reaction plate</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">I19N3Q216</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR+sequencing</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Taq DNA polymerase (Biolase)</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NH4 buffer&#160;</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Sequencing primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxcel</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;"></td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA sequencing service provider</td>
			<td>http://www.htseq.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GRC web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/grc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChromatQuan web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

研究的单氨基酸改变的适应度的影响在HIV-1的Gag-p24的，我们使用寡核苷酸定向诱变，以引入突变到含有HIV-1的COTB-p24的基因23,32,33一个pNL4-3质粒。通过293T细胞的转染产生和48小时后收获病毒母液。我们估计每个病毒的股票的50％组织培养感染剂量（TCID 50）的里德- Muench的方法31。原型的TCID 50和突变病毒介于10月4日至10月五日国际单位/毫升（ 图3A）。 的重组病毒的生长动力学建立在PBMC中从以0.005的MOI单一供体。 RT-qPCR方法每天测量病毒cDNA拷贝数6天。所有突变体和病毒的原型成倍增长2日和4日，随后病毒增长放缓，由病毒RNA拷贝数的增加降低斜率（指示与&lt;STRONG&gt;图3B）。在cDNA中的减少复制0天（相应于接种物）和第2天之间数是由于病毒的吸收，细胞和由第1天洗涤（步骤5.1.6）去除未结合的病毒粒子。在指数生长期中，所有三个突变体具有更慢的生长速率（ 克 ）比原型病毒（ 图3C）。 所有这三个突变体对竞争的原型病毒生长竞争测定为0.005，总MOI。在双重感染病毒培养生长动力学是类似于monoinfection的;病毒指数生长期为2和4天之间，和病毒生长达到约5天（ 图4A）平台期。 病毒生长速率的差异来源于在病毒比率随时间的变化。基于参考的cDNA拷贝数和利用RT-qPCR的，突变的病毒和通过比较峰高的病毒比计算在序列色谱图在核苷酸位点区分这两种病毒（ 图4B）。使用峰高和病毒RNA的拷贝数的方法来确定的生长速率的差异，得到了类似的结果（ 图4C）。所有三种突变体具有比原型病毒与具有最低健身（ 图4C）的突变I256V下复制健身。 <img alt="图1" src="/files/ftp_upload/52610/52610fig1.jpg" /> 图1:在这个文件中提出的协议流程图的HIV-1重组克隆病毒制剂协议建设的关注个股病毒和生成。健身化验协议是建立病毒的生长动力学，并确定相对病毒健身。虚线表示的协议备选流。例如，一个突变可直接引入HIV-1的NL4-3分子克隆，但不产生重组​​克隆。 <img alt="图2" src="/files/ftp_upload/52610/52610fig2.jpg" /> 图2:采用重叠延伸PCR（A）的HIV-1 NL4-3 COTB的Gag-P24重组分子克隆施工图设计嵌合引物。 5&#39;引物半部包含NL4-3矢量序列和3&#39;半部分包含插入序列的两端。 （B）的嵌合引物用于扩增插入片段，COTB的Gag的p24。 （C）的插入片段被用作引物用于第二次PCR以产生新的重组质粒。 <img alt="图3" src="/files/ftp_upload/52610/52610fig3.jpg" /> 图3:外周血单个核细胞中的病毒生长特点 （ 一 ）登录10 TCID 50 </sUB&gt;病毒股票。 （ 二）monoinfections病毒生长动力学开始以MOI = 0.005。 （C）的病毒生长率超过六天，包括从面板（B）中得到的指数生长期（2-4天）。显示的值代表从一个实验的三次重复的平均值。误差线代表95％置信区间。 <img alt="图4" src="/files/ftp_upload/52610/52610fig4.jpg" /> 图4:病毒生长和相对适合测定的 （A）双重感染PBMC培养物的病毒生长的影响。的T242N和原型病毒（B）比率使用RT-qPCR的或测序峰高度的方法测定。 （C）的突变和双重感染的文化原型病毒之间的病毒净增长速度的差异（D），如图（A）。分别计算F D值ROM中的（B）示出的病毒比数据。显示的值代表从一个实验的三次重复的平均值。误差线代表95％置信区间。

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.1K 次观看.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

视频: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

确定使用血凝检测血清中的反应和滴度

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

科研

免疫与感染

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

确定土壤传播的蠕虫感染状况和体质的学龄儿童

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

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