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>
	<li>Domingo, E., Holland, 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=RNA+virus+mutations+and+fitness+for+survival.">RNA virus mutations and fitness for survival.</a> Annu Rev Microbiol. 51, 151-178 (1997).</li><li>Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+viral+emergence.">Mechanisms of viral emergence.</a> Veterinary research. 41 (6), 38 (2010).</li><li>Martinez-Picado, J., Martinez, M. A. <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+reverse+transcriptase+inhibitor+resistance+mutations+and+fitness:+a+view+from+the+clinic+and+ex+vivo.">HIV-1 reverse transcriptase inhibitor resistance mutations and fitness: a view from the clinic and ex vivo.</a> Virus Res. 134 (1-2), 104-123 (2008).</li><li>Troyer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+fitness+impact+of+HIV-1+escape+mutations+to+cytotoxic+T+lymphocyte+(CTL)+response.">Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.</a> PLoS Pathog. 5 (4), e1000365 (2009).</li><li>Rolland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+co-variation+in+HIV-1+Gag+subtype+C:+HLA-mediated+selection+pressure+and+compensatory+dynamics.">Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics.</a> PLoS One. 5 (9), e12463 (2010).</li><li>Abraha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR5-+and+CXCR4-tropic+subtype+C+human+immunodeficiency+virus+type+1+isolates+have+a+lower+level+of+pathogenic+fitness+than+other+dominant+group+M+subtypes:+implications+for+the+epidemic.">CCR5- and CXCR4-tropic subtype C human immunodeficiency virus type 1 isolates have a lower level of pathogenic fitness than other dominant group M subtypes: implications for the epidemic.</a> J Virol. 83 (11), 5592-5605 (2009).</li><li>Quinones-Mateu, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+infection/competition+assay+shows+a+correlation+between+ex+vivo+human+immunodeficiency+virus+type+1+fitness+and+disease+progression.">A dual infection/competition assay shows a correlation between ex vivo human immunodeficiency virus type 1 fitness and disease progression.</a> J Virol. 74 (19), 9222-9233 (2000).</li><li>Ball, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+ex+vivo+fitness+of+CCR5-tropic+human+immunodeficiency+virus+type+1+isolates+of+subtypes.">Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes.</a> B and C. J Virol. 77 (2), 1021-1038 (2003).</li><li>Lanxon-Cookson, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+relative+fitness+measurements+in+pairwise+competition+assays+of+human+immunodeficiency+viruses.">Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses.</a> J Virol Methods. 194 (1-2), 7-13 (2013).</li><li>Garcia-Lerma, J. G., MacInnes, H., Bennett, D., Weinstock, H., Heneine, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitted+human+immunodeficiency+virus+type+1+carrying+the+D67N+or+K219Q/E+mutation+evolves+rapidly+to+zidovudine+resistance+in+vitro+and+shows+a+high+replicative+fitness+in+the+presence+of+zidovudine.">Transmitted human immunodeficiency virus type 1 carrying the D67N or K219Q/E mutation evolves rapidly to zidovudine resistance in vitro and shows a high replicative fitness in the presence of zidovudine.</a> J Virol. 78 (14), 7545-7552 (2004).</li><li>Sharma, P. L., Crumpacker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuated+replication+of+human+immunodeficiency+virus+type+1+with+a+didanosine-selected+reverse+transcriptase+mutation.">Attenuated replication of human immunodeficiency virus type 1 with a didanosine-selected reverse transcriptase mutation.</a> J Virol. 71 (11), 8846-8851 (1997).</li><li>Martinez-Picado, J., Savara, A. V., Sutton, L., D'Aquila, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+fitness+of+protease+inhibitor-resistant+mutants+of+human+immunodeficiency+virus+type+1.">Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1.</a> J Virol. 73 (5), 3744-3752 (1999).</li><li>Wang, 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+HIV-1+reverse+transcriptase+mutants+G190S+and+G190A,+which+confer+resistance+to+non-nucleoside+reverse+transcriptase+inhibitors,+demonstrate+reductions+in+RNase+H+activity+and+DNA+synthesis+from+tRNA(Lys,+3)+that+correlate+with+reductions+in+replication+efficiency.">The HIV-1 reverse transcriptase mutants G190S and G190A, which confer resistance to non-nucleoside reverse transcriptase inhibitors, demonstrate reductions in RNase H activity and DNA synthesis from tRNA(Lys, 3) that correlate with reductions in replication efficiency.</a> Virology. 348 (2), 462-474 (2006).</li><li>Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+immune+escape+mutations+on+HIV-1+fitness+in+the+context+of+the+cognate+transmitted/founder+genome.">Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome.</a> Retrovirology. 9 (89), (2012).</li><li>Ali, A., Yang, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+small+reporter+gene+and+HIV-1+fitness+assay.">A novel small reporter gene and HIV-1 fitness assay.</a> Journal of Virological Methods. 133 (1), 41-47 (2006).</li><li>Maarseveen, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+real-time+PCR+assay+to+determine+relative+replication+capacity+for+HIV-1+protease+variants+and/or+reverse+transcriptase+variants.">A novel real-time PCR assay to determine relative replication capacity for HIV-1 protease variants and/or reverse transcriptase variants.</a> J Virol Methods. 133 (2), 185-194 (2006).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+human+immunodeficiency+virus+type+1+cytotoxic+T-lymphocyte+epitopes:+fitness-balanced+escape.">Evolution of human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitopes: fitness-balanced escape.</a> J Virol. 81 (22), 12179-12188 (2007).</li><li>Anastassopoulou, C. G., Ketas, T. J., Klasse, P. J., Moore, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+CCR5+inhibitors+caused+by+sequence+changes+in+the+fusion+peptide+of+HIV-1+gp41.">Resistance to CCR5 inhibitors caused by sequence changes in the fusion peptide of HIV-1 gp41.</a> Proc Natl Acad Sci U S A. 106 (13), 5318-5323 (2009).</li><li>Abraha, A., Troyer, R. M., Quinones-Mateu, M. E., Arts, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+determine+HIV-1+ex+vivo+fitness.">Methods to determine HIV-1 ex vivo fitness.</a> Methods Mol Biol. 304, 355-368 (2005).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+real-time+PCR+based+assay+to+estimate+the+impact+of+amino+acid+substitutions+on+the+competitive+replication+fitness+of+human+immunodeficiency+virus+type+1+in+cell+culture.">A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.</a> J Virol Methods. 189 (1), 157-166 (2013).</li><li>Brumme, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+replication+capacity+of+NL4-3+recombinant+viruses+encoding+reverse+transcriptase-integrase+sequences+from+HIV-1+elite+controllers.">Reduced replication capacity of NL4-3 recombinant viruses encoding reverse transcriptase-integrase sequences from HIV-1 elite controllers.</a> J Acquir Immune Defic Syndr. 56 (2), 100-108 (2011).</li><li>Nomura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+reductions+in+Gag-protease-mediated+HIV-1+replication+capacity+during+the+course+of+the+epidemic+in.">Significant reductions in Gag-protease-mediated HIV-1 replication capacity during the course of the epidemic in.</a> Japan. J Virol. 87 (3), 1465-1476 (2013).</li><li>Rolland, M. <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+conserved-element+vaccines:+relationship+between+sequence+conservation+and+replicative+capacity.">HIV-1 conserved-element vaccines: relationship between sequence conservation and replicative capacity.</a> J Virol. 87 (10), 5461-5467 (2013).</li><li>Martinez-Picado, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitness+cost+of+escape+mutations+in+p24+Gag+in+association+with+control+of+human+immunodeficiency+virus+type+1.">Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1.</a> J Virol. 80 (7), 3617-3623 (2006).</li><li>Brockman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+and+Compensation+from+Early+HLA-B57-Mediated+Cytotoxic+T-Lymphocyte+Pressure+on+Human+Immunodeficiency+Virus+Type+1+Gag+Alter+Capsid+Interactions+with+Cyclophilin+A.">Escape and Compensation from Early HLA-B57-Mediated Cytotoxic T-Lymphocyte Pressure on Human Immunodeficiency Virus Type 1 Gag Alter Capsid Interactions with Cyclophilin A.</a> J Virol. 81 (22), 12608-12618 (2007).</li><li>Miura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-associated+alterations+in+replication+capacity+of+chimeric+NL4-3+viruses+carrying+gag-protease+from+elite+controllers+of+human+immunodeficiency+virus+type+1.">HLA-associated alterations in replication capacity of chimeric NL4-3 viruses carrying gag-protease from elite controllers of human immunodeficiency virus type 1.</a> J Virol. 83 (1), 140-149 (2009).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of visualized experiments : JoVE. 62 (Apr 20), (2012).</li><li>McClure, J., van't Wout, A. B., Tran, T., Mittler, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-monocyte+colony-stimulating+factor+upregulates+HIV-1+replication+in+monocyte-derived+macrophages+cultured+at+low+density.">Granulocyte-monocyte colony-stimulating factor upregulates HIV-1 replication in monocyte-derived macrophages cultured at low density.</a> J Acquir Immune Defic Syndr. 44 (3), 254-261 (2007).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimatingfifty+percent+endpoints.">A simple method of estimatingfifty percent endpoints.</a> Am J Hyg. 27 (3), 493-497 (1938).</li><li>Nickle, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+and+ancestral+state+HIV+vaccines.">Consensus and ancestral state HIV vaccines.</a> Science. 299 (5612), 1515-1518 (2003).</li><li>Rolland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+and+function+of+ancestral+center-of-tree+human+immunodeficiency+virus+type+1+proteins.">Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins.</a> J Virol. 81 (16), 8507-8514 (2007).</li><li>Bryksin, A. V., Matsumura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlap+extension+PCR+cloning:+a+simple+and+reliable+way+to+create+recombinant+plasmids.">Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids.</a> Biotechniques. 48 (6), 463-465 (2010).</li><li>Spira, A. I., Ho, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+different+donor+cells+on+human+immunodeficiency+virus+type+1+replication+and+selection+in+vitro.">Effect of different donor cells on human immunodeficiency virus type 1 replication and selection in vitro.</a> J. Virol. 69 (1), 422-429 (1995).</li><li>Manocheewa, S., Swain, J. V., Lanxon-Cookson, E., Rolland, M., 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=Fitness+costs+of+mutations+at+the+HIV-1+capsid+hexamerization+interface.">Fitness costs of mutations at the HIV-1 capsid hexamerization interface.</a> PLoS One. 8 (6), e66065 (2013).</li><li>Zakeri, H., Amparo, G., Chen, S. M., Spurgeon, S., Kwok, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peak+height+pattern+in+dichloro-rhodamine+and+energy+transfer+dye+terminator+sequencing.">Peak height pattern in dichloro-rhodamine and energy transfer dye terminator sequencing.</a> Biotechniques. 25 (3), 406-410 (1998).</li><li>Lalonde, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+oligonucleotide+ligation+assay+for+low-level+detection+of+nevirapine+resistance+mutations+in+human+immunodeficiency+virus+type+1+quasispecies.">Sensitive oligonucleotide ligation assay for low-level detection of nevirapine resistance mutations in human immunodeficiency virus type 1 quasispecies.</a> J Clin Microbiol. 45 (8), 2604-2615 (2007).</li></ol>

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

Research

JoVE Journal

Immunology and Infection

11.0K 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.