Name: genotypic inference of hiv-1 tropism using population-based sequencing of v3
Uploaded: 2010-12-27T18:00:00.000+00:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3 at JoVE.com

이 분석의 개발은 바이브 의료 및 건강 연구 (CIHR)의 캐나다 연구소에서 박사 해리건에 대한 임상 바이러스학의 GlaxoSmithKline / CIHR 의자 통해 지원되었다. 화이자와 바이브 건강에서 제공하는 지원.

<ol>
	<li>Feng, Y., Broder, C. C., Kennedy, P. E. <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+entry+cofactor:+functional+cDNA+cloning+of+a+seven+transmembrane,+G+protein-coupled+receptor.">HIV-1 entry cofactor: functional cDNA cloning of a seven transmembrane, G protein-coupled receptor.</a> Science. 272, 872-877 (1996).</li><li>Dragic, T., Litwin, V., Allaway, G. P. <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+entry+into+CD4++cells+is+mediated+by+the+chemokine+receptor+CC-CKR-5.">HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.</a> Nature. 381, 667-673 (1996).</li><li>Dorr, P., Westby, M., Dobbs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+(UK-427,857),+a+potent,+orally+bioavailable,+and+selective+small-molecule+inhibitor+of+chemokine+receptor+CCR5+with+broad-spectrum+anti-human+immunodeficiency+virus+type+1+activity.">Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.</a> Antimicrob. Agents Chemother. 49, 4721-4732 (2005).</li><li>Gulick, R. M., Lalezari, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+for+previously+treated+patients+with+R5+HIV-1+infection.">Maraviroc for previously treated patients with R5 HIV-1 infection.</a> N. Engl. J. Med. 359, 1429-1441 (2008).</li><li>Cooper, D. A., Heera, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+versus+efavirenz,+both+in+combination+with+zidovudine/lamivudine,+for+the+treatment+of+antiretroviral-na&#237;ve+subjects+with+CCR5-tropic+HIV-1.">Maraviroc versus efavirenz, both in combination with zidovudine/lamivudine, for the treatment of antiretroviral-na&#237;ve subjects with CCR5-tropic HIV-1.</a> J. Infect. Dis. 201, 803-813 (2010).</li><li>Low, A. J., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trofile+HIV+co-receptor+usage+assay.">Trofile HIV co-receptor usage assay.</a> Expert Opin. Med. Diagnostics. 3, 181-191 (2000).</li><li>Sing, T., Low, A. J., Beerenwinkel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+HIV+co-receptor+usage+based+on+genetic+and+clinical+covariates.">Predicting HIV co-receptor usage based on genetic and clinical covariates.</a> Antiviral Ther. 12, 1097-1106 (2007).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+HIV+tropism+using+population-based+V3+genotypic+analysis:+a+retrospective+virological+outcome+analysis+using+stored+plasma+screening+samples+from+MOTIVATE-1.">Screening for HIV tropism using population-based V3 genotypic analysis: a retrospective virological outcome analysis using stored plasma screening samples from MOTIVATE-1.</a> , (2009).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cut-points+for+the+determination+of+HIV+co-receptor+usage+to+predict+maraviroc+responses+in+treatment+experienced+(TE)+patients+using+population+V3+genotyping.">Optimization of clinically relevant cut-points for the determination of HIV co-receptor usage to predict maraviroc responses in treatment experienced (TE) patients using population V3 genotyping.</a> , (2009).</li><li>Swenson, L. C., McGovern, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cutoffs+for+determining+HIV+co-receptor+use+by+population+and+"deep"+sequencing+methods.">Optimization of clinically relevant cutoffs for determining HIV co-receptor use by population and "deep" sequencing methods.</a> , (2009).</li><li>Swenson, L., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+for+HIV+tropism+versus+both+population-based+and+"deep"+sequencing.">Phenotypic screening for HIV tropism versus both population-based and "deep" sequencing.</a> , (2009).</li><li>Swenson, L. C., Moores, A., Low, A. J., Thielen, A., Dong, W., Woods, C., Jensen, M. A., Wynhoven, B., Chan, D., Glascock, C., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Detection+of+CXCR4-Using+HIV+by+V3+Genotyping:+Application+of+Population-based+and+'Deep'+Sequencing+to+Plasma+RNA+and+Proviral+DNA.">Improved Detection of CXCR4-Using HIV by V3 Genotyping: Application of Population-based and 'Deep' Sequencing to Plasma RNA and Proviral DNA.</a> Journal of Acquired Immune Deficiency Syndromes. 54, 506-510 (2010).</li><li>Swenson, L. C., Boehme, R., Thielen, A., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+determination+of+HIV-1+tropism+in+the+clinical+setting.">Genotypic determination of HIV-1 tropism in the clinical setting.</a> HIV Therapy. 4, 293-303 (2010).</li></ol>

P. 리차드 해리건은 바이브 의료, Virco, 머크, 애보트, 그리고 퀘스트와 관심의 충돌을했다. 이 동영상 문서는 바이브 의료 후원됩니다.

여기서 제시하는 방법은 tropism 테스트에 적용되는 표준 시퀀싱 방식입니다. 늦은 시간까지 제한하고 바이러스 tropism을 예측하는 순서 HIV 봉투 V3 루프의 임상 응용 프로그램이 있습니다. 이 방법은 최소한 임상 사용하는 다른 검증 assays에 비해 바이러스 tropism을 예측하는 동등 수 있도록, maraviroc (바이브 의료) 임상 시험의 회고 분석에 표시하고 있습니다. tropism 예측에 대한 V3 루프의 genotypic 분석을 수행하기 위해 많은 혜택이 있습니다. 첫번째로 제일 중요 한건,이 절차는 크게 접근을 증가하고 tropism 테스트의 처리 시간을 줄여, 운영 시퀀싱 장비와 모든 시설에서 수행할 수 있습니다. 비교에서, tropism 테스트를위한 황금 표준으로 재직했습니다 phenotypic 향상된 감도 Trofile 분석 (ESTA) (모노그램 Biosciences)는, 남부 캘리포니아에서 한 센터에서 수행됩니다. 추가 혜택이 적은 시작 물질 500copies/mL 최소 요구 플라즈마 바이러스 부하의 요구 사항을 포함합니다. 뿐만 아니라, genotypic 분석을 실행하는 운영 비용은 phenotypic 분석 분들에 비해 상대적으로 낮은 수 있습니다. 특히 리콜 소프트웨어의 사용은 유전자형 분석의 노동 집약적인 부분이 될 경향이 수동으로 순서대로 검토에 대한 요구 사항을 제거합니다. 여기서 제시하는 방법은 중요성에 또 다른 outweighing 특별한 단계로, 매우 간단합니다. 우리는 샘플의 바이러스 인구 내에서 캡처 소수 종족의 확률을 높일 세중의에 PCR 및 시퀀싱을 실행해야합니까. 세 이상의 복제 큰 수행할 수 있으며, 그러나 우리가 찾은이 효과적이고 신뢰할 수있는 모두 수 triplicates. 또한 고감도와 특이성을 유지하면서 같은 반응 RT - PCR과 PCR 첫 라운드를 모두 수행 한 단계 거꾸로 Transcriptase PCR 키트 (Qiagen)를 사용하는 것이 좋습니다.

<table><tbody><tr><td>&lt;strong&gt;RNA extraction using NucliSens easyMAG version 1.0&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NucliSens easyMAG Magnetic Silica</td><td>bioMerieux</td><td>cat. # 280133</td><td>(48 x 0.6 ml)</td></tr><tr><td>NucliSens easyMAG Lysis Buffer</td><td>bioMerieux</td><td>cat. # 280134</td><td>(4 x 1000 ml)</td></tr><tr><td>NucliSens easyMAG Extraction Buffer 1</td><td>bioMerieux</td><td>cat. # 280130</td><td>(4 x 1000 ml)</td></tr><tr><td>NucliSens easyMAG Extraction Buffer 2</td><td>bioMerieux</td><td>cat. # 280131</td><td>(4 x 1000 ml)</td></tr><tr><td>NucliSens easyMAG Extraction Buffer 3</td><td>bioMerieux</td><td>cat. # 280132</td><td>(4 x 1000 ml)</td></tr><tr><td>NucliSens easyMAG version 1.0 </td><td>bioMerieux</td><td></td><td></td></tr><tr><td>Class II A2 biological safety cabinet</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;One-Step Reverse Transcriptase PCR and Second Round PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DEPC-treated water</td><td>Ambion</td><td>cat. # 9922</td><td></td></tr><tr><td>SuperScrip III One-Step RT-PCR System with Platinum Taq High Fidelty</td><td>Invitrogen</td><td>cat#12574-035</td><td>Kit components used:&lt;br/&gt;- SuperScript III RT/ Platinum Taq High Fidelty Enzyme Mix&lt;br/&gt;- 2X Reaction Mix (a buffer containing 0.4mM of each dNTP, 2.4mM MgSO&lt;sub&gt;4&lt;/sub&gt;)</td></tr><tr><td>Expand High Fidelity PCR System</td><td>Roche Group</td><td>cat. # 1 759 078</td><td>Kit components used:- Expand High Fidelity Buffer 10X conc. with 15 mM MgCl2 (lids labelled 2)- Expand HF PCR Enzyme Mix (3.5U/&amp;#x03BC;l)- MgCl&lt;sub&gt;2&lt;/sub&gt; Stock Solution (25mM) (lids labelled 4)</td></tr><tr><td>dNTPs: dATP, dGTP, dCTP, dTTP</td><td>Roche Group</td><td>cat. # 11969064001</td><td>(100 mM)</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>cat. # S0389-500G</td><td></td></tr><tr><td>Bromophenol blue sodium salt</td><td>Sigma-Aldrich</td><td>cat.# B5525</td><td></td></tr><tr><td>Cresol Red</td><td>Sigma-Aldrich</td><td>cat.# 114472-5G</td><td>indicator grade</td></tr><tr><td>Thermocycler </td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Gel Electrophoresis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50X TAE buffer</td><td>Invitrogen</td><td>cat. # 24710030</td><td>(1000 ml) </td></tr><tr><td>SYBR Safe DNA gel stain</td><td>Invitrogen</td><td>cat. # S33102</td><td></td></tr><tr><td>Agarose (ultra pure)</td><td>Invitrogen</td><td>cat. # 15510-027</td><td></td></tr><tr><td>E-Gel Low Range Quantitative DNA Ladder</td><td>Invitrogen</td><td>cat. # 12373-031</td><td></td></tr><tr><td>Reagent Grade Type II water</td><td></td><td></td><td></td></tr><tr><td>Gel apparatus</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Sequencing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ABI PRISM BigDye Terminator Cycle Sequencing Kit</td><td>Applied Biosciences</td><td>cat. # 4337458</td><td>(25000 reactions)</td></tr><tr><td>Hi-Di Formamide</td><td>Applied Biosciences</td><td>cat. # 4311320</td><td></td></tr><tr><td>Sodium Acetate (NaOAc)</td><td>Sigma-Aldrich</td><td>cat. # S-2889</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>Cat.# 03690-100ML</td><td>0.5M, pH=8.0</td></tr><tr><td>TRIZMA Hydrochloride Buffer Solution</td><td>Sigma-Aldrich</td><td>cat. # T-2819</td><td>1 M, pH 9.0</td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>cat. # M-1028</td><td>1 M</td></tr><tr><td>95% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Running Buffer (10X) with EDTA</td><td>Applied Biosystems</td><td>cat. # 4335613</td><td>500 mL</td></tr><tr><td>Polymer Pop-7 (25mL) Or Polymer Pop-7 (10mL)</td><td>Applied Biosystems</td><td>cat. # 44363929 or cat. # 4352759</td><td></td></tr><tr><td>3700/3730 BigDye Terminator v3.1 Sequencing Standard Kit</td><td>Applied Biosciences</td><td>Cat# 4336943</td><td></td></tr><tr><td>Thermocycler </td><td></td><td></td><td></td></tr><tr><td>Centrifuge with plate holders</td><td></td><td></td><td></td></tr><tr><td>3730xl DNA Analyzer</td><td>Applied Biosciences</td><td></td><td></td></tr></tbody></table>

genotypic inference of hiv-1 tropism using population-based sequencing of v3

배경 : 이전 HIV 치료에 CCR5 - 길항제 클래스에서 마약을받을하기 위해 환자 자신의 바이러스성 인구가 대안 coreceptor를 휴대 전화 항목에 대한 CCR5 coreceptor을 사용하지 않는 것 확인하는 HIV의 tropism 테스트를 받아야합니다. tropism 테스트하는 한 가지 방법은 coreceptor와 상호 작용하는 HIV 봉투의 V3 지역의 순서를 검사하는 것입니다. 방법 : 바이러스성 RNA는 혈장에서 추출한 것입니다. V3 지역은 중첩된 리버스 transcriptase - PCR로 증폭 세중의에있다. amplifications 그런 다음 합성 및 소프트웨어, RE_Call를 사용하여 분석하고 있습니다. 시퀀스는 다음 V3 지역에서 바이러스 tropism을 추론하기 위해 geno2pheno 같은 bioinformatic 알고리즘에 제출합니다. 시퀀스들은 geno2pheno 거짓 긍정적인 속도 아래 5.75 %를 일인 경우는 비 R5으로 유추됩니다. 샘플에서 세 시퀀스 중 하나가 아닌 R5로 유추하는 경우, 환자는 CCR5 - 길항제에 응답하지 않을 수 있습니다.

Watch this Scientific Journal Video about Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3 at JoVE.com

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

HIV tropism는 바이러스 봉투의 V3 지역에서 유추하실 수 있습니다. V3는 PCR은 RT - PCR 중첩, 합성을 사용하여 세중의에 증폭하고, bioinformatic 소프트웨어를 사용하여 해석됩니다. 낮은 g2P 점수 1 개 이상의 시퀀스 (S)와 함께 샘플이 아닌 R5 바이러스로 분류됩니다.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

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 ...

<a class="grey_button" href="/video/3538">Espa&ntilde;ol </a>

genotypic-inference-hiv-1-tropism-using-population-based-sequencing

Research

JoVE Journal

Immunology and Infection

12.2K 조회수.  BC Centre for Excellence in HIV/AIDS.  Español 

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

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

하여 HIV - 1 V3 루프 크라운 R2 시퀀스 Immunologically 관련 동적 차 구조 기능의 평가 순이 론적 접는

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

연구

면역학 및 감염병학

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

양방향 레트로 바이러스 통합 사이트 PCR 방법론 및 정량적 데이터 분석 워크 플로

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

유전학

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]).

Genotypic 접근 방법을 사용하여 시퀀스 분석에 의한 HIV-1 Coreceptor 사용 (Tropism)의 예측

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 ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

다음-세대 시퀀싱에 대 한 전체 길이 HIV-1 Proviruses 근처의 증폭

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

결정 3'-테르미니와 에이즈-1 중 초기 단일 가닥 바이러스 성 DNA 분자의 순서 반대로 감염 된 세포에서 전사

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

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.

제한 효소 기반의 복제 방법을 평가하기 위해<em&gt; 시험관</em&gt; HIV-1 서브 타입 C 개그 - MJ4 키메라 바이러스의 복제 능력

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 ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

자원 제한 설정에 대한 경제적 인 HIV-1 약제 내성 모니터링 방법

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

의학

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.

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.

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

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

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

필기록

요약

더 많은 비디오 탐색

이 비디오의 챕터

하여 HIV - 1 V3 루프 크라운 R2 시퀀스 Immunologically 관련 동적 차 구조 기능의 평가 순이 론적 접는

양방향 레트로 바이러스 통합 사이트 PCR 방법론 및 정량적 데이터 분석 워크 플로

Genotypic 접근 방법을 사용하여 시퀀스 분석에 의한 HIV-1 Coreceptor 사용 (Tropism)의 예측

다음-세대 시퀀싱에 대 한 전체 길이 HIV-1 Proviruses 근처의 증폭

결정 3'-테르미니와 에이즈-1 중 초기 단일 가닥 바이러스 성 DNA 분자의 순서 반대로 감염 된 세포에서 전사

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

제한 효소 기반의 복제 방법을 평가하기 위해

자원 제한 설정에 대한 경제적 인 HIV-1 약제 내성 모니터링 방법

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

단일 셀 정량 mRNA와 유인원 면역 결핍 바이러스에 감염 된 CD4에 표면 단백질 표정⁺ T 세포 격리에서 붉은 털 원숭이

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

필기록

요약

더 많은 비디오 탐색

이 비디오의 챕터

하여 HIV - 1 V3 루프 크라운 R2 시퀀스 Immunologically 관련 동적 차 구조 기능의 평가 순이 론적 접는

양방향 레트로 바이러스 통합 사이트 PCR 방법론 및 정량적 데이터 분석 워크 플로

Genotypic 접근 방법을 사용하여 시퀀스 분석에 의한 HIV-1 Coreceptor 사용 (Tropism)의 예측

다음-세대 시퀀싱에 대 한 전체 길이 HIV-1 Proviruses 근처의 증폭

결정 3'-테르미니와 에이즈-1 중 초기 단일 가닥 바이러스 성 DNA 분자의 순서 반대로 감염 된 세포에서 전사

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

제한 효소 기반의 복제 방법을 평가하기 위해

자원 제한 설정에 대한 경제적 인 HIV-1 약제 내성 모니터링 방법

인간 면역 결핍 바이러스의 복제 피트니스를 결정하기위한 인접 쌍 성장 경쟁 분석

단일 셀 정량 mRNA와 유인원 면역 결핍 바이러스에 감염 된 CD4에 표면 단백질 표정+ T 세포 격리에서 붉은 털 원숭이

단일 셀 정량 mRNA와 유인원 면역 결핍 바이러스에 감염 된 CD4에 표면 단백질 표정⁺ T 세포 격리에서 붉은 털 원숭이