Comparison of intra- and inter-population variability for each subject, HIV-1 protein, and sample type (RNA, VC, or VP).

a<p>Average number of nucleotide differences per site between sequences. PR, protease; IN, integrase; RNA, plasma viral RNA; VC, cell virus isolates; VP, plasma virus isolates.</p

Epidemiological and clinical data of study subjects.

a<p>Virus subtype was determined based on sequences from <i>gag</i>, pol, and <i>env</i>-V3 using the REGA HIV-1 Subtyping tool. BF denotes the recombinant BF HIV-1 form.</p

Phenotypic and genotypic prediction of co-receptor usage from total plasma RNA, VC, and VP primary isolates.

a<p>ESTA, Enhance Sensitivity Trofile Assay. This assay has a detection limit of 0.3% for non-R5 variants.</p>b<p>Cut-off values to define non-R5 using sequences were −4.75 for PSSM and ≤3.5 for g2p, respectively. Non-R5 sequences include X4 and X4/R5 dual tropic virus. RNA, plasma viral RNA, VC, cell virus isolates; VP, plasma virus isolates. – Indicates non-determined.</p

Number of sequences obtained for each subject and sample type by DPS.

a<p>Total reads is the total coverage of sequences obtained after direct DPS.</p>b<p>Valid reads are those sequences obtained after cleaning the total reads by selecting unique sequences with >70% homology to HXB2 and manual correction of homopolymer tacks.</p>c<p>Unique haplotypes are defined by similar sequences represented as a proportion ≥1% from the total unique reads. DPS, deep pyrosequencing; PR, protease; IN, integrase; RNA, plasma viral RNA; VC, cell virus isolates; VP, plasma virus isolates.</p

Piqueria trinervia

原著和名: ステビア科名: キク科 = Compositae採集地: 千葉県 船橋市三山2-2-1 東邦大学 (下総 東邦大学)採集日: 1974/9/26採集者: 萩庭丈壽整理番号: JH025454国立科学博物館整理番号: TNS-VS-97545

Phylogenetic trees for HIV-1 <i>gag</i>, protease, integrase and <i>env</i>-V3 sequences extracted from VC, VP, and RNA using DPS.

<p>Symbols represent unique haplotypes extracted from DPS for subjects P20 ○, P21 ▵, P22 ⋄, and P23 □, according to sample type: VC (light symbols), VP (dark symbols), and RNA (empty symbols). Numbers next to symbols indicate haplotype frequencies obtained from the total reads; only values above 20% are indicated in the figure. Node numbers indicate bootstrap values over 75%. (<b>A</b>) <i>gag</i> maximum-likelihood phylogenetic tree based on the TrN model. (<b>B</b>) Integrase maximum-likelihood phylogenetic tree based on the HKY model. (<b>C</b>) Protease maximum-likelihood phylogenetic tree based on the HKY+ G (α = 0.565) model. (<b>D</b>) <i>env</i>-V3 maximum-likelihood phylogenetic tree based on the TrN model.</p

Comparison of the efficiencies of HIV-1 isolation methods from PBMCs or plasma samples.

<p>To determine the efficiency of HIV-1 isolation from PBMCs and plasma, we compared virus recovery from 56 plasma samples and 38 PBMCs samples with viral load ranging from 10 to >10⁶ copies/ml. (<b>A</b>) Efficiency of HIV-1 recovery from PBMCs in percentages per viral load range. (<b>B</b>) Efficiency of HIV-1 recovery from plasma samples in percentages per viral load range. Bars represent mean values. Numbers next to the bars indicate (number of positive samples/total number of samples tested).</p

MOESM1 of Lack of concordance between residual viremia and viral variants driving de novo infection of CD4+ T cells on ART

Additional file 1: Figure S1. Sorting strategy used to purify the different CD4+ T-cell subsets

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy, Viral Evolution and Compartmentalization

<div><p>Background</p><p>Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.</p><p>Methods & Results</p><p>We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No <i>de novo</i> CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.</p><p>Conclusions</p><p>The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.</p></div

Prevalence of CXCR4-using viruses using different tropism assays and settings.

<p>Bar plot showing the mean and 95% confidence intervals of the prevalence of subjects with CXCR4-using viruses using different tropism assays and settings. The Geno2Pheno_[coreceptor] clinical model was only used in pre-treatment bulk sequences derived from plasma RNA; otherwise, the clonal model was used. ESTA, Enhanced-Sensitivity Trofile™ Assay; FPR, Geno2Pheno_[coreceptor] false positive rate used to assign tropism; MT-2, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. * p-value<0.05, two-sided exact binomial tst.</p