CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

be overcome before routine use in cross-sectional settings: 1) streamlining the cost and workflow, 2) ensuring proper classification between multiple-founder recent infections and chronic infections. Methods: For enhancing the cost efficiency of the sequence-based assay, we developed a high-throughput next-generation sequencing platform; a signal-masking bioinformatics pipeline was devised to analyze 18,434 envelope gene segments (HXB2 7212-7601) obtained from 12 incident and 24 chronic patients. To give the assay power to appropriately discriminate multiple-founder recent infections from chronic infections, we formulated a mathematical model which posits the intersequence nucleotide base difference distribution of each subject’s sequence sample as a function of infection duration and the number of founder sequences. This model was tested by analyzing HIV subtype B and C samples from 40 incident subjects with multiple founder viruses. Results: First, the cost-effective pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 out of 24 chronic subjects (96% specificity). Our signal-masking bioinformatics pipeline yielded a process error rate of 5.8 × 10 -4 per base. Sampling simulations showed that the biomarkers were tolerant of the two factors most likely to affect the accuracy: sequencing errors and template resampling. Second, a quantitative guideline for segregating viral lineages was provided by our mathematical model, enabling us to assess when each subject was infected. The infection periods obtained from our model estimates and from Fiebig laboratory staging showed a statistically significant linear relationship (p<0.0005), correctly identifying all 40 individuals with incident infections. Conclusions: The high-throughput platform permits the assay to be cost-effective, and when it is combined with our mathematical model, we can obtain recency signatures from the complex gene pool that arises frommultiple founder viruses. Our sequence-based approach marks significant progress towards accurate determination of HIV incidence from genomic readouts measured from cross-sectional samples from a single blood draw. 255 A Comprehensive Analysis of Primer IDs to Study Heterogenous HIV-1 Populations David Seifert 1 ; ArminTöpfer 1 ; Francesca Di Giallonardo 2 ; Stefan Schmutz 2 ; Huldrych F. Günthard 2 ;Volker Roth 3 ; Niko Beerenwinkel 1 ; Karin J. Metzner 2 1 ETH Zurich, Basel, Switzerland; 2 University Hospital Zurich, Zurich, Switzerland; 3 University of Basel, Basel, Switzerland Background: Haplotyping of HIV-1 populations is an essential step to better understand the evolutionary dynamics of the virus. With the advent of next-generation sequencing (NGS), haplotyping of viral populations has become feasible. Since HIV-1 is highly heterogeneous, several statistical methods have been devised to deal with error-prone NGS data, however, they often do not capture the population correctly. In order to correct for errors, the use of PrimerIDs (primer identifiers) has been proposed. Here, we used PrimerIDs to systematically estimate different enzymatic error rates and to comprehensively study the feasibility of PrimerIDs. Methods: Plasmids containing full-length genomes of 5 HIV-1 clones were separately amplified in bacteria and then transfected into 293T cells. Generated infectious HIV-1 particles were pooled, DNase treated, and a fragment of the pol gene was reverse transcribed with SuperScript III reverse transcriptase (RT) and primers containing random 10-mers. Reverse transcription was performed in six independent replicates. Subsequently, nested PCR was performed using Platinum Taq DNA Polymerase followed by adapter ligation and sequencing with Illumina MiSeq. Results: From an average number of 1.1 million reads, we called consensus sequences for PrimerIDs, each supported by at least 10 sequencing reads, to yield on average 11,000 consensus sequences per replicate. From these consensus sequences, we could call all mutant bases from the five reference viruses. We estimated a RT error rate of 6.23e-4 (95% CI: [6.13e-4, 6.32e-4]). We inferred the recombination rate of the RT to be 3.44e-5 (95% CI: [2.26e-5, 4.92e-5]). The PCR substitution rate of 1.18e-4 (95% CI: [1.14e-4, 1.22e-4]) was determined from those mutants having arisen in the first cycle of the PCR. We calculated the total number of transcribed RNAs to be on the order of 60,000 from the observed collision rate of 2%. We observed no sequence-specific bias in PrimerID frequencies, the same RT efficiencies as compared to commonly used short, specific RT primers, and no effects of primerIDs on the estimated distribution of the five viruses in the mix. Conclusions: PrimerIDs allow for determining error rates in RT-PCR-NGS protocols and are applicable to study HIV-1 heterogeneity when attention is paid to collision rates. Given these advantages, the protocol is still labor- and cost-intensive and does not significantly improve on the variance of frequency estimates. 256 Near Full Length HIV-1 Sequencing to Understand HIV Phylodynamics in Africa in Real Time Siva Danaviah; Justen Manasa; EduanWilkinson; Sureshnee Pillay; Zandile Sibisi; Sthembiso Msweli; Deenan Pillay; Tulio de Oliveira University of KwaZulu-Natal, Durban, South Africa Background: HIV transmission continues in Africa at alarming rates despite biological and behavioural interventions. Understanding the drivers of HIV transmission and evolution and translating the results into effective interventions is a key component of halting the epidemic. Recent technological advancement in complete genome sequencing has expanded the breadth and speed of genomic analyses currently possible. We have constructed a high-throughput genomics and bioinformatics pipeline that has successfully generated high quality complete HIV genomes in a hyper-endemic region of South Africa (SA), through the PANGEA_HIV Consortium Methods: HIV RNA was extracted from plasma from patients failing antiretroviral therapy, within the Africa Centre (AC) research area and 4 overlapping regions spanning the 9.7kb complete HIV genome were amplified in a one-step RT-PCR strategy optimised for subtype C virus. Pooled amplicons were sequenced on an Illumina MiSeq. Fragments were quality controlled with SMALT software and assembled using two independent strategies (de novo and mapping to reference) in Geneious. Resulting consensus sequences were aligned against published HIV complete genomes from South Africa (n=300). Bayesian and maximum likelihood trees with branch support were reconstructed in PhyML and MrBayes. Results: Amplification success rate of complete genomes, on samples with viral loads >10,000 c/ml was 85%. Near complete HIV genomes were generated for 117/117 samples sequenced thus far, with all nine open reading frames, the U5 / partial R region of the 5’ LTR and partial U3 of the 3’ LTR represented. Coverage of the HIV genome averaged 99.9% with a mean depth of coverage of 15 539 times (range = 21-48 767 times). Phylogenetic reconstruction confirmed that the AC strains were all HIV-1 subtype C where 36/117 sequences clustered with other complete genomes from SA. The discrete AC clusters (n=22) suggested multiple independent introductions of subtype C into the surveillance area and onward transmission within the population.

Poster Abstracts

Maximum likelihood tree of 117 Africa Centre near full length HIV-1 complete genomes and 300 HIV-1 C genomes from South Africa. The tree is rooted on reference strains of subtypes B & D, branch support (bootstrap > 90) are marked with an *.

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CROI 2015