CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

257 Pan-HIV Next-Gen Sequencing Strategy for Viral Surveillance Michael G. Berg 1 ; JulieYamaguchi 1 ; Elodie Alessandri-Gradt 2 ; Jean-Christophe Plantier 2 ; Catherine Brennan 1 1 Abbott Laboratories, Abbott Park, IL, US; 2 Virology Unit, National Reference for HIV, Rouen, France

Background: The complexity of HIV strains has increased significantly due to natural evolution and inter-subtype recombination; recombination is now a worldwide problem. Thus, complete genome sequencing is essential to monitor HIV diversity accurately within populations. Next-generation sequencing (NGS) has the potential to revolutionize strategies for HIV surveillance. We have developed a universal method that permits full genome sequencing of all HIV-1 groups (M, N, O, and P) and HIV-2. Methods: Reverse transcription primers, designed in conserved regions of HIV and spaced at 1.5-2kb intervals, fuse viral sequences to a common adaptor (SMART) sequence. This same adaptor is added to the 3’ end of the cDNA to permit PCR amplification of libraries, which are then tagmented with Nextera XT for multiplexing and sequencing on an Illumina MiSeq. HIV sequences are extracted and assembled in CLC-Bio software (Qiagen) and classified by phylogenetic analysis using PHYLIP and SIMPLOT. Results: Broad application of the approach was demonstrated using a panel of virus isolates (n=47) derived from cell culture that included 27 group M (different subtypes and CRFs), 16 O, 2 N, 1 P, and 1 HIV-2. In a single run multiplexing 23 libraries, 100% genome coverage was obtained for each at a median depth of 2100X, with HIV reads comprising 9.4% (median) of the total. A Cameroonian HIV-1 non-subtype B specimen was used to optimize the protocol for plasma. An NGS run of 8 high titer (>5.0 log copies/ml) clinical specimens, infected with diverse group M subtypes, yielded 96-100% coverage for each at a median 433X depth. Method sensitivity, demonstrated by serial dilution, showed that >50% of a genome could be obtained from a clinical specimen with a viral load >3.8 log copies/ml (100 RNA copies input). Due to inherent variability in sample background, coverage varied widely among specimens with viral loads <4.5 log . Nevertheless, sufficient sequence was obtained for strain classification. From the 55 novel full-length HIV sequences determined in this study, 5 are unique recombinants. Conclusions: The HIV-SMART approach harnesses the specificity of HIV-directed priming without a prior i knowledge of the viral strain present. This technology provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome, illustrating the utility of NGS for viral surveillance. 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.

Oral 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 *. Conclusions: This is the first report, to the best of our knowledge, of a high-throughput complete HIV genome sequencing and analysis pipeline in Africa. The genetic diversity of HIV variants in this population is high and is mediated primarily by multiple introductions of HIV. Interventions therefore must be cognizant of the dynamics that drive these independent introductions in order to impact on going HIV transmission. 254 Present Applications of a High-Throughput, Single Measure HIV Genomic Incidence Assay SungYong Park 1 ;Tanzy Love 2 ; Nolan Goeken 1 ; Robert Bolan 3 ; Alan S Perelson 4 ; Michael Dube 1 ; Ha Youn Lee 1 1 Keck School of Medicine at University of Southern California, Los Angeles, CA, US; 2 University of Rochester School of Medicine and Dentistry, Rochester, CA, US; 3 Los Angeles Gay and Lesbian Center, Los Angeles, CA, US; 4 Los Alamos National Laboratory, Los Alamos, CA, US Background: Annual HIV incidence is the primary assessor for monitoring the epidemic’s rise and decline. In pursuit of an accurate assay, we have proposed a genomic incidence assay utilizing fingerprints harbored in the HIV sequence population, which showed over 95% accuracy among 182 incident and 43 chronic samples. Still, two major hurdles must

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CROI 2015