Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay
In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in compa...
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Published in | Journal of acquired immune deficiency syndromes (1999) Vol. 74; no. 2; p. 221 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
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01.02.2017
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Abstract | In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA).
Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid antigen positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1-V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with highly active antiretroviral therapy in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution.
IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r = 0.94; P < 0.0001).
VOA-UDSA may be an alternative readout for that currently used for QVOA. |
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AbstractList | In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA).
Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid antigen positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1-V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with highly active antiretroviral therapy in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution.
IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r = 0.94; P < 0.0001).
VOA-UDSA may be an alternative readout for that currently used for QVOA. |
Author | Baldoni, Pedro L Zhou, Shuntai Lee, Sook-Kyung Spielvogel, Ean Hudgens, Michael G Margolis, David M Swanstrom, Ronald Archin, Nancie M |
Author_xml | – sequence: 1 givenname: Sook-Kyung surname: Lee fullname: Lee, Sook-Kyung organization: Department of Biochemistry and Biophysics, UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC; †Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC; and Departments of ‡Medicine; §Microbiology and Immunology, UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC – sequence: 2 givenname: Shuntai surname: Zhou fullname: Zhou, Shuntai – sequence: 3 givenname: Pedro L surname: Baldoni fullname: Baldoni, Pedro L – sequence: 4 givenname: Ean surname: Spielvogel fullname: Spielvogel, Ean – sequence: 5 givenname: Nancie M surname: Archin fullname: Archin, Nancie M – sequence: 6 givenname: Michael G surname: Hudgens fullname: Hudgens, Michael G – sequence: 7 givenname: David M surname: Margolis fullname: Margolis, David M – sequence: 8 givenname: Ronald surname: Swanstrom fullname: Swanstrom, Ronald |
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SubjectTerms | Anti-Retroviral Agents - therapeutic use CD4-Positive T-Lymphocytes - virology DNA Primers - genetics Genetic Variation Genotype High-Throughput Nucleotide Sequencing HIV Infections - drug therapy HIV Infections - virology HIV-1 - classification HIV-1 - genetics HIV-1 - isolation & purification Humans Phylogeny |
Title | Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay |
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