{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T08:38:15Z","timestamp":1772613495457,"version":"3.50.1"},"reference-count":32,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T00:00:00Z","timestamp":1772409600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"United States National Institute of Health","award":["R01 AI134419-01"],"award-info":[{"award-number":["R01 AI134419-01"]}]},{"name":"Molecular Profiling and Computational Biology Core of the University of Washington\/Fred Hutch Center for AIDS Research","award":["P30 AI027757"],"award-info":[{"award-number":["P30 AI027757"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UM1AI068632"],"award-info":[{"award-number":["UM1AI068632"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UM1AI068616"],"award-info":[{"award-number":["UM1AI068616"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UM1AI106716"],"award-info":[{"award-number":["UM1AI106716"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000071","name":"NICHD","doi-asserted-by":"publisher","award":["HHSN275201800001I"],"award-info":[{"award-number":["HHSN275201800001I"]}],"id":[{"id":"10.13039\/100000071","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>In people taking antiretroviral therapy (ART) for HIV infection, the methods to characterize latent and active HIV reservoirs remain costly and labor-intensive. Our objective was to develop a relatively low-cost technique to amplify and sequence the proviruses that persist during ART along with the site in the human genome where each provirus is integrated. We developed a novel HIV-specific Multiple Displacement Amplification (HIV-MDA) assay that specifically amplifies HIV-1 proviruses and their associated integration site. Upon comparison of our HIV-MDA to an established commercial kit designed to amplify cellular DNA, we found that the HIV-MDA (1) typically yielded a greater number of HIV integration site (HIV IS) sequences per 150,000 cells analyzed; (2) improved rates of proviral DNA amplification; and (3) amplified HIV IS at a fraction of the cost (13.6 times less expensive). Thus, the HIV-MDA method appears to be a more sensitive and cost-effective approach to sequencing HIV IS and the associated proviruses compared to a commercial kit.<\/jats:p>","DOI":"10.3390\/v18030311","type":"journal-article","created":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T16:06:59Z","timestamp":1772467619000},"page":"311","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Evaluation of a Novel Relatively Low-Cost Method to Derive HIV-1 Integration Sites and Proviral Sequences"],"prefix":"10.3390","volume":"18","author":[{"given":"Samantha R.","family":"Hardy","sequence":"first","affiliation":[{"name":"Center for Global Infectious Disease Research, Seattle Children\u2019s Research Institute, University of Washington, Seattle, WA 98109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sheila","family":"Styrchak","sequence":"additional","affiliation":[{"name":"Center for Global Infectious Disease Research, Seattle Children\u2019s Research Institute, University of Washington, Seattle, WA 98109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2994-9693","authenticated-orcid":false,"given":"Tim","family":"De Meyer","sequence":"additional","affiliation":[{"name":"Department of Data Analysis and Mathematical Modelling, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurens","family":"Lambrechts","sequence":"additional","affiliation":[{"name":"HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tine","family":"Struyve","sequence":"additional","affiliation":[{"name":"HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Basiel","family":"Cole","sequence":"additional","affiliation":[{"name":"HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liesbet","family":"Termote","sequence":"additional","affiliation":[{"name":"HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sherry","family":"McLaughlin","sequence":"additional","affiliation":[{"name":"Center for Global Infectious Disease Research, Seattle Children\u2019s Research Institute, University of Washington, Seattle, WA 98109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James I.","family":"Mullins","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington, Seattle, WA 98109, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linos","family":"Vandekerckhove","sequence":"additional","affiliation":[{"name":"HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9566-8959","authenticated-orcid":false,"given":"Lisa M.","family":"Frenkel","sequence":"additional","affiliation":[{"name":"Center for Global Infectious Disease Research, Seattle Children\u2019s Research Institute, University of Washington, Seattle, WA 98109, USA"},{"name":"Departments of Pediatrics and Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98101, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.celrep.2017.09.081","article-title":"Identification of genetically intact HIV-1 proviruses in specific CD4(+) T cells from effectively treated participants","volume":"21","author":"Hiener","year":"2017","journal-title":"Cell Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2803","DOI":"10.1097\/QAD.0b013e328340a239","article-title":"Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: Implications for eradication","volume":"24","author":"Chun","year":"2010","journal-title":"AIDS"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"13193","DOI":"10.1073\/pnas.94.24.13193","article-title":"Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy","volume":"94","author":"Chun","year":"1997","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1126\/science.278.5341.1295","article-title":"Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy","volume":"278","author":"Finzi","year":"1997","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Aamer, H.A., McClure, J., Ko, D., Maenza, J., Collier, A.C., Coombs, R.W., Mullins, J.I., and Frenkel, L.M. (2020). Cells producing residual viremia during antiretroviral treatment appear to contribute to rebound viremia following interruption of treatment. PLoS Pathog., 16.","DOI":"10.1371\/journal.ppat.1008791"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1038\/s41591-021-01590-5","article-title":"Research priorities for an HIV cure: International AIDS Society Global Scientific Strategy 2021","volume":"27","author":"Deeks","year":"2021","journal-title":"Nat. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1126\/science.1256304","article-title":"HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection","volume":"345","author":"Wagner","year":"2014","journal-title":"Science"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1016\/j.cell.2015.01.020","article-title":"HIV-1 integration landscape during latent and active infection","volume":"160","author":"Cohn","year":"2015","journal-title":"Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"988","DOI":"10.1172\/JCI124291","article-title":"Intact HIV-1 proviruses accumulate at distinct chromosomal positions during prolonged antiretroviral therapy","volume":"129","author":"Einkauf","year":"2019","journal-title":"J. Clin. Investig."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1004","DOI":"10.1016\/j.cell.2016.06.039","article-title":"Multiple origins of virus persistence during natural control of HIV infection","volume":"166","author":"Boritz","year":"2016","journal-title":"Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1126\/science.1254194","article-title":"HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells","volume":"345","author":"Maldarelli","year":"2014","journal-title":"Science"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3727","DOI":"10.1038\/s41467-021-24080-1","article-title":"In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia","volume":"12","author":"Cole","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"25891","DOI":"10.1073\/pnas.1910334116","article-title":"Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors","volume":"116","author":"Patro","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1016\/j.cell.2021.12.011","article-title":"Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses","volume":"185","author":"Einkauf","year":"2022","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e0012222","DOI":"10.1128\/jvi.00122-22","article-title":"Deep sequencing analysis of individual HIV-1 proviruses reveals frequent asymmetric long terminal repeats","volume":"96","author":"Joseph","year":"2022","journal-title":"J. Virol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1016\/j.cell.2013.09.020","article-title":"Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure","volume":"155","author":"Ho","year":"2013","journal-title":"Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1038\/nm.4156","article-title":"Defective proviruses rapidly accumulate during acute HIV-1 infection","volume":"22","author":"Bruner","year":"2016","journal-title":"Nat. Med."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1038\/s41586-019-0898-8","article-title":"A quantitative approach for measuring the reservoir of latent HIV-1 proviruses","volume":"566","author":"Bruner","year":"2019","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Patro, S.C., Niyongabo, A., Maldarelli, F., and Kearney, M.F. (2021). New approaches to multi-parametric HIV-1 genetics using multiple displacement amplification: Determining the what, how, and where of the HIV-1 reservoir. Viruses, 13.","DOI":"10.3390\/v13122475"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1016\/j.chom.2019.08.003","article-title":"HIV rebound is predominantly fueled by genetically identical viral expansions from diverse reservoirs","volume":"26","author":"Vrancken","year":"2019","journal-title":"Cell Host Microbe"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"257","DOI":"10.7326\/0003-4819-125-4-199608150-00001","article-title":"Clinical and epidemiologic features of primary HIV infection","volume":"125","author":"Schacker","year":"1996","journal-title":"Ann. Intern. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"638","DOI":"10.1089\/apc.2006.0169","article-title":"Maternal HIV-1 DNA load and mother-to-child transmission","volume":"21","author":"Arvold","year":"2007","journal-title":"AIDS Patient Care STDs"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e159569","DOI":"10.1172\/JCI159569","article-title":"Antigen specificities and proviral integration sites differ in HIV-infected cells by timing of antiretroviral treatment initiation","volume":"134","author":"Joy","year":"2024","journal-title":"J. Clin. Investig."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"veae019","DOI":"10.1093\/ve\/veae019","article-title":"Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations-Application to HIV-1 quasispecies","volume":"10","author":"Westfall","year":"2024","journal-title":"Virus Evol."},{"key":"ref_25","unstructured":"Oksanen, J., Simpson, G., Blanchet, F., Kindt, R., Legendre, P., Minchin, P., O\u2019Hara, R., Solymos, P., Stevens, M., and Szoecs, E. (2024). vegan: Community Ecology Package, CRAN."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"15499","DOI":"10.1073\/pnas.0808028105","article-title":"A procedure for highly specific, sensitive, and unbiased whole-genome amplification","volume":"105","author":"Pan","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2173","DOI":"10.1038\/s41467-025-57332-5","article-title":"Blood and tissue HIV-1 reservoirs display plasticity and lack of compartmentalization in virally suppressed people","volume":"16","author":"Pardons","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1038\/s41467-019-08431-7","article-title":"Longitudinal HIV sequencing reveals reservoir expression leading to decay which is obscured by clonal expansion","volume":"10","author":"Pinzone","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1016\/j.chom.2016.04.010","article-title":"What integration sites tell us about HIV persistence","volume":"19","author":"Hughes","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"110739","DOI":"10.1016\/j.celrep.2022.110739","article-title":"Extensive characterization of HIV-1 reservoirs reveals links to plasma viremia before and during analytical treatment interruption","volume":"39","author":"Cole","year":"2022","journal-title":"Cell Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/978-1-0716-1871-4_23","article-title":"Near-Full-Length Single-Genome HIV-1 DNA sequencing","volume":"2407","author":"Lee","year":"2022","journal-title":"Methods Mol. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e102","DOI":"10.1093\/nar\/gkad790","article-title":"HIV-PULSE: A long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization","volume":"51","author":"Lambrechts","year":"2023","journal-title":"Nucleic Acids Res."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/3\/311\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T05:20:25Z","timestamp":1772601625000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/3\/311"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,2]]},"references-count":32,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["v18030311"],"URL":"https:\/\/doi.org\/10.3390\/v18030311","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,2]]}}}