NDLI: Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C

Content Provider	Springer Nature : BioMed Central
Author	Buchholtz, N. V. E. J. Nühn, M. M. de Jong, T. C. M. Stienstra, T. A. T. Reddy, K. Ndung’u, T. Ndhlovu, Z. M. Fisher, K. Palmer, S. Wensing, A. M. J. Symons, J. Nijhuis, M.
Abstract	Introduction HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C. Methods Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability. Results The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2–6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region. Conclusions We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.
Related Links	https://virologyj.biomedcentral.com/counter/pdf/10.1186/s12985-024-02300-6.pdf
Ending Page	11
Page Count	11
Starting Page	1
File Format	HTM / HTML
DOI	10.1186/s12985-024-02300-6
Journal	Virology Journal
Issue Number	1
Volume Number	21
Language	English
Publisher	BioMed Central
Publisher Date	2024-01-31
Access Restriction	Open
Subject Keyword	Virology HIV-1 IPDA Subtype B and C Intact-reservoir Quantification Global cure
Content Type	Text
Resource Type	Article
Subject	Virology Infectious Diseases
Journal Impact Factor	4/2023
5-Year Journal Impact Factor	3.8/2023

Sl.	Authority	Responsibilities	Communication Details
1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
5	Website/Portal (Helpdesk)	Queries regarding NDLI and its services	support@ndl.gov.in
6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
7	National Digital Library of India Club (NDLI Club)	Queries related to NDLI Club formation, support, user awareness program, seminar/symposium, collaboration, social media, promotion, and outreach	clubsupport@ndl.gov.in
8	Digital Preservation Centre (DPC)	Assistance with digitizing and archiving copyright-free printed books	dpc@ndl.gov.in
9	IDR Setup or Support	Queries related to establishment and support of Institutional Digital Repository (IDR) and IDR workshops	idr@ndl.gov.in

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