Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies
ABSTRACT Human T lymphotropic virus type 1 (HTLV-1) chronic infection is maintained through mitotic proliferation of the infected CD4+ T cells, where the viral genome is integrated as a provirus in its host genome. HTLV-1 integration sites (ISs) have a part in HTLV-1-associated pathologies, with dis...
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American Society for Microbiology
2025-05-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03208-24 |
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| author | Vincent Guiraud Jérôme Alexandre Denis Sofia Ben Attia Erwan Ablin Ronan Legrand Véronique Morel Claire Lacan Sylvain Choquet Rabab Debs Margaux Cheval Cindy Marques Alexandre Le Joncour Jean-Christophe Corvol Olivier Benveniste Valérie Pourcher Anne-Geneviève Marcelin Agnès Gautheret-Dejean Clotilde Bravetti Vincent Calvez |
| author_facet | Vincent Guiraud Jérôme Alexandre Denis Sofia Ben Attia Erwan Ablin Ronan Legrand Véronique Morel Claire Lacan Sylvain Choquet Rabab Debs Margaux Cheval Cindy Marques Alexandre Le Joncour Jean-Christophe Corvol Olivier Benveniste Valérie Pourcher Anne-Geneviève Marcelin Agnès Gautheret-Dejean Clotilde Bravetti Vincent Calvez |
| author_sort | Vincent Guiraud |
| collection | DOAJ |
| description | ABSTRACT Human T lymphotropic virus type 1 (HTLV-1) chronic infection is maintained through mitotic proliferation of the infected CD4+ T cells, where the viral genome is integrated as a provirus in its host genome. HTLV-1 integration sites (ISs) have a part in HTLV-1-associated pathologies, with distinct IS patterns associated with malignant proliferation or inflammatory diseases. However, IS determination remains challenging because most assays rely on complex biological and biocomputing protocols. We present an IS assay that solely relies on PCR and Sanger sequencing, which allowed HTLV-1 IS determination in four patients with various HTLV-1-associated pathologies. We adapted an IS assay derived from a panhandle PCR, with several modifications to increase yield. Absence of bias regarding IS retrieval was confirmed using TCRγ clonality. IS analysis was performed in four HTLV-1-positive patients: two with polymyositis, one with adult T-cell leukemia/lymphoma (ATLL), and one with HTLV-1-associated myelopathy (HAM). Overall yield was around 20%, with a mean sequence length downstream the IS of 336 ± 230 bp (range, 44–1,024 bp). There was no major bias in clonal determination, as IS results matched clonality assessed using a TCRγ assay. The IS assay revealed distinct clonal patterns depending on HTLV-1 pathology: dominated by a large clone for ATLL, oligo- or polyclonal for polymyositis and polyclonal for HAM. As a conclusion, we present an easy-to-implement integration site assay for HTLV-1 that allows a relatively unbiased IS analysis regarding clonal populations. This assay could be useful to further explore IS involvement in HTLV-1 associated pathologies.IMPORTANCEHuman T lymphotropic virus type 1 (HTLV-1) chronic infection is due to the mitotic proliferation of infected CD4+ T cells, where the proviral DNA is integrated in its host DNA. HTLV-1 integration seems to play a non-negligible part in HTLV-1-associated pathologies. However, most HTLV-1 integration studies originate from a few centers, mostly because integration site (IS) protocols rely on high-cost experimental materials and advanced bioinformatic analysis. We have developed an IS assay that solely relies on Taq polymerase and Sanger sequencing, with no need for costly biological material nor complex bioinformatic skills. This assay was successfully performed on four HTLV-1-positive patients with distinct pathologies (ATLL, HAM, and polymyositis) and distinct material (blood and cerebrospinal fluid). All four patients originated from distinct areas in Africa and the Caribbean Sea Island. This assay has a relatively high yield, around 20%. It provided similar results regarding HTLV-1 clonality compared with a TCRγ assessment, which indicated that IS recovery was likely unbiased. |
| format | Article |
| id | doaj-art-5de93065e9194437b1e06e2c48d46f22 |
| institution | OA Journals |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-5de93065e9194437b1e06e2c48d46f222025-08-20T02:11:31ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-05-0113510.1128/spectrum.03208-24Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologiesVincent Guiraud0Jérôme Alexandre Denis1Sofia Ben Attia2Erwan Ablin3Ronan Legrand4Véronique Morel5Claire Lacan6Sylvain Choquet7Rabab Debs8Margaux Cheval9Cindy Marques10Alexandre Le Joncour11Jean-Christophe Corvol12Olivier Benveniste13Valérie Pourcher14Anne-Geneviève Marcelin15Agnès Gautheret-Dejean16Clotilde Bravetti17Vincent Calvez18Virology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Endocrine and Oncological Biochemistry, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Paris, FranceVirology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceVirology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Endocrine and Oncological Biochemistry, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Paris, FranceDepartment of Clinical Hematology, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Clinical Hematology, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Clinical Hematology, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Neurology, Public Hospital Network of Paris, INSERM, National Center for Scientific Research, Paris Brain Institute, Pitié-Salpêtrière Hospital, Center for Clinical Investigation Neurosciences, Sorbonne University, Paris, FranceDepartment of Neurology, Public Hospital Network of Paris, INSERM, National Center for Scientific Research, Paris Brain Institute, Pitié-Salpêtrière Hospital, Center for Clinical Investigation Neurosciences, Sorbonne University, Paris, FranceDepartment of Internal Medicine and Clinical Immunology, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Internal Medicine and Clinical Immunology, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceDepartment of Neurology, Public Hospital Network of Paris, INSERM, National Center for Scientific Research, Paris Brain Institute, Pitié-Salpêtrière Hospital, Center for Clinical Investigation Neurosciences, Sorbonne University, Paris, FranceDepartment of Internal Medicine and Clinical Immunology, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceInfectious Diseases Department, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Paris, FranceVirology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceVirology Department, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Paris, FranceDepartment of Biological Hematology, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceVirology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, FranceABSTRACT Human T lymphotropic virus type 1 (HTLV-1) chronic infection is maintained through mitotic proliferation of the infected CD4+ T cells, where the viral genome is integrated as a provirus in its host genome. HTLV-1 integration sites (ISs) have a part in HTLV-1-associated pathologies, with distinct IS patterns associated with malignant proliferation or inflammatory diseases. However, IS determination remains challenging because most assays rely on complex biological and biocomputing protocols. We present an IS assay that solely relies on PCR and Sanger sequencing, which allowed HTLV-1 IS determination in four patients with various HTLV-1-associated pathologies. We adapted an IS assay derived from a panhandle PCR, with several modifications to increase yield. Absence of bias regarding IS retrieval was confirmed using TCRγ clonality. IS analysis was performed in four HTLV-1-positive patients: two with polymyositis, one with adult T-cell leukemia/lymphoma (ATLL), and one with HTLV-1-associated myelopathy (HAM). Overall yield was around 20%, with a mean sequence length downstream the IS of 336 ± 230 bp (range, 44–1,024 bp). There was no major bias in clonal determination, as IS results matched clonality assessed using a TCRγ assay. The IS assay revealed distinct clonal patterns depending on HTLV-1 pathology: dominated by a large clone for ATLL, oligo- or polyclonal for polymyositis and polyclonal for HAM. As a conclusion, we present an easy-to-implement integration site assay for HTLV-1 that allows a relatively unbiased IS analysis regarding clonal populations. This assay could be useful to further explore IS involvement in HTLV-1 associated pathologies.IMPORTANCEHuman T lymphotropic virus type 1 (HTLV-1) chronic infection is due to the mitotic proliferation of infected CD4+ T cells, where the proviral DNA is integrated in its host DNA. HTLV-1 integration seems to play a non-negligible part in HTLV-1-associated pathologies. However, most HTLV-1 integration studies originate from a few centers, mostly because integration site (IS) protocols rely on high-cost experimental materials and advanced bioinformatic analysis. We have developed an IS assay that solely relies on Taq polymerase and Sanger sequencing, with no need for costly biological material nor complex bioinformatic skills. This assay was successfully performed on four HTLV-1-positive patients with distinct pathologies (ATLL, HAM, and polymyositis) and distinct material (blood and cerebrospinal fluid). All four patients originated from distinct areas in Africa and the Caribbean Sea Island. This assay has a relatively high yield, around 20%. It provided similar results regarding HTLV-1 clonality compared with a TCRγ assessment, which indicated that IS recovery was likely unbiased.https://journals.asm.org/doi/10.1128/spectrum.03208-24HTLV-1Integration SiteISLApanhandle PCRclonal proliferation |
| spellingShingle | Vincent Guiraud Jérôme Alexandre Denis Sofia Ben Attia Erwan Ablin Ronan Legrand Véronique Morel Claire Lacan Sylvain Choquet Rabab Debs Margaux Cheval Cindy Marques Alexandre Le Joncour Jean-Christophe Corvol Olivier Benveniste Valérie Pourcher Anne-Geneviève Marcelin Agnès Gautheret-Dejean Clotilde Bravetti Vincent Calvez Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies Microbiology Spectrum HTLV-1 Integration Site ISLA panhandle PCR clonal proliferation |
| title | Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies |
| title_full | Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies |
| title_fullStr | Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies |
| title_full_unstemmed | Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies |
| title_short | Development of a new high-yield integration site assay reveals disease-specific patterns across HTLV-1-associated pathologies |
| title_sort | development of a new high yield integration site assay reveals disease specific patterns across htlv 1 associated pathologies |
| topic | HTLV-1 Integration Site ISLA panhandle PCR clonal proliferation |
| url | https://journals.asm.org/doi/10.1128/spectrum.03208-24 |
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