Guiraud Vincent, Denis Jérôme Alexandre, Ben Attia Sofia, Ablin Erwan, Legrand Ronan, Morel Véronique, Lacan Claire, Choquet Sylvain, Debs Rabab, Cheval Margaux, Marques Cindy, Le Joncour Alexandre, Corvol Jean-Christophe, Benveniste Olivier, Pourcher Valérie, Marcelin Anne-Geneviève, Gautheret-Dejean Agnès, Bravetti Clotilde, Calvez Vincent
Virology Department, INSERM, Pierre Louis Institute of Epidemiology and Public Health (IPLESP), Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Sorbonne University, Paris, France.
Department of Endocrine and Oncological Biochemistry, Pitié-Salpêtrière Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France.
Microbiol Spectr. 2025 Mar 20;13(5):e0320824. doi: 10.1128/spectrum.03208-24.
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.
人类嗜T淋巴细胞病毒1型（HTLV-1）慢性感染通过受感染CD4+T细胞的有丝分裂增殖得以维持，病毒基因组作为前病毒整合到宿主基因组中。HTLV-1整合位点（ISs）在HTLV-1相关疾病中起作用，不同的IS模式与恶性增殖或炎症性疾病相关。然而，IS的确定仍然具有挑战性，因为大多数检测依赖于复杂的生物学和生物计算协议。我们提出了一种仅依赖PCR和桑格测序的IS检测方法，该方法可用于确定4例患有各种HTLV-1相关疾病患者的HTLV-1 IS。我们对源自锅柄PCR的IS检测方法进行了改进，通过一些修改提高了产量。使用TCRγ克隆性证实了在IS检索方面不存在偏差。对4例HTLV-1阳性患者进行了IS分析：2例患有多发性肌炎，1例患有成人T细胞白血病/淋巴瘤（ATLL），1例患有HTLV-1相关脊髓病（HAM）。总体产量约为20%，IS下游的平均序列长度为336±230 bp（范围为44-1,024 bp）。在克隆确定方面没有重大偏差，因为IS结果与使用TCRγ检测评估的克隆性相匹配。IS检测根据HTLV-1疾病显示出不同的克隆模式：ATLL以一个大克隆为主，多发性肌炎为寡克隆或多克隆，HAM为多克隆。总之，我们提出了一种易于实施的HTLV-1整合位点检测方法，该方法允许对克隆群体进行相对无偏差的IS分析。该检测方法可能有助于进一步探索IS在HTLV-1相关疾病中的作用。重要性人类嗜T淋巴细胞病毒1型（HTLV-1）慢性感染是由于受感染CD4+T细胞的有丝分裂增殖，前病毒DNA整合到宿主DNA中。HTLV-1整合似乎在HTLV-1相关疾病中起不可忽视的作用。然而，大多数HTLV-1整合研究来自少数几个中心，主要是因为整合位点（IS）协议依赖于高成本的实验材料和先进的生物信息学分析。我们开发了一种仅依赖Taq聚合酶和桑格测序的IS检测方法，无需昂贵的生物材料和复杂的生物信息学技能。该检测方法在4例患有不同疾病（ATLL、HAM和多发性肌炎）和不同材料（血液和脑脊液）的HTLV-1阳性患者中成功进行。所有4例患者来自非洲和加勒比海岛屿的不同地区。该检测方法产量相对较高，约为20%。与TCRγ评估相比，它在HTLV-1克隆性方面提供了类似的结果，这表明IS回收可能是无偏差的。
