Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion

Kou Hiraga; Kenta Tezuka; Koh Nagata; Ki-Ryang Koh; Hitomi Nakamura; Yasuko Sagara; Rieko Sobata; Masahiro Satake; Michikazu Tanio; Hiroo Hasegawa; Masumichi Saito; Kiyonori Miura; Takuo Mizukami; Isao Hamaguchi; Madoka Kuramitsu

doi:10.1016/j.jviromet.2024.115071

Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion

J Virol Methods. 2024 Nov 21:332:115071. doi: 10.1016/j.jviromet.2024.115071. Online ahead of print.

Authors

Affiliations

¹ Research Center for Biological Products in the Next Generation, National Institute of Infectious Diseases, Tokyo, Japan; Research Center for Biosafety, Laboratory Animal and Pathogen Bank, National Institute of Infectious Diseases, Tokyo, Japan.
² Research Center for Biological Products in the Next Generation, National Institute of Infectious Diseases, Tokyo, Japan.
³ Department of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
⁴ Department of Hematology, Osaka General Hospital of West Japan Railway Company, Osaka, Japan.
⁵ Japanese Red Cross Kyushu Block Blood Center, Fukuoka, Japan.
⁶ Central Blood Institute, Blood Service Headquarters, Japanese Red Cross Society, Tokyo, Japan.
⁷ Department of Laboratory Medicine, Nagasaki University Hospital, Nagasaki, Japan.
⁸ Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan; Center for Emergency Preparedness and Response, National Institute of Infectious Diseases, Tokyo, Japan.
⁹ Research Center for Biological Products in the Next Generation, National Institute of Infectious Diseases, Tokyo, Japan; Department of Clinical Laboratory, Subaru Health Insurance Society Ota Memorial Hospital, Gunma, Japan.
¹⁰ Research Center for Biological Products in the Next Generation, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address: mkura@niid.go.jp.

PMID: 39577671
DOI: 10.1016/j.jviromet.2024.115071

Abstract

The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions. We analyzed HTLV-1-positive asymptomatic carriers (ACs) and AC samples at high risk for developing ATL due to high proviral load (ATL high-risk (HR) ACs) using dPCR. Deletions were identified in 11.8 % (4/34, all Type 1) of ACs and 33.3 % (7/21, Type 1:1, Type 2:6) of ATL HR ACs. dPCR analysis revealed that in three ATL samples, all exhibited Type 1 defective characteristics, and two showed extremely low ratios in the pol region. Clonality analysis of these two samples revealed high monoclonality, indicating monoclonal expansion of ATL cells with defective proviruses. These findings demonstrate that our method effectively detects defective proviruses in both ACs and ATL, providing a valuable tool for understanding the genomic characteristics of proviruses in these conditions.

Keywords: Defective provirus; Digital PCR; HTLV-1.