Nakao Ryo, Matsuno Keita, Qiu Yongjin, Maruyama Junki, Eguchi Nao, Nao Naganori, Kajihara Masahiro, Yoshii Kentaro, Sawa Hirofumi, Takada Ayato, Sugimoto Chihiro
Unit of Risk Analysis and Management, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan; Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan; Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Japan.
Ticks Tick Borne Dis. 2017 Jan;8(1):103-111. doi: 10.1016/j.ttbdis.2016.10.005. Epub 2016 Oct 15.
Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated I. scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of I. scapularis-associated virus-1, which was reported in a recent metagenomic study of I. scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks.
蜱虫携带多种微生物，其中一些是人类和动物的病原体。基因组测序技术的最新进展表明，蜱虫中存在大量以前未被识别的微生物。从蜱虫建立的连续细胞系可能在分离此类微生物中发挥关键作用；然而，已知蜱虫细胞本身也携带共生微生物。本研究旨在鉴定在最常用的蜱虫细胞系之一ISE6的培养上清液中检测到的假定RNA病毒序列，ISE6细胞系源自肩突硬蜱的胚胎。从培养上清液中纯化的病毒颗粒用于RNA提取，随后进行Illumina测序。对读取的序列进行从头组装，并通过tBLASTx搜索对所得的重叠群进行注释。结果表明，ISE6细胞中至少存在四个系统发育上不同谱系的五个假定病毒序列。在ISE6细胞中发现的主要病毒序列，命名为肩突硬蜱伊弗病毒，是伊弗病毒科的成员，该病毒科是一组感染节肢动物的病毒。我们还鉴定了布尼亚病毒科的L和M片段，它们无法归类到五个已知属中的任何一个，以及一种与果蝇A病毒相关的潜在衣壳蛋白。除了这些以前未被识别的病毒外，ISE6还被发现携带肩突硬蜱相关病毒-1的假定基因组序列，该序列在最近对肩突硬蜱本身的宏基因组研究中已有报道。通过RT-PCR在ISE6细胞和培养上清液中均检测到了所有五个假定病毒序列。电子显微镜分析显示，ISE6细胞的培养上清液中存在直径为50-70nm不等的球形病毒粒子。需要进一步研究来调查ISE6相关病毒在蜱虫中的潜在作用。
