Utilization of RNA in situ Hybridization to Understand the Cellular Localization of Powassan Virus RNA at the Tick-Virus-Host Interface
Utilization of RNA in situ Hybridization to Understand the Cellular Localization of Powassan Virus RNA at the Tick-Virus-Host Interface
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DOI：
10.3389/fcimb.2020.00172
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发表时间：
2020-04-28
影响因子：
5.7
通讯作者：
Thangamani, Saravanan
中科院分区：
文献类型：
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作者：
Hermance, Meghan E.;Thangamani, Saravanan
皮肤是附着的、进食的蜱与宿主之间的界面;因此，它是抵抗与蜱唾液一起递送到脊椎动物宿主的入侵病原微生物的第一道防线。蜱传病原体成功传播的核心是宿主免疫应答和早期蜱介导的免疫调节之间的复杂相互作用，所有这些最初都发生在皮肤界面。这项工作的重点是证明使用RNA原位杂交（RNA ISH）作为一种工具，用于了解病毒RNA的细胞定位在进食部位的Powassan病毒（POWV）感染的肩突硬蜱。POWV RNA的强阳性染色经常在真皮病灶中检测到，偶尔在经POWV感染的蜱喂食24小时后的皮下病灶中检测到。此外，双链显色RNA ISH染色证明了POWV RNA与小家鼠F4/80 RNA、CD 11 c RNA、波形蛋白RNA、Krt 14 RNA和CD 3 β RNA在POWV感染蜱的摄食部位的共定位。在未来的研究中，RNA ISH可用于验证在蜱-病毒-宿主皮肤界面进行的转录组学分析，并将为感染蜱喂养期间暂时表达的特定基因签名提供细胞分辨率。这样的系统生物学方法将有助于创造一个更精细的理解影响病毒在皮肤界面传播的细胞和分子相互作用。
Skin is the interface between an attached, feeding tick and a host; consequently, it is the first line of defense against invading pathogenic microorganisms that are delivered to a vertebrate host together with tick saliva. Central to the successful transmission of a tick-borne pathogen are complex interactions between the host immune response and early tick-mediated immunomodulation, all of which initially occur at the skin interface. The focus of this work was to demonstrate the use of RNA in situ hybridization (RNA ISH) as a tool for understanding the cellular localization of viral RNA at the feeding site of Powassan virus (POWV)-infected Ixodes scapularis ticks. Intense positive staining for POWV RNA was frequently detected in dermal foci and occasionally detected in hypodermal foci after 24 h of POWV-infected tick feeding. Additionally, duplex chromogenic RNA ISH staining demonstrated co-localization of POWV RNA with Mus musculus F4/80 RNA, CD11c RNA, vimentin RNA, Krt14 RNA, and CD3 epsilon RNA at the feeding site of POWV-infected ticks. In future studies, RNA ISH can be used to validate transcriptomic analyses conducted at the tick-virus-host cutaneous interface and will provide cellular resolution for specific gene signatures temporally expressed during infected tick feeding. Such a systems biology approach will help create a more refined understanding of the cellular and molecular interactions influencing virus transmission at the cutaneous interface.