Utilization of RNA in situ Hybridization to Understand the Cellular Localization of Powassan Virus RNA at the Tick-Virus-Host Interface.

Meghan E Hermance,Saravanan Thangamani

doi:10.3389/fcimb.2020.00172

Abstract

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 CD3ε 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.

Highlights

Mammalian skin serves as a mechanical and immunological barrier to protect the host from injury and infection (Pasparakis et al, 2014)
The attached I. scapularis tick body in these tissue sections served as an internal negative control because the M. musculus ubiquitin C (Ubc), Polr2a, FIGURE 3 | Distribution of Powassan virus (POWV) RNA, Mus musculus vimentin RNA, and M. musculus keratin 14 (Krt 14) RNA in the Ixodes scapularis nymph feeding site at 24 h post-tick attachment. (A) POWV-infected nymph feeding site showing POWV RNA and M. musculus vimentin RNA
The present study is the first to demonstrate the use of RNA in situ hybridization (RNA ISH) technology for precise cellular localization of viral RNA at the tick feeding site

Summary

Introduction

Mammalian skin serves as a mechanical and immunological barrier to protect the host from injury and infection (Pasparakis et al, 2014). This complex organ consists of an intricate network of epithelial cells, stromal cells, resident immune cells, migratory immune cells, blood and lymphatic vessels, peripheral nerves, and soluble mediators of the immune response (Wikel, 2013; Pasparakis et al, 2014; Nguyen and Soulika, 2019). To acquire its necessary blood meal, an ixodid tick must remain attached to the skin of a vertebrate host and complete its multi-day feeding process without being deterred by the complex and redundant host cutaneous defense mechanisms. Studies have demonstrated that a variety of tick salivary proteins, as well as some non-proteinaceous molecules, can modulate the cutaneous immune defenses of the host (Ribeiro et al, 2006; Oliveira et al, 2011; Kazimírová et al, 2017)

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Conclusion