Dissecting Flavivirus Biology in Salivary Gland Cultures from Fed and UnfedIxodes scapularis(Black-Legged Tick)

Abstract

The Ixodes scapularis tick transmits a number of pathogens, including tick-borne flaviviruses (TBFVs). In the United States, confirmed human infections with the Powassan virus (POWV) TBFV have a fatality rate of ∼10% and are increasing in incidence. Tick salivary glands (SGs) serve as an organ barrier to TBFV transmission, and little is known regarding the location of TBFV infection in SGs from fed ticks. Previous studies showed I. scapularis vanin (VNN) involved with TBFV infection of I. scapularis ISE6 embryonic cells, suggesting a potential role for this gene. The overall goal of this study was to use SG cultures to compare data on TBFV biology in SGs from fully engorged, replete (fed) ticks and from unfed ticks. TBFV multiplication was higher in SGs from fed ticks than in those from unfed ticks. Virus-like particles were observed only in granular acini of SGs from unfed ticks. The location of TBFV infection of SGs from fed ticks was observed in cells lining lobular ducts and trachea but not observed in acini. Transcript knockdown of VNN decreased POWV multiplication in infected SG cultures from both fed and unfed ticks. This work was the first to identify localization of TBFV multiplication in SG cultures from a fed tick and a tick transcript important for POWV multiplication in the tick SG, an organ critical for TBFV transmission. This research exemplifies the use of SG cultures in deciphering TBFV biology in the tick and as a translational tool for screening and identifying potential tick genes as potential countermeasure targets. IMPORTANCE Tick-borne flaviviruses (TBFVs) are responsible for more than 15,000 human disease cases each year, and Powassan virus lineage 2 (POWV-L2) deer tick virus has been a reemerging threat in North America over the past 20 years. Rapid transmission of TBFVs in particular emphasizes the importance of preventing tick bites, the difficulty in developing countermeasures to prevent transmission, and the importance of understanding TBFV infection in tick salivary glands (SGs). Tick blood feeding is responsible for phenomenal physiological changes and is associated with changes in TBFV multiplication within the tick and in SGs. Using SG cultures from Ixodes scapularis female ticks, the primary aims of this study were to identify cellular localization of virus-like particles in acini of infected SGs from fed and unfed ticks, localization of TBFV infection in infected SGs from fed ticks, and a tick transcript (with associated metabolic function) involved in POWV-L2 infection in SG cultures.

Sharing the Ride: Ixodes scapularis Symbionts and Their Interactions

“…Four recent developments provide the groundworks for rapidly advancing our understanding of I. scapularis biology and microbial interactions. These are: (1) application of RNA interference (RNAi) gene silencing methods for functional analysis of tick proteins; (2) the release of the annotated I. scapularis genome (Gulia-Nuss et al, 2016); (3) a contained, artificial feeding system for nymphal and adult I. scapularis (Oliver et al, 2016); and (4) ex vivo organ culture of midgut and salivary glands (Grabowski et al, 2017(Grabowski et al, , 2019.…”

“…Symbiont-tick interactions can also be studied in specific tissues using the recently described ex vivo organ culture system described by Grabowski et al (2017Grabowski et al ( , 2019. Dissected midgut, salivary glands, and synganglion were maintained in a metabolically active state for 9-10 days.…”

Characterization of flavivirus infection in salivary gland cultures from male Ixodes scapularis ticks

“…and then mounted on glass slides [28,30] using Prolong Gold Antifade mounting agent (ThermoFisher Scientific). The preparations were held for 24 h at RT prior to being imaged using an LSM710 confocal microscope (Zeiss).…”

“…Mock-infected or LGTV-infected male SG cultures were collected at 3 hpi, 168 hpi, and 216 hpi. 4 SG pairs were collected per timepoint and infection type and were subsequently processed for study by transmission electron microscopy as previously described [28].…”

“…Type I acini are agranular and believed to be responsible for osmoregulation [22][23][24]. Types II and III acini are comprised of cells with a variety of granules which contain the pharmacopeia of bioactive molecules [25,26] that constitute tick saliva [22,[27][28][29]. We recently demonstrated that ex vivo SG cultures derived from female I. scapularis ticks can support TBFV replication and that virus localizes to cells of granular acini [30].…”