The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research

Grabowski, Jeffrey M.; Offerdahl, Danielle K.; Bloom, Marshall E.

doi:10.1021/acsinfecdis.7b00274

Cited by 16 publications

(29 citation statements)

References 99 publications

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“…Viability and TBFV multiplication in SG cultures from fed and unfed ticks. The studies undertaken required a careful determination of the viability of short-term SG cultures from both fully engorged, replete (fed) I. scapularis ticks and unfed I. scapularis ticks (41,45). Thus, we compared the levels of metabolic activity of cultures from fed and unfed ticks.…”

Section: Resultsmentioning

confidence: 99%

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

Grabowski¹,

Nilsson²,

Fischer

et al. 2019

mBio

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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.

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Section: Resultsmentioning

confidence: 99%

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

Grabowski¹,

Nilsson²,

Fischer

et al. 2019

mBio

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“…This is possibly due to the ability of tick SGs to harbor virus that can be quickly released into the saliva. Furthermore, as we showed previously, ex vivo SG cultures provide a highly controlled model for studying infection and related processes [ 28 , 30 , 36 ].…”

Section: Discussionmentioning

confidence: 98%

“…previously, ex vivo SG cultures provide a highly controlled model for studying infection and related processes [28,30,36].…”

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 99%

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

Kendall¹,

Grabowski²,

Rosenke³

et al. 2020

PLoS Negl Trop Dis

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Infected Ixodes scapularis (black-legged tick) transmit a host of serious pathogens via their bites, including Borrelia burgdorferi, Babesia microti, and tick-borne flaviviruses (TBFVs), such as Powassan virus (POWV). Although the role of female I. scapularis ticks in disease transmission is well characterized, the role of male ticks is poorly understood. Because the pathogens are delivered in tick saliva, we studied the capacity of male salivary glands (SGs) to support virus replication. Ex vivo cultures of SGs from unfed male I. scapularis were viable for more than a week and maintained the characteristic tissue architecture of lobular ducts and acini. When SG cultures were infected with the TBFVs Langat virus (LGTV) or POWV lineage II (deer tick virus), the production of infectious virus was demonstrated. Using a green fluorescent protein-tagged LGTV and confocal microscopy, we demonstrated LGTV infection within SG acinus types II and III. The presence of LGTV in the acini and lobular ducts of the cultures was also shown via immunohistochemistry. Furthermore, the identification by in situ hybridization of both positive and negative strand LGTV RNA confirmed that the virus was indeed replicating. Finally, transmission electron microscopy of infected SGs revealed virus particles packaged in vesicles or vacuoles adjacent to acinar lumina. These studies support the concept that SGs of male I. scapularis ticks support replication of TBFVs and may play a role in virus transmission, and further refine a useful model system for developing countermeasures against this important group of pathogens.

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“…Put differently, each virus appears to possess its own mechanism, distinctive not only among related viruses, but among various cell types as well. Global techniques spanning tissues of both mammal and arthropod vectors [63] will be invaluable in unraveling the biology of these pathogens, and eventually devising ways to combat them.…”

Section: Discussionmentioning

confidence: 99%

PERK-Mediated Unfolded Protein Response Signaling Restricts Replication of the Tick-Borne Flavivirus Langat Virus

Lewy

Offerdahl

Grabowski

et al. 2020

Viruses

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The unfolded protein response (UPR) maintains protein-folding homeostasis in the endoplasmic reticulum (ER) and has been implicated as both beneficial and detrimental to flavivirus infection. Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a sensor of the UPR, is commonly associated with antiviral effects during mosquito-borne flavivirus (MBFV) infection, but its relation to tick-borne flavivirus (TBFV) infection remains largely unexplored. In this study, we identified changes in UPR and autophagic activity during Langat virus (LGTV) infection.LGTV robustly activated UPR and altered autophagic flux. Knockdown of endogenous PERK in human cells resulted in increased LGTV replication, but not that of closely related Powassan virus (POWV). Finally, on examining changes in protein levels of components associated with UPR and autophagy in the absence of PERK, we could show that LGTV-infected cells induced UPR but did not lead to expression of C/EBP homologous protein (CHOP), an important downstream transcription factor of multiple stress pathways. From these data, we hypothesize that LGTV can antagonize other kinases that target eukaryotic initiation factor 2α (eIF2α), but not PERK, implicating PERK as a potential mediator of intrinsic immunity. This effect was not apparent for POWV, a more pathogenic TBFV, suggesting it may be better equipped to mitigate the antiviral effects of PERK.

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The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research

Cited by 16 publications

References 99 publications

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

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

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

PERK-Mediated Unfolded Protein Response Signaling Restricts Replication of the Tick-Borne Flavivirus Langat Virus

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