GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

Hameeda Sultana; Waqas Ahmed; Girish Neelakanta

doi:10.1016/j.isci.2024.110391

. 2024 Jun 27;27(8):110391. doi: 10.1016/j.isci.2024.110391

GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

Hameeda Sultana ^1,^2,^∗, Waqas Ahmed ¹, Girish Neelakanta ¹

PMCID: PMC11301069 PMID: 39108711

Summary

Exosomes/extracellular vesicles (EVs) are essential for the successful transmission of flaviviruses from vector to vertebrate host. Arthropod-EVs are envisioned as important target for blocking the transmission of vector-borne viral diseases. In this study, we show that the selective inhibition of EVs secretion by sphingomyelinase inhibitor, GW4869 significantly reduces vector efficiency and competence in acquiring and transmitting tick-borne flaviviruses. We show that GW4869 reduces EVs release from Langat Virus (LGTV)-infected Ixodes scapularis adult tick salivary glands (SGs). GW4869 treatment showed reduced dissemination of LGTV in SGs and other tissues within ticks. Decreased release of SG-EVs directly correlated with reduced tick blood-feeding efficiency, engorgement weights, and reduction in LGTV acquisition/transmission. Our data indicates that LGTV infection significantly improves molting/fitness, and survival efficiency of ticks and GW4869 alone affects the repletion rates of blood-feeding naïve-ticks. Overall, we provide evidence for GW4869 potential use as therapeutic agent in the tick control and prevention of tick-borne flaviviral transmission.

Subject areas: Entomology, Virology, Cell biology

Graphical abstract

Highlights

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GW4869 treatment reduces the efficiency of LGTV replication and dissemination in ticks
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GW4869 blocks exosomes secretion from tick adult salivary glands
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GW4869 treatment reduces pathogen acquisition and transmission
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GW4869 treatment reduced tick molting efficiency and repletion and survival rates

Entomology; Virology; Cell biology

Introduction

Ticks are generally referred as specialized obligate hematophagous arachnids of medical and veterinary importance.¹^,²^,³ Ixodes scapularis ticks typically go through four life stages which includes eggs, larvae, nymphs, and adults. The larvae, nymphs and adult ticks require blood meal to facilitate the developmental process and egg laying.¹^,²^,³^,⁴ In the three life stages of larvae, nymphs and adult, ticks may acquire a pathogen through blood meal, but the nymphs and adults are the most likely to transmit pathogens efficiently to the vertebrate host.¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹ The relatively long-life span of ticks makes them very important vectors of parasites, bacteria, and viruses.¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹² Viruses transmitted by ticks are referred in this study as tick-borne viruses (TBVs). Some medically important TBVs includes tick-borne encephalitis virus (TBEV), Powassan virus (POWV), Heartland virus, Kyasanur Forest Disease virus (KFDV), Bourbon virus, Colorado tick fever virus (CTFV), severe fever with thrombocytopenia syndrome virus (SFTS) and Crimean Congo hemorrhagic fever virus (CCHFV).⁵^,⁶^,⁷^,⁸^,⁹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷ Tick-borne viral infections range from asymptomatic to severe symptomatic cases that may be fatal in many incidents.³^,⁵^,⁶^,¹²^,¹⁴^,¹⁸^,¹⁹^,²⁰^,²¹ Langat virus (LGTV), a naturally attenuated tick-borne virus (similar to TBEV) is used as a model pathogen to study the effects of highly virulent tick-borne viruses.¹¹^,¹⁴^,²²^,²³^,²⁴^,²⁵

Prevention of TBV-infections has relied mainly on reducing vector-human contact and vaccine development.²^,³^,⁴^,⁷^,⁹^,¹²^,¹³^,¹⁵^,¹⁶^,²⁶^,²⁷ However, there is growing interest in the possibility of reducing vector competence in acquiring, sustaining and/or transmitting TBVs.²^,³^,⁴^,⁷^,⁹^,¹²^,¹³^,¹⁵^,¹⁶^,²⁶^,²⁷ Studies of this nature have looked at various aspects of tick biology, such as tick immunity, TBV-tick interactions, and tick-human host interactions.²^,³^,⁴^,⁷^,⁹^,¹²^,¹³^,¹⁵^,¹⁶^,²⁶^,²⁷ Studies on tick-human interface has focused on the role of saliva in the transmission of TBVs.⁴^,⁷^,⁸^,⁹^,¹¹^,¹²^,¹³^,¹⁶^,¹⁷^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³^,³⁴ These studies are based on the principle that vector competence is dependent on the density of TBVs present in tick saliva and the efficiency of tick feeding.⁴^,⁷^,⁸^,⁹^,¹¹^,¹²^,¹³^,¹⁶^,¹⁷^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³^,³⁴ Saliva assisted transmission (SAT) is the phenomenon where the tick saliva facilitates the transmission of pathogens through interactions between salivary molecules and vertebrate host.⁴^,⁷^,⁸^,⁹^,¹¹^,¹²^,¹³^,¹⁶^,¹⁷^,²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³^,³⁴^,³⁵^,³⁶^,³⁷^,³⁸ Some studies have focused on the immunization of vertebrates with tick saliva to induce strong immune reactions on tick infestation, reducing the length and efficiency of tick feeding, while others have focused on specific components of tick saliva.⁷^,⁸^,⁹^,¹²^,¹⁶^,¹⁷^,²⁶

Exosomes/Extracellular Vesicles (EVs), the membrane bound lipid rich vesicles secreted by all cells are involved in cell-to-cell communications.¹²^,¹³^,¹⁶^,²⁵^,²⁶^,³⁰^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³ Ticks are reported to secrete EVs such as exosomes in their saliva that may modulate host immune responses and facilitate pathogen transmission.¹²^,¹³^,¹⁶^,²⁵^,²⁶^,³⁰^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³ The rate of EVs secretion as well as its cargo is dependent on physiological changes in a cell.¹²^,¹³^,¹⁶^,²⁶^,⁴⁰^,⁴¹^,⁴²^,⁴³ Infected ticks do secrete EVs packed with infectious viral RNA and proteins/poly-proteins in their saliva.¹²^,¹³^,¹⁶^,²⁶^,⁴⁰^,⁴¹^,⁴²^,⁴³ Furthermore, exosomes with infectious viral RNA genomes are sufficient for the transmission of pathogens.²⁵^,⁴⁰^,⁴¹^,⁴²^,⁴³ Our hypothesis focuses on ways of exploiting tick EVs in prevention and control of tick-borne pathogens. Although, it is important to focus on specific exosomal components, it is essential to also consider the effects of inhibiting EVs secretion to achieve vector competence. GW4869 is a commercially available sphingomyelinase inhibitor that is known to block the release of exosomes and affect exosome biogenesis.²⁵^,⁴⁰^,⁴¹^,⁴²^,⁴³

Our previous studies have shown that treating murine cortical neurons with GW4869 reduced the Zika virus (ZIKV) loads and its transmission efficiency through neuronal exosomes.⁴³ We demonstrated that GW4869 reduced ZIKV loads by inhibiting the expression and activity of neutral sphingomyelinase-2/SMPD3, an essential molecule for the release of exosomes.⁴³ In arthropods, we reported that LGTV-mediated the suppression of I. scapularis sphingomyelinase D (IsSMase), resulting in an accumulation of sphingomyelin lipid that perhaps facilitated membrane associated viral replication and exosome biogenesis.²⁵ Similarly, the LGTV modulation of IsSMase was reversed upon GW4869 treatment.²⁵ These findings suggest an important role of GW4869 in blocking flavivirus replication and transmission via tick salivary exosomes, and its potential role as a therapeutic agent against flaviviral infections.²⁵^,³⁰ We have shown that GW4869 treatment (in vitro) significantly reduced viral loads/replication in tick/mosquito/mouse cortical neuronal cells.²⁵^,³⁰^,⁴⁰^,⁴¹^,⁴²^,⁴³ In addition, upon GW4869 treatment the efficiency of the transmigration of viral RNA/proteins from one cell type to another via infectious exosomes was affected.²⁵^,³⁰^,⁴⁰^,⁴¹^,⁴²^,⁴³ In this current study, we have determined the effects of GW4869 treatment (in vivo) on the vector competence of I. scapularis ticks upon LGTV infection. In addition, the effects of GW4869 treatment on the efficiency of EVs release in adult tick salivary glands (SGs), nymphal tick feeding, virus-acquisition and transmission (from infected murine host to ticks or from infected ticks to mouse, respectively) were addressed. Furthermore, this study delineates the role of EVs in virus transmission and vector competence measured by determining the tick molting and survival rates. Overall, our study suggests that GW4869 is a potential candidate in controlling virus-infected ticks and in blocking tick-borne virus dissemination within ticks and transmission from the infected ticks to the naive vertebrate hosts.

Results

Simultaneous or pre-treatment of GW4869 inhibitor reduces the efficiency of Langat Virus dissemination within nymphal ticks and to the salivary glands

We determined the potential effects of GW4869 treatment in unfed nymphs. To standardize the working concentration of GW4869 dose in unfed ticks (in vivo), uninfected I. scapularis nymphs were first pre-tested with varying doses (10–250 μM) of GW4869 inhibitor. Most of the unfed uninfected ticks from 200 to 250 μM treatment were unhealthy or died during or after incubations. Few unfed nymphs treated (for 4 or 24 h) with two different doses of GW4869 inhibitor (50 or 150 μM, low or high dose) followed by LGTV infection (by synchronous method, for additional 24 h) showed variability in the viral loads (Figure S1A). Furthermore, at 24 h post GW4869-treatment (with doses of 10, 20 and 50 μM) showed no effects on IsSMase transcript levels in unfed uninfected and untreated ticks (Figure S1B). To address viral dissemination within the tick body, we analyzed unfed nymphs that were either simultaneously treated with GW4869 inhibitor (150 μM) or with 1.5% DMSO (as mock control) and synchronously infected with 2 x 10⁶ PFU/mL LGTV suspension for 0, 3 or 17-day post infection (p.i.). In another condition, unfed nymphs were pre-treated first with GW4869 inhibitor (150 μM, for 4 h) or 1.5% DMSO (for 4 h), followed by synchronous infection with 2 x 10⁶ PFU/mL of LGTV for 3 or 17-day p.i. of ticks. We found no significant (p > 0.05) differences in LGTV loads (both in Midguts [MGs] or in Salivary glands [SGs]) at day 0 post simultaneous incubations with GW4869 treatment and LGTV infection (Figure 1A). However, simultaneous incubations with GW4869 and LGTV showed significantly reduced viral loads in MGs and SGs of nymphal ticks at 3 days p.i. (Figure 1B). Also, at 17 days p.i., significant (p < 0.05) reduction in LGTV loads were noted in SGs of GW4869-treated ticks when compared to their respective mock control (Figure 1C). No significant (p > 0.05) differences were observed in MGs and carcass (C) collected from ticks that were simultaneously treated and infected with LGTV (17 days p.i.), when compared to their respective mock controls (Figure 1C). Pre-treatment with GW4869 inhibitor (for 4 h), followed by LGTV infection showed significantly reduced LGTV loads in C and SGs at 3 days p.i., when compared to their respective mock control groups (Figure 1D). However, at 17 days p.i., LGTV loads were not significant in any tested tissues (MGs, C, or SGs) upon pre-treatment with GW4869 followed by LGTV-infection (Figure 1E). These data show that GW4869 treatment affects viral loads and dissemination during early time points of LGTV infection in unfed ticks.

Efficiency of LGTV dissemination within *I. scapularis* nymphal ticks is affected upon GW4869 treatment

(A–C) LGTV loads in midgut (MG), salivary glands (SGs) or carcass (C) of nymphs that were simultaneously exposed to LGTV (1 MOI) and either mock (1.5% DMSO) or GW4869 (150 μM) and incubated for 0 (A), 3 (B) or 17 (C) days post infection (DPI), respectively.

(D and E) LGTV loads in MG, C and SGs of nymphs that were pretreated with either mock/GW4869 followed by LGTV infection and incubated for 3 (C) or 17 (D) days p.i., respectively. LGTV loads were normalized to total RNA. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents a pool of either two (A–C) or three (D and E) ticks in the simultaneous or pretreatment groups, respectively. p value less than 0.05 is considered statistically significant and ns indicates not significant.

Treatment with GW4869 affects Langat virus dissemination within tick bodies that acquired blood meal from infected murine host

To understand the effects of GW4869 treatment in reducing LGTV dissemination within ticks that naturally acquired the viral loads, we performed an acquisition experiment to allow uninfected unfed nymphal ticks (treated with GW4869, 150 μM for 24 h) to naturally acquire the pathogen by feeding on LGTV-infected (5 x 10e4 pfu/mouse) murine host. We found that upon repletion, GW4869-treated post fed ticks had smaller body sizes (as revealed by the images in Figure 2A) and showed significantly (p < 0.05) reduced body weights in comparison to the mock control group of post fed ticks (Figures 2A and 2B). Also, we dissected MGs and SGs from these post fed ticks and found significantly (p < 0.05) reduced LGTV loads in GW4869-treated post fed ticks (both MGs and SGs) in comparison to their respective mock control groups (Figure 2C). In addition, we collected mock and GW4869-treated (150 μM for 24 h) unfed nymphal ticks during blood feeding (DF ticks) on LGTV-infected murine host. Intriguingly, QRT-PCR analysis determined significantly (p < 0.05) reduced LGTV loads in GW4869-treated DF ticks MGs and SGs in comparison to their respective mock control groups of DF ticks MGs and SGs (Figure 2D). These data suggest that GW4869 treatment severely affects pathogen dissemination within ticks that also naturally acquire the viral loads. The reduced dissemination of LGTV to SGs of GW4869-treated ticks suggested lower viral replication, diminished establishment, and colonization of the pathogen in SGs that may directly affect the LGTV transmission to the vertebrate host.

GW4869 treatment affects viral dissemination within ticks that fed on LGTV-infected mice

Photographs (A) showing reduced body sizes of ticks treated with GW4869 (150 μM) in comparison to mock controls (1.5% DMSO-treated) and fully fed on LGTV-infected mice to acquire the viral loads.

(B) Quantification of body weights of GW4869 or mock-treated ticks is shown. QRT-PCR analysis showing LGTV loads in midguts (MG) or salivary glands (SGs) of fully engorged repleted ticks (C) or ticks collected during feeding (D) on LGTV-infected mice. In both post fed or during feeing (DF) groups, ticks were first treated with either GW4869 (150 μM) or mock control (1.5% DMSO) followed by feeding on LGTV-infected mice to acquire the viral loads. LGTV loads were normalized to total RNA. Circles/triangles denote the mock-treated groups, whereas squares/inverted triangles represent the GW4869-treated groups. Each circle/square/triangle/inverted triangle represents one MG or a pair of SGs dissected from post-fed ticks (C) or during feeding ticks (D). p value less than 0.05 is considered statistically significant and ns indicates not significant.

Simultaneous GW4869 treatment and Langat virus infection results in reduced viral loads and decreased release of extracellular vesicles from salivary glands of adult ticks

Adult ticks were simultaneously treated with GW4869 (150 μM) or mock solution (1.5% DMSO) and followed by LGTV (2 x 10⁶ PFU/mL) infection (for 3 days p.i.). QRT-PCR analysis revealed no significant differences in LGTV loads detected in MGs and C of adult ticks. However, in SGs significantly (p < 0.05) reduced LGTV loads were evident when compared to its respective mock control group (Figure 3A). To successfully isolate EVs from GW4869-treated adult ticks (in vivo), we first dissected SGs (7–8 pairs) from uninfected and untreated nymphal ticks and isolated EVs that were measured using the Spectradyne’s nCS1 particle analyzer. In comparison to 1 x PBS (control, with no EVs), we determined high concentrations of EVs isolated from nymphal SGs (Figure S2). Next, we addressed the effects of GW4869 treatment (150 μM) and simultaneous LGTV-infection (2 x 10⁶ PFU/mL, for 3 days p.i.) on EVs released from unfed adult tick SGs (in vivo). EVs-derived from GW4869-treated and LGTV-infected adult tick SGs (five pairs of female SGs collected in 1 x PBS), showed significantly reduced LGTV loads when compared to the loads noted in mock control group (Figure 3B). We also noted significantly reduced number of EVs from GW4869-treated and LGTV-infected unfed adult tick SGs in comparison to the mock control (Figure 3C). The concentration measurements of EVs using the nCS1 particle analysis revealed that LGTV-infected and GW4869-treated adult tick SGs-derived EVs were less in number (N = 3460 and 3440) (Figures 3D and 3E) and concentration (4.45 x 10⁹ ± (8.03 x 10⁷, 7.83 x 10⁷)/mL and 3.21 × 10⁹ ± (5.69 x 10⁷, 5.56 x 10⁷)/mL), when compared to the EVs derived from mock-treated and LGTV-infected adult tick SGs. This data suggests that simultaneous GW4869 treatment and LGTV infection of adult ticks, affects the viral dissemination and replication within SGs that may decrease the EVs release or may reduce the viral loads transported within the EVs from adult tick SGs, thereby perhaps affecting the viral transmission to the vertebrate host.

LGTV loads and EVs released from salivary glands isolated from *I. scapularis* female ticks are reduced upon GW4869 treatment

(A) QRT-PCR showing LGTV loads in midgut (MG), carcass (C), and salivary glands (SGs) of adult ticks that were simultaneously exposed to LGTV (1 MOI) and to either mock (1.5% DMSO) or GW4869 (150 μM) and incubated for 3 days post-infection.

(B) LGTV loads shown from SGs-derived EVs isolated from female ticks incubated at 3 days post LGTV infection with simultaneous treatment with mock/GW4869.

(C) Concentration of EVs-derived from SGs isolated from female ticks incubated for 3 days post LGTV infection and mock/GW4869 treatment is shown.

(D and E) Graphical representation showing the quantification of EVs-derived from SGs isolated from female ticks after 3 days post LGTV infection and mock/GW4869 treatment. Four-five independent replicates were considered for QRT-PCR or for determining EVs concentration. LGTV loads were normalized to total RNA. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents tissues from one adult tick (in A and B) and salivary glands collected from two female ticks (C), respectively. p value less than 0.05 is considered statistically significant and ns indicates not significant.

GW4869 treatment reduces Langat virus acquisition from infected murine host into naive ticks and affects the efficiency of nymphal tick feeding

Vector competence is dependent on the vector’s ability to efficiently acquire, multiply and transmit a pathogen. In this regard, feeding efficiency is very important in the transmission of tick-borne flaviviruses. Based on the observed effects of GW4869 treatment on EVs release in adult tick SGs, we performed a dose response experiment (with two different doses of 50 or 150 μM of GW4869- low and high dose) to determine tick feeding and LGTV acquisition from murine host to nymphal ticks. Nymphal ticks treated (for 24 h) with lower dose (of 50 μM) or higher dose (of 150 μM) of GW4869 and untreated (control) or mock-treated (1.5% DMSO) control were allowed to feed on LGTV-infected mice (obtained from two different vendors, Jackson Laboratories or Charles River Laboratories) (Figures 4 and 5), respectively. We found that GW4869-treated nymphs (from both lower or higher dose, of independent experiments) had significantly reduced body weights (Figures 4A, 4B, 5A, and 5B). Differences in tick body sizes are shown with micrographs (Figures 4A and 5A). Significant reduction in tick body sizes was apparent in nymphs treated with GW4869, when compared to their respective mock controls (Figures 4B and 5B). Furthermore, significant reduction in tick viral loads were noted in GW4869-treated nymphs that were fed on LGTV-infected mice in comparison to their respective mock control nymphs that were also fed on LGTV-infected mice (Figures 4C and 5C). In addition, the significantly reduced viral loads in GW4869-treated nymphs correlated with dramatically increased IsSMase transcript levels that might result due to the decreased LGTV loads (Figures 4D and 5D). Mice used in tick feeding acquisition experiments were confirmed for LGTV detection in blood (Figures 4E and 5E). These data suggests that reduced blood feeding and pathogen acquisition by ticks (from murine host) may impact the efficiency of replication, colonization, and subsequent dissemination of LGTV in GW4869 inhibitor treated nymphal ticks. Since, reduced LGTV loads in GW4869-treated ticks consistently correlated with increased IsSMase transcript levels, we silenced I. scapularis IsSMase by RNA-interference in tick cells. A fragment of 229 bp of IsSMase was PCR amplified and cloned into L4440 vector and the generation of dsRNA is shown (Figure S3). We found no morphological changes in tick cells transfected with IsSMase-dsRNA in comparison to the mock-dsRNA treated tick cells (Figure S4). We determined that dsRNA-mediated silencing of IsSMase significantly (p < 0.05) reduced its transcript levels in tick cells (Figure S5A). Reduced IsSMase levels increased the LGTV loads in tick cells (Figure S5B), and in tick cell derived EVs (Figure S5C).

Treatment with low dose of GW4869 reduces feeding efficiency and LGTV acquisition in nymphal ticks

(A) Micrographs showing sizes of post-fed ticks collected (after repletion) from either control (no treatment) or GW4869 (50 μM) treated nymphs.

(B) Nymphal body weights are shown (in milligrams, mg) from either control (no treatment) or GW4869- treated ticks collected at post-repletion. Each circle/square represents the weight of a single nymph per group.

(C) LGTV loads in control/GW4869-treated post fed nymphs is shown from acquisition.

(D) IsSMase transcript levels from control/GW4869-treated post fed nymphs is shown.

(E) Agarose gel image showing LGTV transcripts in infected blood isolated from mice used for tick feeding studies. Mice used for feeding of untreated nymphs were labeled as control and mice that were used for feeding GW4869-treated (50 μM) ticks are labeled as GW4869 group. M represents DNA marker, PC indicate positive control, and NC denotes negative control. The expected band size for LGTV transcript (140 bp) is shown with black arrow. LGTV and IsSMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered statistically significant.

Treatment with higher dose of GW4869 reduces feeding efficiency and LGTV acquisition in nymphal ticks

(A) Micrograph showing sizes of mock (1.5% DMSO) or GW4869 (150 μM) treated-nymphs collected post repletion.

(B) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869- treated ticks collected at post-repletion. Each circle/square represents one nymphal tick.

(C) LGTV loads in mock/GW4869- treated post fed nymphs is shown.

(D) IsSMase transcript levels are shown from either mock/GW4869 treated post-fed nymphs.

(E) Agarose gel image showing LGTV transcript (of 140 bp, indicated with arrow) in mice that were used for tick feeding studies. Mice used for feeding of untreated nymphs were labeled as control and mice that were used for feeding GW4869-treated (150 μM) ticks were labeled as GW4869. M represents DNA marker, PC indicate positive control, and NC denotes negative control. LGTV and IsSMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered statistically significant.

GW4869 treatment reduces Langat virus transmission from infected nymphal ticks to murine vertebrate host and affects the feeding efficiency of ticks

Upon examining that GW4869 treatment significantly reduces tick efficiency to acquire LGTV loads from murine host, we determined the effects of GW4869 treatment on viral transmission from LGTV-infected ticks to naive mice. To generate infected nymphal ticks, naive larvae were fed on LGTV-infected mice and allowed to molt into LGTV-infected nymphs. Freshly molted nymphs were randomly selected and screened to confirm the LGTV infection of 60% or above. LGTV-infected nymphal ticks (after 10–12 weeks of molting) were treated with GW4869 (150 μM) or with mock control (1.5% DMSO) for 24 h and were allowed to feed on naive/uninfected mice until tick repletion. Like acquisition experiments, mice were obtained from two different vendors (Jackson Laboratories and Charles River Laboratories) for transmission experiments (Figures 6 and 7). GW4869-treated nymphs showed significantly lower body weights (Figures 6A and 7A), thereby showing effects of the inhibitor on blood feeding efficiency. No differences were noted for the repletion rates and times (days 4, 5 and 6) for these LGTV-infected feeding ticks from both GW4869 or mock-treated groups (Figure S6). In addition, GW4869 treated nymphs had significantly reduced LGTV loads in comparison to their respective mock groups (Figures 6B and 7B). This data indicated that GW4869 not only reduces feeding efficiency, but also aids in decreased viral transmission and replication in ticks. No differences were noted in IsSMase transcript levels in repleted ticks due to pre-existing reduced viral loads that correlates with GW4869 treatment (Figures 6C and 7C). Furthermore, we found significantly reduced LGTV loads in murine blood and tissues such as livers and brains isolated from mice that allowed feeding of GW4869-treated ticks when compared to the viral loads noted in murine blood and tissues isolated from mice that allowed feeding of the mock-treated ticks (Figures 6D and 7D). Mice from both vendors showed significantly reduced viral loads in liver suggesting reduced viral transmission from GW4869-treated and LGTV-infected ticks in comparison to their respective mock control ticks (Figures 6D and 7D). These observations suggest that the GW4869 treatment of nymphal ticks reduces pathogen transmission to the vertebrate host. Independent of LGTV infection, we tested if, GW4869 treatment (50 μM) has any direct effects on uninfected ticks feeding and repletion. We allowed feeding of uninfected nymphal ticks on uninfected/naive mice. We found no differences in body sizes or body weights of GW4869-treated or mock-treated uninfected ticks that fully engorged and repleted from uninfected/naive mice (Figures S7A and S7B). However, we noted that on days 4 and 5, the repleted number of ticks were significantly (p < 0.05) reduced in GW4869-treated group of ticks when compared to the mock-treated group of ticks (Figure S7C). These data suggests that independent of LGTV infection, GW4869 treatment slows blood feeding and directly affects the engorgement rates of uninfected ticks. Overall, reduced LGTV loads, lower EVs release in adult SGs, reduced viral acquisition and transmission suggest that treatment with GW4869 could influence the exosome biogenesis that hampers tick efficiency to acquire or transmit tick-borne viruses.

Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor A, Charles River Laboratories)

(A) Nymphal body weights are shown (in milligrams, mg) from either mock (1.5% DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion. Each circle/square represents the weight of one nymph.

(B) LGTV loads from mock/GW4869-treated post fed nymphs is shown.

(C) IsSMase transcript levels in mock/GW4869 treated post fed nymphs is shown.

(D) Viral loads in tissues are shown from four independent mice that were used for mock/GW4869-treated LGTV-infected nymphs feeding. LGTV and IsSMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered satistically significant and ns indicates not significant.

Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories)

(A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion. Each circle/square represents the weight of one nymph.

(B) LGTV loads from mock/GW4869-treated post-fed nymphs are shown.

(C) IsSMase transcript levels in mock/GW4869 treated post fed nymphs are shown.

(D) LGTV loads in tissues are shown from four independent mice that were used for mock/GW4869-treated LGTV-infected nymphs feeding. LGTV and IsSMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered satistically significant and ns indicates not significant.

GW4869 treatment reduces molting efficiency and survival rates in ticks

Pathogen infection and persistent co-existence in ticks does impact the biology and fitness of this medically important vector.²^,³^,⁵^,⁶^,¹⁴^,¹⁵^,¹⁸^,²³^,⁴⁴^,⁴⁵^,⁴⁶ We determined the effects of LGTV infection and GW4869 treatment on tick molting efficiency and fitness/survival rates. First, we determined the effects of LGTV infection and GW4869 treatment (50 μM) on molting rates of larvae into the nymphal stage. At day 26 of incubation, the number of LGTV-infected larvae molting into nymphs were higher in comparison to the uninfected control or to the GW4869-treated LGTV-infected tick groups (Figure 8A). No differences were noted for later days (of 28 and 34) during molting period between any of these groups (Figure 8A). Next, we performed an acquisition experiment to allow uninfected larvae treated with GW4869 inhibitor (at 150 μM) or DMSO control (1.5%) to feed on LGTV-infected mice. No significant differences were noted in body weights between GW4869- or mock-treated fed larval ticks (Figure S8A). Also, LGTV loads or IsSMase transcript levels of larval ticks treated with GW4869 inhibitor showed no significant differences in comparison to their respective mock controls (Figures S8B and S8C). PCR analysis confirmed that all mice were positive for LGTV infection (Figure S8D). Also, we noted significant increase in survival of LGTV-infected molted nymphs when compared to the molting and survival numbers observed for uninfected control or GW4869-treated LGTV-infected nymphal ticks (Figure 8B). Furthermore, GW4869 inhibitor did not showed any effects on nymphs molting into adult stage or the survival of these adult ticks (Figure 8C). However, viral loads were significantly reduced in these GW4869-treated (150 μM) and LGTV-infected group of molted adult ticks in comparison to their mock-treated and LGTV-infected control ticks (Figure 8D). Overall, these data shows that GW4869 significantly reduces the tick molting and survival rates and affects the efficiency of the transtadial transmission of LGTV from nymphs to adult ticks.

GW4869 treatment reduces molting efficiency and survival rates of LGTV-infected ticks

(A) Molting rates of untreated uninfected or LGTV-infected or GW4869 (50 μM)-treated and LGTV-infected *I. scapularis* larvae into nymphs is shown.

(B) Total survival rates of untreated uninfected or LGTV-infected or GW4869 (50 μM)-treated and LGTV-infected larvae molting into nymphs are shown.

(C) Total survival rates of mock (1.5% DMSO) or GW4869 (150 μM)- treated and LGTV-infected nymphs molting into adults are shown.

(D) LGTV loads in adult ticks molted from mock/GW4869-treated and LGTV-infected nymphs is shown. LGTV loads were normalized to total RNA. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one tick. p value less than 0.05 is considered statistically significant and ns indicates not significant.

(E) Model showing the effects of the GW4869 treatment of ticks on LGTV transmission to the vertebrate host. Graphical representation shows that LGTV-infected and GW4869-treated (150 μM) ticks are hampered in blood feeding (with reduced tick body weights). GW4869-treated and LGTV-infected ticks fed partially showing reduced feeding efficiency and decreased viral transmission into the vertebrate host. Treatment of GW4869 affected EVs release that corelated with significant reduction in LGTV loads from these ticks. The GW4869 treatment resulted in inefficient tick feeding, lower EVs biogenesis, decreased viral loads during acquisition and transmission, and reduced molting/survival rates. The combination of all these factors eventually affected vector fitness and competence.

Discussion

Arthropod exosomes are an essential part of the successful transmission of flaviviruses between the vector and the vertebrate host.¹²^,¹³^,¹⁶^,²⁶^,²⁸^,⁴⁰^,⁴¹^,⁴²^,⁴³ Exosomes have been reported to play an important role in the successful transmission of tick or mosquito-borne flaviviruses.³¹^,⁴⁰^,⁴⁷ GW4869 is known to be a selective inhibitor of neutral sphingomyelinases and our previous studies showed that treatment with GW4869, reduces the efficiency of LGTV/ZIKV/DENV (dengue) transmission via infectious exosomes in vitro, by reducing the viral loads in both infected tick/mosquito cells, cortical neuronal cultures or from exosomes-derived from these respective cells.¹²^,¹³^,¹⁶^,²⁵^,²⁶^,³⁰^,⁴⁰^,⁴¹^,⁴²^,⁴³ However, questions remained on the GW4869 effects on ticks (in vivo), on the release of EVs from tick SGs (in vivo) and whether GW4869 influences the acquisition and transmission efficiency of flaviviruses in ticks (in vivo). The current study therefore investigated all these effects of GW4869 treatment on EVs secretion (in vivo). We have postulated EVs to aid in the dissemination of flaviviruses from the tick midgut to the SGs and into the secreted saliva during transmission or blood feeding of ticks.¹²^,¹⁶^,²⁶^,⁴¹^,⁴² Our previous finding that GW4869 treatment (of 1 μM) reduced LGTV loads in tick cells (in vitro), promoted us to investigate the transmission and dissemination efficiency of LGTV in GW4869-treated (150 μM) I. scapularis nymphal and adult ticks (in vivo). We assume that GW4869 inhibitor could play an important therapeutic role. Therefore, we performed an experiment as simultaneous GW4869 treatment and LGTV infection or pre-treatment with GW4869 inhibitor (150 μM, for 4h) followed by LGTV-infection to replicate the in vivo system and possible natural occurrence, respectively. GW4869 treatment was observed to significantly reduce the efficiency of the dissemination or replication of LGTV within nymphal and adult tick body tissues such as MGs and SGs (at 3 days p.i.) but this effect declined over longer incubation days (of 17 days p.i.). However, it is interesting to note that simultaneous incubations with both GW4869 inhibitor and LGTV-infection maintained reduced viral loads in SGs at 17 days p.i., thus suggesting a prolonged inhibitory effect of GW4869 in SGs and to maintain low viral loads. These data also suggest that the prolonged treatment of ticks with GW4869 may inhibit the dissemination of LGTV from tick SGs into saliva and may hinder pathogen transmission from infected ticks to the naive vertebrate host. Our data with 0 days of simultaneous post incubations with GW4869 and LGTV-infection suggests that virus dissemination is affected only upon longer incubations such as 3–17 days and not at early timepoints such as day 0. The observation of no changes at 17 days p.i. with the pre-treatment of GW4869 followed by LGTV infection suggest a decrease in the life-span of this inhibitor.

To support the data from the synchronous infection of LGTV, we tested the effects of GW4869 by treating (150 μM) ticks with this inhibitor and allowing these ticks to acquire the pathogen loads from LGTV-infected murine host (mimicking the natural route of infection). The data from GW4869-treated unfed nymphal ticks blood feeding on LGTV-infected murine vertebrate host (as natural route) further strengthened our simultaneous or pre-treatment studies with GW4869 inhibitor and LGTV-infection. The reduced body sizes and weights of repleted fed ticks indicated that GW4869 treatment affects tick blood feeding. The reduced LGTV loads in MGs and SGs of these post fed ticks (fully engorged and repleted ticks) or in during feeding ticks (DF ticks still infested on mice and acquiring the blood meal) further supported the role of GW4869 as a potential candidate for blocking LGTV acquisition from infected vertebrate host. The rapid treatment of ticks during itch, scratching and attachment would perhaps be the best timing for GW4869 therapeutic effects, however the pretreatment of ticks (in yards and other locations) may be a potential use of GW4869. We have shown that the pretreatment of ticks (for 3 or 17 days) showed significantly reduced LGTV loads in salivary glands of these ticks. We believe that if GW4869 inhibitor is used as a component in repellents (as body sprays or yard sprays) it could be also a potential therapeutic approach to control tick-borne virus transmission from ticks to human and animals (including pets). The pretreatment or simultaneous use of external body sprays with GW4869 (as repellents) may rapidly affect the tick attachment, feeding and pathogen transmission. This data strongly supports GW4869 longer use as a possible candidate in the formulations to develop novel repellents to target ticks in nature.

Furthermore, our novel study reports for the first time that GW4869 treatment also significantly reduced the secretion of EVs from adult tick SGs that corelated with dramatically reduced viral loads in those SGs and perhaps into the secreted saliva. These findings show that GW4869 does not only reduces the efficiency of flavivirus dissemination or replication but also significantly reduces the availability of infectious EVs that can facilitate the transmission of tick-borne viruses. The effects of GW4869 treatment on sphingomyelinase activity in tick cells perhaps reduces the availability of sphingomyelin lipids needed for LGTV replication at lipid rafts.²⁵^,³⁰ The observation of reduced sphingomyelinase activity in tick cells correlated with reduced levels of EVs secretion from tick salivary glands. This could result in reduced LGTV loads in secreted EVs. The findings from this study support our previous observations that mosquito-borne ZIKV loads are dramatically reduced upon GW4869 treatment (20 μM) in mouse embryonic cortical neurons.⁴³ GW4869 reduced ZIKV loads over a time course and in a dose response of treatment (24, 48 and 72 h p.i. and 5, 10 and 20 μM dose).⁴³ These effects of GW4869 were revealed in both neurons and EVs derived from those neuronal cells.⁴³ Taken together, these data suggest that GW4869 is effective in reducing tick- and mosquito-borne flavivirus replication by regulating neutral sphingomyelinase enzymes that could affect the exosome biogenesis pathways resulting in reduced EVs release.

EVs have been suggested to play a key role in tick feeding.¹¹^,¹²^,¹³^,¹⁶^,²⁵^,²⁶^,²⁹^,³⁰^,⁴¹^,⁴² This role of arthropod EVs is essential for the successful acquisition or transmission of pathogens.¹¹^,¹²^,¹³^,¹⁶^,²⁵^,²⁶^,²⁹^,³⁰^,³⁵^,⁴¹^,⁴²^,⁴⁸^,⁴⁹ To confirm the effects of GW4869 treatment on EVs release from ticks (in vivo), we determined, if this effect will impact tick feeding efficiency and pathogen acquisition or transmission from infected-murine host to naive ticks (acquisition) or from infected-ticks to naive murine host (transmission), respectively. Our observations that uninfected/LGTV-infected ticks exposed to GW4869 treatment acquired or transmitted reduced pathogen loads suggested low ability of vector fitness and competence of these ticks. Although, there were no observable differences in the repletion rates or times of feeding for these GW4869-treated and LGTV-infected ticks (that either acquired or transmitted pathogen burden) but significant differences were noted in the tick body sizes and weights upon the repletion of these GW4869-treated ticks that either acquired/transmitted the pathogen burden. The reduced body weights of GW4869-treated nymphs (in both our acquisition and transmission studies from two different vendors) are an indication of decreased amount of blood in these fully engorged repleted ticks that also correlated with dramatic reduction in viral burden. The GW4869-treated and LGTV-infected ticks took approximately the same length of time as respective mock-treated group of ticks to acquire/transmit the pathogen. However, the dramatic differences in reduced body weights further suggest decreased efficiency of these GW4869-treated ticks in blood feeding that perhaps affects the acquisition/transmission of LGTV. Independent of LGTV infection, we found that GW4869 treatment affected uninfected nymphal tick blood feeding rates. These data further indicated a role for GW4869 as a target for controlling ticks and tick-borne diseases. Furthermore, these results indicates that GW4869 treatment dramatically affects EVs biogenesis in ticks and that EVs plays an essential role in tick feeding and pathogen acquisition/transmission. Our data suggests that concentrations of EVs may reduce the efficiency of tick blood feeding. The reduced EVs concentration in the saliva may impact tick blood feeding. Our results suggest that the efficiency of virus acquisition is dependent on the efficiency of blood feeding. Reduced viral loads may influence the efficiency of viral dissemination from gut to SGs due to decreased EVs biogenesis. Reduced blood-feeding affected viral loads in ticks and in adult tick SGs-derived EVs. In addition to reduced blood feeding efficiency, the GW4869-treated nymphs were shown to acquire significantly lower LGTV loads that correlated with higher expression of IsSMase. Therefore, GW4869 treatment in ticks restored the decreased levels of IsSMase upon LGTV infection. Higher expression of IsSMase in GW4869-treated nymphs (collected from acquisition experiment) provide an in vivo validation of the fact that the suppression of IsSMase transcription is required during viral replication. No differences in IsSMase transcript level were noted in ticks during the transmission of LGTV, further suggesting a role for IsSMase in tick immune response. In such scenario, where pathogen burden is reduced, there is no requirement for induced tick immune response and hence IsSMase levels are lower or unaffected in ticks during transmission. To understand the role of IsSMase, we silenced this tick molecule which is an ortholog of spider’s venomous sphingomyelinase protein. In our previous study,²⁵ we had found reduced IsSMase transcript levels upon LGTV infection in tick cells and in LGTV-infected unfed or 24 h partially fed ticks. Our current IsSMase silencing studies showed increasing levels of LGTV in tick cells and in tick cell-derived EVs, thereby further suggesting IsSMase as a tick immune molecule.

Our current study provides first in vivo evidence that the inhibition of EVs release via GW4869 treatment has a significant effect on pathogen acquisition (from infected murine host into ticks) and transmission (from infected-tick to the naive vertebrate host). It is noteworthy that GW4869 treatment dramatically reduced tick body weights, and LGTV loads in nymphal ticks. The finding of no effects of GW4869 treatment on larval tick feeding or LGTV acquisition suggest that this inhibitor is more effective to control nymphal and adult tick population in the field. Although, the effects of GW4869 on reduced repletion rates of larvae to molted nymphs further corroborated these findings. The blood volume or adsorbing moisture amount could be quite low in larval ticks in comparison to the nymphs or adult ticks. Therefore, we believe that higher concentration of GW4869 may be needed to target the larval ticks. These differences observed in the reduced impact of GW4869 on the engorgement of ticks (between low dose or high dose treatments from Figures 4 and 5) could be due to the technical challenges with using mice from two different vendors (with differences in their housing/microbiota or variations in immune responses to pathogens), or due to differences in ticks batches (molted nymphs were used from different batches), or differences in times after molting or their molting efficiency, fitness, and starvation/thirst levels of these ticks. However, the data collected from the body weights, viral loads, and IsSMase levels from both these experiments were significant. The replication of LGTV in murine tissues consistently showed reduced viral loads in livers of mice obtained from two commercial vendors. Consistent reduction of LGTV loads in livers, suggest a decrease dissemination of viruses within the periphery of mice. Lower replication of tick-borne viruses in liver further indicates reduced peripheral viremia and neuroinvasion into brain.⁴² This data is also consistent with our previous published study where we found highest LGTV loads in livers when compared to other tested murine tissues.²² Additionally, viral loads (transmission experiment) were consistently lower in livers of naive mice infested with GW4869-treated and LGTV-infected ticks. No significant changes in mice blood and other tissues (spleen and brains) could be due to the biological differences in mice that were obtained from two different vendors. Also, different batches of infected ticks used in each of these transmission experiment (Figures 6, 7, and 8) could be the reason for inconsistency in the transmission of LGTV loads to mice tissues such as spleens and brains. Results of transmission experiment demonstrates that GW4869-treatment impacts the viral loads even when infection has already been well established in ticks. Also, GW4869 treatment is shown to directly impact viral replication/loads in LGTV-infected nymphs after transmission to murine host. These results suggest that reduced efficiency in LGTV transmission is due to the combined effects of lower availability of infectious exosomes as well as reduced feeding efficiency in ticks.

The interactions between ticks and pathogens they carry is postulated to be mutually beneficial.²⁰^,³¹^,³²^,³³^,³⁴^,⁴⁴^,⁴⁵^,⁴⁶^,⁵⁰^,⁵¹ In this study, we demonstrate for the first time that LGTV-infected larvae molted significantly faster and showed higher molting rates when compared to the uninfected larvae molting. This data suggests a mutualistic/beneficial relationship between larval ticks and LGTV where fitness and molting efficiency from larvae to nymphs is improved in the presence of a tick-borne viral pathogen. This improved fitness and molting efficiency may facilitate higher viral transmission from infected ticks to naive vertebrate host when newly emerged nymphs seek a host for blood meal. The reversal of this phenomena was noted upon GW4869 treatment where molting and survival rates of LGTV-infected ticks was inhibited. Enhanced molting rates in LGTV-infected larvae suggests a possible role for neutral sphingomyelinases. We are currently exploring how neutral sphingomyelinases contribute to this molting process and how this mechanism of enhanced EVs biogenesis can be exploited in vector competence and in vector population control.¹²^,¹⁶^,²⁶^,³⁰^,³²^,⁵² On-going work in our laboratory is currently addressing the importance of these sphingomyelinases. In addition to the effects of viral infection on molting and survival, we noted the transtadial transmission of LGTV from larvae to nymphs or from nymphs to adult ticks. In this study, we establish that infectious nymphs used in transmission experiments remained infectious after molting into adult ticks. The reduced LGTV loads observed in GW4869-treated nymphs perhaps affected the molting/survival process, and thus resulted in emerged adult ticks with significantly lower viral loads. No differences in survival rates of adult ticks could be due to the biological variation between male and female ticks. We cannot exclude the possibility of acquiring such survival data from large batch of ticks (to control male-female and tick-tick variations that could be greater in adult ticks) and perhaps with higher dose of GW4869 treatment. Our proposed model (Figure 8E) summarizes the effects of GW4869 treatment on vector competence and its dependence on EVs biogenesis. In summary, our findings suggest that GW4869 treatment inhibits the secretion of EVs and hampers EVs biogenesis. This reduction in EVs biogenesis, especially in the SGs and saliva reduces the efficiency of tick blood feeding, virus replication and dissemination within tick, and virus acquisition from vertebrate host to ticks and transmission from infected ticks to the vertebrate host. Additionally, the enhancement of IsSMase upon GW4869 treatment is inferred to modify tick fitness and efficiency of tick feeding, molting and survival. Our observation that showed downregulation of IsSMase upon LGTV infection and its upregulation by GW4869-treatment is critical in controlling pathogen transmission and suggest IsSMase as an important antiviral molecule in ticks. Our results suggest that EVs are essential in vector competence and survival, in tick-borne virus acquisition, dissemination within the vector, and during pathogen transmission from infected ticks to naive vertebrate hosts. Overall, our study highlights GW4869 as potential agent in the prevention and control of tick-borne flaviviruses. This study provides a platform for the exploration of this potential inhibitor as a novel therapeutic candidate in vector control and to target the transmission of tick-borne pathogens.

Limitations of the study

In this work, we have provided evidence of inhibiting the tick exosomes in vivo with GW4869 and how this inhibition affects the pathogen acquisition from LGTV-infected murine host or transmission from an infected tick to a naive vertebrate host. We also provide insights on how GW4869 hampers the tick repletion, molting and survival rates and suggest this inhibitor as a potential component in controlling ticks and tick-borne diseases. Two of the limitations that will be addressed in the immediate future are how GW4869 can be used as a therapeutic? and what is the evidence that GW4869 may have application(s) as a repellent in the yard? More work is therefore needed to address these important questions and our future studies will be focused on these issues.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Virus strain

Langat Virus, TP21	BEI Resources, NIAID, NIH	NR-51658

Biological samples

Ixodes scapularis (larvae, nymph, adult- male and female) live ticks	BEI Resources, NIAID, NIH	NR-44115 (Larvae), NR-44116 (Nymphs) NR-42510 (Adult)

Chemicals

GW4869 inhibitor	Santa Cruz Biotechnology, Inc., USA	sc-218578A
DMSO	Sigma, USA	Cat # D5879-500ML
Phosphate Buffered Saline (10X PBS), pH 7.2	Sigma, USA	Cat # 806552
Leibovitz's L-15 Medium, powder	Gibco	Cat # 41300-039
Tryptose phosphate broth	MP Biomedicals, USA	Cat # ICN1682149
Bovine Cholesterol Lipoprotein Concentrate:	MP Biomedicals, USA	Cat # ICN19147625
FBS	VWR, USA	Cat # 89510-186
iTaq Universal SYBR Green Supermix	BioRad, USA	Cat # L001752 B
2X Universal SYBR Green fast qPCR Mix	ABclonal, USA	Cat # RM21203
Lipofectamine 2000 Transfection reagent	(Invitrogen, Thermo/Fisher Scientific)	Cat # 11668500

Critical commercial assays

Aurum Total RNA Mini kit	BioRad, USA	Cat # 732-6820
iScript cDNA synthesis kit	BioRad, USA	Cat # 1708891
Gel extraction kit	Qiagen, USA	Cat # 28704
MEGA script RNAi kit	Invitrogen, Thermo/Fisher Scientific, USA	Cat # AM1626

Experimental models: cell lines

Tick cell line ISE6	ATCC	Cat # CRL-11974

Experimental models: organisms/strains

Ticks- Ixodes scapularis	BEI Resources, NIAID, NIH	NR-44115, NR-44116, and NR-42510
Mice C57BL/6J	Jackson Laboratories, USA	Cat # 000664

Oligonucleotides

Primers for qPCR	This paper	Integrated DNA Technology
Primers for RNAi analysis	This paper	Integrated DNA Technology

Software and algorithms

GraphPad Prism	GraphPad, USA	Version 6
Cytation7 imaging system	BioTek/Agilent	https://www.agilent.com

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Resource availability

Lead contact

Further information about the protocols and requests for resources and/or reagents should be directed to and will be fulfilled by the lead contact, Hameeda Sultana (hsultana@utk.edu).

Materials availability

This study did not generate any new or unique reagents.

Data and code availability

•
Any additional information or data is available from the lead contact upon request.
•
This paper does not report any original code.
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Additional data is provided as supplemental information.

Experimental model and study participant details

Virus

Virus dilution was prepared from the laboratory virus stocks of 1 x 10⁸ plaques forming units (pfu/mL). Mice in the LGTV-infected group were injected intraperitoneal with 0.1 mL (50,000 pfu/mL) of diluted virus suspension in 1 x PBS containing 1% gelatin (SIGMA Aldrich). Uninfected or naive ticks were allowed to feed on LGTV-infected mice to acquire the pathogen from murine host.

Ticks

Unfed nymphs or adult (male and female) ticks obtained from BEI resources/Center for Disease Prevention and Control (CDC) were maintained in our laboratory environmental chamber (set at 23°C temperature and 95% relative humidity with a 14/10 light/dark cycle). The bottom of the humidity chamber was covered with potassium sulfate and 1 inch of water was added above that. Tick vials were placed on the top of the rack and were kept out of this solution. Ticks were held at 73°F.

Tick cell line

The I. scapularis ISE6 tick cell line was purchased from ATCC and grown in L15B300 medium prepared from Leibovitz’s L-15 Medium powder with 5% tryptose phosphate broth, 5% heat-inactivated FBS, and 0.1% bovine lipoprotein concentrate, pH 7.2. Cells were maintained at 34°C incubator with no CO2.

Method details

GW4869 inhibitor treatments and synchronous infection of nymphal or adult ticks

Ticks were maintained in our laboratory as described.²²^,²⁵^,²⁸^,⁴¹^,⁴²^,⁴⁴^,⁴⁶^,⁵¹^,⁵³^,⁵⁴^,⁵⁵ To generate LGTV-infected nymphal/adult ticks (used only in simultaneous or pre-treatment studies or in testing GW4869 doses to use in this study), synchronous infection with LGTV was performed using a previously published protocol with few modifications.²⁵^,⁵⁵ Briefly, ticks were submerged (for 40 min at 34°C) in 1 mL of 2 x 10⁶ PFU/mL LGTV suspended in Leibovitz L-15B300 medium containing 5% FBS. Ticks were washed twice in 1 x PBS and transferred into sterile tubes after drying (using paper towel) and kept at 23°C for either 3 or 17-day post infection, respectively. For GW4869 treatment, 10 mM of GW4869 lyophilized powder was dissolved in 100% DMSO stock solution and stored as aliquots at −80°C. For each experiment, 1 mL solution of appropriate GW4869 concentration (ranging from 1 to 150 μM) and its equivalent DMSO concentration (as mock) was prepared in Leibovitz L-15B300 medium supplemented with 5% FBS. For simultaneous treatment of nymphal ticks, LGTV (2 x 10e6 PFU/ml) was mixed with either GW4869 (150 μM) or with 1.5% DMSO (mock control) in Leibovitz L-15B300 medium with 5% FBS. Ticks (nymphs or adults) were incubated in respective solution for 40 min at 34°C. In case of pretreatment, nymphal ticks were first submerged in GW4869 (150 μM) or mock (1.5% DMSO) solutions for 40 min at 34°C followed by incubation with LGTV (2 x 10e6 PFU/ml) suspension (for additional 40 min at 34°C). In both cases, ticks were washed (in cold 1 x PBS), thoroughly dried and incubated in environmental chamber for either 3- or 17-day post infection.²⁵^,⁵⁵ Any dead ticks were excluded from either experiment (synchronous or pretreatment). For total RNA extractions, we used dissected tissues such as salivary glands, midguts, or carcass (SGs, MGs and C, respectively) from 2 nymphs or one adult (for simultaneous treatment with GW4869 and LGTV infection) or 3 nymphs (in pretreatment with GW4869, followed by LGTV-infection). In both treatments, 4 replicates per group were considered for analysis.

RNA extractions, cDNA synthesis, and QRT-PCR analysis

Total RNA was extracted from GW4869/mock-treated (50 or 150 μM or 1.5% DMSO) post-fed nymphs (collected from acquisition or transmission experiments), molted nymphs/adult ticks, dissected tissues (SGs, MGs, and C) of nymphal/adult ticks pre-treated/simultaneously-treated with GW4869/mock (150 μM or 0.5% DMSO) and infected with LGTV or from mouse blood, liver, spleen and brain samples (collected from acquisition or transmission experiments). For RNA extraction from dissected tick tissues (SGs, MGs and C), we used 1 adult tick as one sample. In total, 5 adult ticks were dissected (for each respective group) to obtain 5 independent samples. RNA was extracted using Aurum Total RNA Mini Kit and by following the manufacturer’s instructions.²²^,³⁹^,⁴⁶^,⁵³^,⁵⁵ Extracted RNA (1 μg) was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad, USA). Synthesized cDNA served as template for quantitative real-time PCR (QRT-PCR) reactions that were performed using SYBR Green super mix and CFX-OPUS (Bio-Rad). QRT-PCR was performed as follows: 95°C for 3 min followed by 40 cycles of 95°C for 10 s, 58°C for 10 s, 72°C for 30 s and final denaturation at 95°C for 10 s. Published gene-specific primers were used to detect LGTV-RNA,²²^,²⁵^,⁴²^,⁵⁵ IsSMase transcripts,²⁵ and tick beta-actin levels.²²^,²⁵^,⁴²^,⁵⁵^,⁵⁶^,⁵⁷ LGTV loads were normalized to total RNA while IsSMase levels were normalized to tick beta-actin. Standard curves were prepared for each gene using 10-fold serially diluted standards ranging from 1 to 0.00001 ng/μL of known quantities of respective fragments.

Isolation and nano-quantification of EVs-derived from adult tick SGs

LGTV-infected, I. scapularis unfed female ticks were dissected to collect SGs. SGs were collected from two adult ticks (as two pairs) and used as one replicate. For EVs isolation, SGs were incubated at 4°C in cold 1 x PBS (filtered) for overnight. The resulting suspension was collected and centrifuged at 1000 x g for 15 min and the pellets were discarded. Resulting supernatant was processed for EVs isolation by ultracentrifugation method.²⁵^,⁴⁰^,⁴¹^,⁴²^,⁴³ Isolated EVs were briefly stored at 4°C (for 1 h) or quantified immediately with nCS1 Spectradyne nanoparticle analyzer and following the manufacturer’s recommendations as described.⁴¹ Isolated EVs from tick SGs were diluted as 1: 2000 in 1% Tween 20 prepared in 1 x PBS (filtered). Diluted samples (5 μL) were loaded on to nCS1 microfluidic cartridge TS-400 with a size range of 65–400 nm (in diameters). Loaded cartridges were inserted in analyzer to quantify EVs in each sample and data was collected from four independent replicates and as described.⁴¹

Animal infections and tick feeding experiments

All protocols used in mice studies were approved by the Institutional Animal Care and Use Committee (IACUC) and adheres to the recommendations of the NIH ‘Guide for Care and use of Laboratory animals (USA)’. Husbandry and tranquilization are performed based on approved protocol number (2805-0321) and as described.²²^,²⁵^,³⁹^,⁵⁵ All tick feeding experiments were performed using six-week-old C57BL/6 mice received from two different vendors (Charles River or Jackson Laboratories, USA). For tick acquisition experiments, mock (0.5 or 1.5% DMSO) or GW4869 (50 or 150 μM, lower or higher dose)- treated (for 40 min at 34°C) larval/nymphal ticks were allowed to recover for 24 h after treatment and then fed on LGTV-infected mice. Mice were infected (intraperitoneal) with 5 x 10⁴ PFU/mouse of LGTV diluted virus and a day before tick feeding.²²^,²⁵ LGTV-infected mice (3 mice/group) were infested with GW4869/mock-treated naive/uninfected nymphs and allowed to feed until repletion (4–5 days post infestation) and acquire the pathogen loads. All mice were euthanized after 6 days post infection (or 5 days after tick infestation). Blood was collected to confirm LGTV infection in mice (from both vendors) used for feeding of uninfected ticks (during acquisition experiment). For transmission experiments (from both vendors), LGTV-infected post-fed larvae (generated by feeding naive/uninfected larvae on LGTV-infected mice, in an independent acquisition experiment and maintained in our laboratory) were allowed to molt into infected-nymphs. Freshly molted nymphs were screened to confirm LGTV infection in tick colony. LGTV-infected nymphs were pre-treated with GW4869 (150 μM, in both transmission experiments with mice from two different vendors) or with 1.5% DMSO (mock control) and allowed for 24 h recovery. GW4869 or mock-treated and LGTV-infected nymphs were allowed to feed on uninfected mice (5 mice/group) and transmit the pathogen loads to the murine host. Ticks were allowed to feed until repletion. Mice were euthanized after 5 days of tick infestation, and blood or other mice tissues (liver, spleen, and brain) were collected to analyze LGTV burden or pathogen transmission from LGTV-infected ticks to naive mice. In addition, to determine the inhibitory effects, which is independent of LGTV-infection, we collected GW4869-treated (50 μM) or mock-treated (0.5% DMSO) ticks and allowed these ticks to feed on naive/uninfected mice. Repleted ticks were collected after 5 days of tick infestation on these uninfected mice. Ticks collected upon repletion were weighed to estimate feeding efficiency and were either maintained at 23°C and 95% humidity (to allow for molting into adult ticks) or stored at −80°C until used for RNA extractions.

Estimation of tick body weights, and molting/repletion/survival rates

Upon repletion, post-fed nymphal tick body weights were determined using Sartorius Cubis II analytical balance (DWS, USA), as an estimation of feeding efficiency. GW4869-treated or LGTV-infected or uninfected post-fed larvae or nymphs from acquisition experiments were divided into tubes of 10 and allowed to molt into nymphal or adult ticks and processed for estimation of molting and survival rates. Tubes were checked daily, and the number of molted and/or dead ticks were recorded. Number of ticks that successfully molted to next developmental stage were used to determine the molting/survival rates. For repletion/molting rates analysis, repleted/molted ticks were collected (between days 4–6 for repleted ticks or days 26–34 for molted ticks) and the repletion/molting numbers were recorded.

dsRNA fragment cloning and synthesis, and tick cell transfection studies

First, we amplified a fragment of IsSMase (299 bp) from I. scapularis tick cDNA and cloned it into KpnI and BglII enzyme restriction sites of the L4440 double T7 Script II vector. The fragment of interest was PCR amplified by using specific primers comprising the KpnI and BglII restriction enzyme sites (CGAGATCTGAACGTGTTGCTGTCCATCG-forward Primer and TGGGTACCAGACCTTGTCGACGTAGCTC- Reverse Primer). PCR amplified IsSMase fragment was purified using gel extraction kit (Qiagen, USA) and following user’s manual instructions. The amplified fragment is cloned into L4440 double T7 Script II vector in BglII-KpnI enzyme restriction sites. MEGA script RNAi kit (Invitrogen, Thermo/Fisher Scientific, USA) is used to synthesize the dsRNA complementary to IsSMase sequence. The agarose gel electrophoresis image for three subsequent elution of dsRNA is shown. For dsRNA transfections and silencing of IsSMase, we plated 5 x 10e5 ISE6 tick cells in complete L-15B300 media containing 5% FBS (exo-free-FBS), and cells were allowed to adhere overnight. Lipofectamine reagent (Invitrogen, Thermo/Fisher Scientific) was mixed with 750 ng of dsRNA for transfection of tick cells. Tick cells were transfected with either mock-dsRNA control or IsSMase-dsRNA. To recover cells from transfection stress, 2X L-15 recovery media (with 10% FBS) was added, after 6 h of post transfection. Followed by recovery and 24 h of dsRNA treatment, tick cells were infected with LGTV (MOI 1, for 48 h p.i.) and collected as 72 h post transfection for RNA extractions. Numerous phase contrast images were collected (at 0, 4 and 72 h post transfection, p.t.), using the 10-x magnification and a scale bar of 200 μm for each group. Representative images captured using the Cytation7 imaging System are shown. Tick cells were collected at 48 h p.i. (or 72 h post transfection) for further analysis. QRT-PCR was performed to determine silencing efficiency of IsSMase transcripts by using specific primers.

Quantification and statistical analysis

We used Microsoft excel and GraphPad Prism 7 software (https://www.graphpad.com/) and Microsoft Excel 2010 (https://www.microsoft.com) to analyze all data collected in this study. Statistical analysis was performed by the non-paired, two-tail Student’s t test or ANOVA analysis to compare the group’s variations. Error bars represent mean (+SD) values, and p value < 0.05 was considered statistically significant in all analyses.

Acknowledgments

We would like to sincerely acknowledge the useful resource of ticks (nymphs and adults) from CDC/BEI. The following reagents were provided by Center for Disease Control and Prevention for distribution by BEI Resources, NIAID, and NIH: I. scapularis Nymph (Live), NR-44116, Larvae (Live), NR-44115, and I. scapularis Adult (Live), NR-42510. We are extremely thankful to colleagues at CDC for help with tick rearing and shipments. We also would like to acknowledge the support from BEI resources. “The following reagent was obtained through BEI Resources, NIAID, NIH: Langat Virus, TP21, NR-51658. This study has been supported by funding from National Institute of Allergy and Infectious Diseases/National Institutes of Health (Award number R01AI141790 to HS).

Author contributions

H.S., W.A., and G.N. performed the experiments. H.S. and G.N. discussed, analyzed, and interpreted the data in several settings. G.N. infected and tranquilized mice and dissected salivary glands. H.S. and G.N. obtained the unfed/uninfected I. scapularis nymphal ticks from BEI/CDC. H.S. wrote the article, revised, modified, and finalized the article. All authors read and edited the complete article. H.S. collected all required materials and reagents, conceived, designed, and coordinated the entire study, organized all the data, and compiled, and supervised overall investigations.

Declaration of interests

The authors declare that there is no competition or conflict of interest exists.

Published: June 27, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110391.

Supplemental information

Document S1. Figures S1–S8

mmc1.pdf^{(2.8MB, pdf)}

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S8

mmc1.pdf^{(2.8MB, pdf)}

Data Availability Statement

•
Any additional information or data is available from the lead contact upon request.
•
This paper does not report any original code.
•
Additional data is provided as supplemental information.

[bib1] 1.Adegoke A., Kumar D., Budachetri K., Karim S. Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum. Front. Cell. Infect. Microbiol. 2022;12 doi: 10.3389/fcimb.2022.1037387. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib4] 4.Wikel S.K. Tick-host-pathogen systems immunobiology: an interactive trio. Front. Biosci. 2018;23:265–283. doi: 10.2741/4590. [DOI] [PubMed] [Google Scholar]

[bib5] 5.de la Fuente J., Antunes S., Bonnet S., Cabezas-Cruz A., Domingos A.G., Estrada-Peña A., Johnson N., Kocan K.M., Mansfield K.L., Nijhof A.M., et al. Tick-Pathogen Interactions and Vector Competence: Identification of Molecular Drivers for Tick-Borne Diseases. Front. Cell. Infect. Microbiol. 2017;7:114. doi: 10.3389/fcimb.2017.00114. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib7] 7.Nuttall P.A. Tick saliva and its role in pathogen transmission. Wien Klin. Wochenschr. 2023;135:165–176. doi: 10.1007/s00508-019-1500-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Nuttall P.A., Labuda M. Dynamics of infection in tick vectors and at the tick-host interface. Adv. Virus Res. 2003;60:233–272. doi: 10.1016/s0065-3527(03)60007-2. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Nuttall P.A., Paesen G.C., Lawrie C.H., Wang H. Vector-host interactions in disease transmission. J. Mol. Microbiol. Biotechnol. 2000;2:381–386. [PubMed] [Google Scholar]

[bib10] 10.Hassett E.M., Thangamani S. Ecology of Powassan Virus in the United States. Microorganisms. 2021;9 doi: 10.3390/microorganisms9112317. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib11] 11.Kazimírova M., Thangamani S., Bartikova P., Hermance M., Holikova V., Stibraniova I., Nuttall P.A. Tick-Borne Viruses and Biological Processes at the Tick-Host-Virus Interface. Front. Cell. Infect. Microbiol. 2017;7:339. doi: 10.3389/fcimb.2017.00339. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

Hameeda Sultana

Waqas Ahmed

Girish Neelakanta

Summary

Graphical abstract

Highlights

Introduction

Results

Simultaneous or pre-treatment of GW4869 inhibitor reduces the efficiency of Langat Virus dissemination within nymphal ticks and to the salivary glands

Figure 1.

Treatment with GW4869 affects Langat virus dissemination within tick bodies that acquired blood meal from infected murine host

Figure 2.

Simultaneous GW4869 treatment and Langat virus infection results in reduced viral loads and decreased release of extracellular vesicles from salivary glands of adult ticks

Figure 3.

GW4869 treatment reduces Langat virus acquisition from infected murine host into naive ticks and affects the efficiency of nymphal tick feeding

Figure 4.

Figure 5.

GW4869 treatment reduces Langat virus transmission from infected nymphal ticks to murine vertebrate host and affects the feeding efficiency of ticks

Figure 6.

Figure 7.

GW4869 treatment reduces molting efficiency and survival rates in ticks

Figure 8.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Data and code availability

Experimental model and study participant details

Virus

Ticks

Tick cell line

Method details

GW4869 inhibitor treatments and synchronous infection of nymphal or adult ticks

RNA extractions, cDNA synthesis, and QRT-PCR analysis

Isolation and nano-quantification of EVs-derived from adult tick SGs

Animal infections and tick feeding experiments

Estimation of tick body weights, and molting/repletion/survival rates

dsRNA fragment cloning and synthesis, and tick cell transfection studies

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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