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Published in final edited form as: Ticks Tick Borne Dis. 2023 Dec 21;15(2):102301. doi: 10.1016/j.ttbdis.2023.102301

Cytoarchitecture of Ex Vivo Midgut Cultures of Unfed Ixodes scapularis Infected with a Tick-Borne Flavivirus

Missiani Ochwoto a, Danielle K Offerdahl a, Jacqueline M Leung b, Cindi L Schwartz b, Dan Long c, Rebecca Rosenke c, Philip E Stewart a, Greg A Saturday c, Marshall E Bloom a,*
PMCID: PMC10923016  NIHMSID: NIHMS1956137  PMID: 38134511

Abstract

A bite from an infected tick is the primary means of transmission for tick-borne flaviviruses (TBFV). Ticks ingest the virus while feeding on infected blood. The traditional view is that the virus first replicates in and transits the tick midgut prior to dissemination to other organs, including salivary glands. Thus, understanding TBFV infection in the tick midgut is a key first step in identifying potential countermeasures against infection. Ex vivo midgut cultures prepared from unfed adult female Ixodes scapularis ticks were viable and remained morphologically intact for more than 8 days. The midgut consisted of two clearly defined cell layers separated by a basement membrane: an exterior network of smooth muscle cells and an internal epithelium composed of digestive generative cells. The smooth muscle cells were arranged in a stellate circumferential pattern spaced at regular intervals along the long axis of midgut diverticula. When the cultures were infected with the TBFV Langat virus (LGTV), virus production increased by two logs with a peak at 96 h post-infection. Infected cells were readily identified by immunofluorescence staining for the viral envelope protein, nonstructural protein 3 and dsRNA. Microscopy of the stained cultures suggested that generative cells were the primary target for virus infection in the midgut. Infected cells exhibited an expansion of membranes derived from the endoplasmic reticulum; a finding consistent with TBFV infected cell cultures. Electron microscopy of infected cultures revealed virus particles in the basolateral region between epithelial cells. These results demonstrated LGTV replication in midgut generative cells of artificially infected, ex vivo cultures of unfed adult female I. scapularis ticks.

Keywords: Tick Midgut, Tick-borne flaviviruses, ex-vivo cultures, Langat virus

1. Introduction

The global incidence of infection with tick-borne flaviviruses (TBFV) has increased 400% in the last three decades (Kellman et al., 2018; McAuley et al., 2017). Human infections are common in some parts of the world and can be associated with serious neurological disease, hemorrhagic fever, and sometimes death. The viruses are usually transmitted by the bite of an infected ixodid tick as it takes a blood meal, but there are other less frequent modes of infection (Mlera and Bloom, 2018).

The majority of the TBFV are BSL-3 or BSL-4 agents; however, Langat Virus (LGTV); that was originally isolated from a pool of Ixodes granulatus ticks in Malaysia (Smith, 1956), is naturally attenuated making it a suitable BSL-2 model for TBFV (Grabowski and Kissinger, 2020; Offerdahl et al., 2012). Like other TBFV, LGTV is an RNA virus with a single-stranded positive sense genome of 11kb. The genome codes for three structural and seven nonstructural proteins. In an infected cell, replication proceeds in the cytoplasm accompanied by a dramatic proliferation of membranes derived from the endoplasmic reticulum and the formation of vesicle-like replication compartments (Offerdahl et al., 2012). Virions mature in the Golgi and trans-Golgi membrane network, then exit the cell (Holbrook et al., 2001).

The TBFV are estimated to spend 90% of their evolutionary lifespan in ticks (Mitzel et al., 2007) and can be transstadially and transovarially transmitted (Ahmed et al., 2022). However, the complex biology of the virus within the tick has not been extensively examined. The traditional view is that the midgut is the first organ and potential barrier that the virus encounters upon entry into the tick during the bloodmeal. The midgut of an unfed ixodid tick, such as Ixodes scapularis, is a simple organ. The ixodid tick midgut consists of a central cavity (ventriculus) connected to the rectal sac by the hindgut and several diverticula (Franta et al., 2010; Remedio et al., 2013; Simo and Park, 2014; Starck et al., 2018). The diverticula are located towards the posterior end and occupy most of the internal cavity of the tick body (Starck et al., 2018). The cellular structure of an unfed ixodid tick midgut is also relatively simple; there is an external web-like smooth muscle layer facing the hemocoel (Remedio et al ., 2013), a basement membrane and an internal layer comprised primarily of resting polarized, cuboidal epithelial cells with a microvillous brush border facing the lumen (Franta et al., 2010). These resting cells are denoted as stem or generative cells, but for consistency, we will use the term generative cells in this study. After taking a blood meal, the generative cells mature to digestive cells which detach from the basement membrane into the lumen where they ingest and digest the constituents of the blood meal (Akov, 1982; Kleiboeker et al., 1999; Koh et al., 1991). Residual digestive cells from the previous blood meal can often be observed in the midgut lumen of an unfed tick (Starck et al., 2018). The contractions of smooth muscle cells churn the blood meal throughout the blind caeca of the diverticula (Akov, 1982).

The traditional view is that a virus must infect the tick midgut cells, replicate, and be released from the midgut prior to dissemination to other tick organs (Franz et al., 2015; Füzik et al., 2018; Lejal et al., 2019). Although virus-midgut interaction plays an important role in the biology of TBFV infections, the detailed process by which TBFV infect and exit the tick midgut remains unclear.

Earlier work from this laboratory established ex vivo tick organ cultures as a suitable model for studying virus infection in a highly controlled fashion (Grabowski et al., 2017). In this study, we have investigated LGTV infection in midgut cultures derived from unfed adult female I. scapularis ticks. We describe the cytoarchitecture of the tick midgut and show that LGTV infects generative cells and smooth muscle cells of these ex vivo midgut cultures. The infected generative cells have increased membranous structures and virus particles were identified in the basolateral spaces between generative cells.

2. Materials and Methods

2.1. Tick acquisition and dissection

Unfed adult female I. scapularis ticks were obtained from the Oklahoma State University tick rearing facility (Stillwater, OK, USA) and were maintained in humidified bell jars containing a saturated potassium chloride solution (Mitzel et al., 2007). The ticks were surface-sterilized and dissected as previously described (Grabowski and Kissinger, 2020), and midgut was separated from the trachea, salivary glands, synganglion and other internal organs. The midguts were washed twice in Dulbecco’s phosphate-buffered saline (DPBS, Life Technologies, Paisley, UK) before transfer to Nunc Lab-tek chamber slides (Cat #154941,ThermoFisher, Ashville, NC, USA) containing 400 μl of L15C300 tick medium (Oliver et al., 2014) supplemented with 5% fetal bovine serum, 5% tryptose phosphate Broth, 0.1% bovine lipoprotein, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of Amphotericin B (Gibco® Antibiotic-Antimycotic, Thermo Fisher) (Kellman et al., 2018).

2.2. Confirmation of viability

The viability of midgut cultures was examined using an inverted microscope to physically observe the peristaltic contractions of the midgut diverticula, and it was confirmed by resazurin salt-based reagent (Alamar Blue, ThermoFisher) as previously reported (Grabowski and Kissinger, 2020). Briefly, individual midguts were transferred into wells of 96-well plates containing 250 μl complete medium with 1:10 dilution resazurin salt-based reagent (Alamar Blue, ThermoFisher). Wells contained either 5×105 ffu of virus or no virus. Medium containing the resazurin salt but no midgut served as an additional negative control. The samples were incubated at 34 °C without CO2. Absorbance at a wavelength of 570 nm was measured using Molecular Device SpectraMax plus 384 plate reader and Softmax Pro v6.5 software (Molecular Devices, LLC. CA, USA). Absorbance measurements were recorded at 3 h post dissection (hpd) and every 24 h thereafter until the incubation was terminated. Viability experiments were performed using no less than four uninfected and four infected midguts. Complete medium with resazurin salt-based reagent but no midgut served as a negative control. Samples were monitored daily until the absorbance of the midgut containing samples was equal to the negative control.

2.3. Histopathology preparation of uninfected and LGTV infected midgut cultures

Samples prepared for histopathology were freshly dissected midguts as well as midguts cultured with and without LGTV for designated time intervals. The specimens were washed using DPBS and then fixed in 10% neutral buffered formalin (Cancer Diagnostic, Durham, NC, USA) for a minimum of 24 h. The samples were dehydrated in a graded series of ethanol (50–100%) for 1h 45 min, followed by clearing for 45 min, wax infiltration for 1 h and 30 min before being embedded in pureAffin paraffin polymer (Cancer Diagnostic). The midgut blocks were sectioned at 5 μm thickness in cross sectional or longitudinal orientation, using a Leica RM2265 microtome. Sections were mounted on slides, and then subjected to either standard hematoxylin and eosin (H&E) staining or periodic acid Schiff (PAS) staining to stain mucosubstances of the tick midgut, primarily the basement membrane prior to being viewed on an Olympus model (BX51) microscope. Images were captured by an Olympus DP70 digital camera at 40X, 100X, 200X and 400X magnifications.

2.4. Infection of ex vivo midgut cultures

For infection of the midgut cultures, the L15C300 tick medium was carefully aspirated and replaced with either 400 μl of medium containing 5×105 focus forming units (ffu) 2nd passage LGTV TP21 or medium alone. The TP21 strain of LGTV (Smith, 1965) was cultured in African green monkey kidney cells (Vero, ATCC, version CCL-81). For infection of the midgut cultures, the L15C300 tick medium was carefully aspirated and replaced with either 400 μl of medium from Vero cells containing 5×105 focus forming units (ffu) second passage LGTV TP21 or medium alone. After rocking at 34 °C for 1 h, the viral inoculum was carefully removed, and culture was washed twice using DPBS. A fresh 400 μl aliquot of medium was then added, and the midgut was incubated at 34 °C without CO2 throughout the experiment.

2.5. Quantitation of virus production in ex vivo midgut cultures

Midgut cultures were infected with 5×105 ffu LGTV, and controls included uninfected cultures, as well as virus in medium to assess titer of residual virus inoculum. Supernatants were collected at 3 h post infection (hpi), and every 24 h thereafter until 168 hpi. Viral titration was done using an immunofocus assay in Vero cells as previously described (Offerdahl et al., 2012).

2.6. Localization of viral replicative intermediates (dsRNA, NS3 and E proteins) by immunofluorescence

Samples were prepared for immunofluorescence after 72 hpi. L15C300 tick medium was carefully removed, and wells were washed twice with DPBS. The cultures were fixed with 4% paraformaldehyde with 5% sucrose in PBS for 20 min at room temperature (RT) prior to permeabilization with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, Missouri, USA) in PBS for 15 min at RT. After washing with DPBS, free aldehydes were quenched with 50 mM glycine (Thermo Scientific, Geel, Belgium) in PBS for 10 min followed by washing twice with DPBS. The cultures were blocked by incubation in 2% bovine serum albumin (ICN Biomedicals Inc, Auroa, Ohio, USA) in PBS for 1 h at 37 °C. After washing, cultures were incubated overnight at RT with a 1:1000 dilution of primary antibody. The unbound antibody was removed by washing three times with DPBS with 5 min incubations per wash. A 1:1000 dilution of the fluorescent conjugated secondary antibody was added. Samples were protected from light and incubated for 2 h at 37 °C. In general, all incubations were done while rocking the midgut culture using an orbital shaker (Bellco Biotechnology- USA) that was set at X4, to ensure even distribution of the reagent. After the last washing step, the specimens were carefully transferred to 0.15 mm microscope cover glasses (Fisher Scientific, Pittsburgh, USA) where Prolong Gold Antifade was used for mounting (Life Technologies-Invitrogen, Eugene, Oregon, USA); a 1 mm thick microscope glass slide was added and incubated overnight in the dark at RT.

The following primary antibodies against viral intermediates were used: mouse monoclonal antibody ds-RNA J2 (anti-double stranded RNA, English & Scientific Consulting, Szirak, Hungary); mouse anti-LGTV envelope monoclonal antibody 11H12 (a kind gift from Dr. Connie Schmaljohn, NIAID Integrated Research Facility, Fort Detrick Frederick, MD, USA) (Iacono-Connors et al.,1996); and chicken polyclonal anti-NS3 (sequence CZRDIREFVSYASGRR) (Aves labs, Davis, CA, USA). All primary antibodies were employed at a 1:1000 dilution. The following secondary antibodies were used: Alexa Fluor 488 and Alexa Fluor 594 conjugated to either anti-mouse or anti-chicken specific IgG (Life Technologies). Polymerized actin in smooth muscle cells was labeled with Alexa Fluor 488 phalloidin (Life Technologies) and cell nuclei were stained with 1:100 dilution of Hoescht NucBlue stain (Life Technologies-Invitrogen) for 30 min. To stain the endoplasmic reticulum (ER), mouse monoclonal Dylight 488-conjugated protein disulfide isomerase 1D3 (PDI, Enzo Life Sciences Farmingdale, NY, USA) was used.

Images of the midguts were acquired with a Carl Zeiss LSM 880 confocal microscope driven by ZEN Black v2.3 software using a PlanFluor 10X/0.30 numerical aperture (NA), PlanApo 20X/0.80NA or a PlanApo 63X/1.40NA oil immersion objective.

2.7. Localization of virus particles using electron microscopy

After 72 hpi, infected and uninfected tick midguts were washed with PBS and fixed with 4% paraformaldehyde with 2.5% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer. Samples were processed in a Biowave microwave oven (Ted Pella, Inc Redding, CA USA). Briefly, samples were rinsed with 0.1 M sodium cacodylate buffer, fixed with 0.5% OsO4 + 0.8% K4Fe(CN)6 in a cycle of 2 min on, 2 min off run 5 times. Samples were rinsed with distilled water and the same cycle (5× [2 min on, 2 min off]) was performed with 1% aqueous tannic acid, rinsed with distilled water and the same cycle (5× (2 min on, 2 min off)) used with aqueous 1% uranyl acetate. Samples were then dehydrated in a graduated ethanol series, switched into propylene oxide, followed by microwave-assisted embedding into epon/araldite with polymerization at 60°C overnight. Seventy nanometer (70 nm) sections were examined at 80 keV using an HT7800 transmission electron microscope (Hitachi High Technologies Inc. Hitachinaka, Japan) and digital images were captured using a XR81-B camera (AMT).

3. Results

3.1. Viability of infected and uninfected tick midgut cultures

We began our studies by confirming viability of the ex vivo LGTV-infected and uninfected midgut cultures. Using an inverted microscope, both infected and uninfected midguts in resazurin salt-based reagent exhibited visible peristaltic contractions up to 144 h post dissection (Grabowski et al., 2017). The rate of these contractions diminished over time. Using optical density of resazurin salt-based reagent, we determined that both LGTV-infected and uninfected midguts were metabolically active for 216 h after dissection with peak metabolic activity observed between 72 hpd and 192 hpd (data not shown). Notably, there was no statistically significant difference between infected or uninfected groups, results consistent with previous work (Grabowski et al., 2017).

3.2. Quantification of virus production in infected tick midgut culture

We next quantified the production of LGTV in the cultures. Midgut cultures were exposed to LGTV and incubated for a total of 168 h (Fig. 1). Every 24 h, the supernatant was collected and replaced with fresh medium until the end of the incubation at which time all the samples were titrated on Vero cells. The amount of virus in the cultures increased by almost 1.5 log10 between 48 and 96 hpi (Fig. 1). No virus was detected in uninfected midgut cultures. The amount of virus in a well containing virus but no midgut showed a steep decline from 0 hpi to 24 hpi after which point no viable virus could be identified. The results showed the peak for virus production in the midgut cultures to be between 72 and 96 hpi. Based on these results of viability and viral production, we elected to do our subsequent analyses between 72 and 168 h (minimum and endpoint time).

Fig 1. Replication of LGTV in ex vivo cultures of midgut from adult female Ixodes scapularis ticks.

Fig 1.

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Midgut cultures were inoculated with 5×105 ffu of LGTV. Supernatant was collected at the indicated time points and the amount of virus was determined by immunofocus assay. Maximal virus production occurred between 72 and 96 h post infection. No viral production was observed in wells containing virus without the midgut (virus no midgut) or in midgut cultured without virus (data not shown).

3.3. Basic structure of uninfected and infected unfed tick midgut

Having confirmed that ex vivo cultures of midgut were viable and supported LGTV production, we examined the basic cytoarchitecture of the midgut of unfed I. scapularis. To appreciate the scale of the organs, Fig. 2a and b respectively show an uninfected, female adult I. scapularis tick and a dissected midgut.

Fig. 2. Basic structure of unfed female Ixodes scapularis tick midgut.

Fig. 2.

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a. An unfed, female I. scapularis tick on a millimeter ruler. b. A midgut dissected from a female I. scapularis tick. The central ventriculus (CV) and numerous diverticula (DV) are evident. scale bar = 100 μm. c. and d. are serial sections from a fixed and embedded freshly dissected midgut showing longitudinal profiles of a midgut diverticulum stained with either (c) hematoxylin and eosin (H&E) or (d) periodic acid Schiff stains. Denoted are lumen (LM), basal membrane (BM), smooth muscle cells (SMC), residual digestive cells (RDC) and the single layer of generative cell (GC) epithelium. e. and f. Sections from uninfected (e) or LGTV-infected (f) ex vivo midgut cultured for 72 h. stained with H&E. Scale bar c–f = 20 μm

Sections of uninfected midgut diverticula revealed a central lumen surrounded by an epithelial monolayer of cuboidal generative cells (Fig. 2c–e). In some sections of the midgut, large cells adjacent and interior to the epithelium were occasionally observed (Fig. 2 c–d); these resembled the residual digestive cells (RDC) from the previous instar reported in Rhipicephalus sanguineus (sensu stricto) midguts by Remedio et al. (2013). Although both are hard ticks, there may be differences between the midgut anatomy of R. sanguineus (a metastriate tick) and I. scapularis (a prostriate tick). The epithelium rested on a thin basement membrane, which was demonstrable by periodic acid–Schiff (PAS) stain (Fig. 2d). External to the basement membrane was a layer of flattened smooth muscle cells (Fig. 2c–f). The smooth muscle cells had small nuclei and were sparsely distributed compared to generative cells that had large nuclei. Smooth muscle cells resided on the hemocoelic side of the basement membrane (Fig. 2c–f). We observed no obvious major structural differences between the freshly dissected midguts (Fig. 2c–d) and the ex vivo cultured midguts (Fig. 2e).

Midguts cultured for 72 h either with LGTV or in control medium and stained with either H&E (Fig. 2e–f, respectively) or PAS (data not shown) did not reveal any obvious differences using standard light microscopy. In both situations, we observed an internal epithelium of cuboidal generative cells separated from a layer of smooth muscle cells separated by a definable basement membrane.

We next examined freshly dissected midguts by confocal microscopy after applying a phalloidin stain for polymerized actin, a characteristic of smooth muscle cells, and a stain for cell nuclei (Hoechst NucBlue). The pattern of phalloidin labeling revealed an annular network of smooth muscle cells (Fig. 3a–c), and the nuclei of these cells were apparent. Numerous nuclei not associated with the smooth muscle cells were associated with the generative cell epithelium.

Fig. 3. Confocal microscopy of freshly dissected uninfected midgut diverticula of female Ixodes scapularis.

Fig. 3.

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a. Nuclei of midgut cells stained with Hoescht NucBlue (magenta, note: in subsequent figures nuclear staining is pseudocolored in blue) identified cells that form the annular circumferential pattern and those distributed between the annular pattern. b. Staining with phalloidin (green) for polymerized actin revealed a regularly spaced annular network of smooth muscle cells, processes of which bridge the annular networks. c. Nuclear staining identified nuclei of the smooth muscle cellular network as well as the germinal cell epithelium, the cells of which do not stain as strongly with phalloidin. scale bar = 50 μm.

3.4. Localization of LGTV intermediates (dsRNA, NS3 and E proteins) in infected midgut cultures

To localize the site of LGTV infection, we employed confocal microscopy on cultures of midgut ≥72 hpi. We used three markers for TBFV infection in cells: the presence of envelope E protein, the double-stranded, replicative form RNA (dsRNA) and nonstructural protein 3 (NS3) (Le Breton et al., 2011; Offerdahl et al., 2012). In addition, cells supporting TBFV infection characteristically exhibit an expansion of membranes derived from the endoplasmic reticulum (ER), a phenomenon which can be visualized by staining for the ER marker protein disulfide isomerase (PDI) (Offerdahl et al., 2012; Rothan et al., 2019). Thus, in an effort to precisely identify which midgut cells were supporting LGTV infection, we stained for E, NS3, dsRNA and PDI, as well as using Hoechst (NucBlue) to label cell nuclei and phalloidin to stain the smooth muscle cells.

When infected midgut cultures were stained for these markers, we readily identified cells positive for E-protein (Fig. 4), dsRNA (Fig. 5) and NS3 (Fig. 5 and 6). Cells staining positive for ER expansion (increased PDI staining, Fig. 4) also stained for E protein. The cells bearing these hallmarks of LGTV infection were in a pattern that resembled that of the generative cells and smooth muscle cells (Fig. 5). As time after initial exposure to virus lengthened from 72 h (Fig. 5d) to 144 h (Fig. 6b), evidence of replication was noted in adjacent cells, suggesting possible cell-to-cell spread of virus. Confocal microscopy was used to acquire a z-series of the virus infected midgut at 144 hpi (Movie 1). Staining of LGTV dsRNA was visible in phalloidin-positive cells and generative cells in the acquired z-series. No cells positive for markers of virus infection were identified in uninfected cultures (Fig. 6). Taken together, these observations led us to conclude that LGTV infection in the midgut cultures was occurring in the generative cells and possibly on the smooth muscle cells of the luminal epithelium.

Fig. 4. Immunofluorescence of Ixodes scapularis midgut cultures.

Fig. 4.

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Dissected midguts from unfed female I. scapularis ticks were cultured either (a–c) uninfected or (d–f) infected with Langat virus (LGTV). After 72 h of incubation, the cultures were stained with the nuclear marker Hoescht NucBlue (a & d, blue), for LGTV E protein (b & e, red), and with PDI, a marker for endoplasmic reticulum (ER) derived membranes (c &f, green). A fraction of the generative cells in the infected midguts were positive for LGTV E protein, and exhibited increased staining with PDI, indicating an increase of ER derived membranes in the infected cells. Scale bar = 150 μm.

Fig. 5. Immunofluorescence of uninfected and infected Ixodes scapularis midgut stained for Langat virus (LGTV), dsRNA and viral NS3.

Fig. 5.

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Dissected midguts from unfed female I. scapularis ticks were cultured either uninfected (a–c) or infected with LGTV (d–f). After 72 h of incubation, the cultures were stained for LGTV NS3 protein (a & d, red), for double-stranded RNA (b & e, green), as well as the nuclear marker Hoechst NucBlue (c &f, blue). A merge of all the stains (c & f) revealed dsRNA and NS3 overlapping indicating that virus replication occurs in generative cells, and smooth muscle cells forming the annular circumferential pattern (see also Supplemental Fig. 1). Scale bar = 10 μm.

Fig. 6. Cytoarchitecture of female Ixodes scapularis midgut cultures showing polymerized actin.

Fig. 6.

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Dissected midguts from unfed adult I. scapularis were cultured either a. uninfected, or b. infected with Langat virus (LGTV). After 144 h of incubation, the cultures were stained for polymerized actin which is abundant in smooth muscle cells (phalloidin, green), for LGTV NS3 protein (red), and for nuclei with Hoechst NucBlue (blue). Scale bar = 50 μm.

3.5. Ultrastructure of infected and uninfected midgut cultures.

In the previous experiments, we confirmed earlier findings that LGTV replicated in midgut cultures from adult I. scapularis ticks and identified markers for virus replication in the epithelial layer of generative cells and smooth muscle cells. To more precisely characterize the infection and to seek virus particles, we turned to electron microscopy using 72 hpi midgut cultures.

Ultrastructural examination of dissected, uninfected midguts confirmed some of the results we obtained by light microscopy. Specifically, low magnification electron micrographs of a midgut diverticulum revealed a central lumen with a simple epithelium of generative cells, a basement membrane and an outer layer of flattened cells facing the hemocoelic compartment (Fig. 7a). Cells in the outermost layer contained densely packed, parallel fibers consistent with polymerized actin filaments, indicating that these were in fact smooth muscle cells (Fig. 7b). On the other side of the basement membrane was the single layer of generative epithelial cells, which presented a cuboidal profile (Fig. 7c) and a luminal brush border of microvilli (Fig. 7d).

Fig. 7. Electron microscopy of 72 hpi uninfected (a–d) and Langat virus (LGTV) infected midgut (e–h) cultures from female Ixodes scapularis.

Fig. 7.

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a–d uninfected midgut culture, a. A low magnification section of cultured diverticulum showing the lumen, the single layer of generative cell epithelium, the basal membrane and the smooth muscle cells (SMC). Scale bar is 12 μm. b. A higher magnification of the insert indicated in panel a, showing the nucleus and the cytoplasm of a generative cell, Basement membrane and the polymerized actin, a characteristic of a smooth muscle cells. Scale bar is 1.4 μm. c. Section in which the opposing generative cell layer occludes the midgut lumen, the SMC on the outer region is separated from the generative cell by the basement membrane. d. A higher magnification of the inset in panel c, showing the brush border microvilli and profiles of endoplasmic reticulum (ER). Scale bar is 1.2 μm e. A section of LGTV infected cultured tick midgut diverticulum showing a generative cell nucleus, lumen and SMC. Scale bar is 12 μm. f. a section of infected culture showing membranous expansion of the ER in a generative cell, the basement membrane that separate SMC and generative cell. g. a section of the infected diverticula showing a single layer of generative cells and the microvilli. Scale bar is 1.4μm. h. a higher magnification of the inset in panel g, showing microvilli of infected tick lumen and expanded ER membranes. scale bar is 1.2 μm

Denoted are L = lumen, MV = microvilli, M = mitochondria, SMC = smooth muscle cell, A = actin, BM = basement membrane, N = nucleus, GC = generative cell, ER = endoplasmic reticulum

However, the cells in which virus particles (Fig. 8) were found contained increased numbers of membranous structures (Fig. 7 e–h) as compared to cells from uninfected cultures (Fig. 7 a–d). These findings were consistent with the increased staining for PDI, reflecting expansion of ER-derived membranes (Rothan and Kumar, 2019) (Fig. 4). The smooth muscle cells did not contain unusual membrane structures in sections we surveyed, and their basement membrane remained intact (Fig. 7 e–h).

Fig. 8. Identification of virus particles in 72 hpi cultures of female Ixodes scapularis midgut infected with Langat virus (LGTV).

Fig. 8.

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a. Section of infected midgut culture (72 hpi) revealing cell with ER membrane expansion typical of a LGTV-infected cell. The nucleus is indicated. Scale bar is 1 μm. b. Magnified inset from (a) showing possible virus particles (black arrows), 46.5 μm in diameter, in a membranous structure and a single structure resembling a particle budding into a membranous compartment (*). Scale bar is 200 nm. c. Image showing the basolateral region of several adjacent cells, the adjacent basement membrane, and the nucleus and polymerized actin filaments of a smooth muscle cell (SMC). Expanded endoplasmic reticulum membranes and vacuoles containing dense inclusions, particles are observed. Scale bar is 800 nm. d. Magnified inset from (c) showing virus particles (black arrows) in the basolateral space and convoluted cell membranes adjacent to the basement membrane. The nucleus and polymerized actin filaments of the SMC are clearly evident at this magnification. Scale bar is 500 nm. e. Membranous structures of ER showing the viruses located in the basolateral region. Scale bar is 800 nm. f. magnified insert from e, showing the diameter of the virus particles to be 47.9 nm in diameter. Scale bar is 200 nm. Denoted are A = actin, BM = basement membrane, N = nucleus, ER = endoplasmic reticulum, V = vacuole, G = glycogen.

Virus particles were observed in the generative cells of the infected cultures (Fig. 8). The particles measured approximately ~47 nm (Fig. 8b), consistent with LGTV particles identified in previous studies (Zhou et al., 2018). In some instances, the particles were enclosed within membrane-bound vesicles (Fig. 8b) or appeared to be in the basolateral region between generative cells (Fig. 8). No particles were observed in smooth muscle cells that were in the same region of infected generative cells or in cells of uninfected midgut cultures.

4. Discussion

The current dogma indicates the first organ system that a pathogen encounters upon infecting a tick is the digestive system (Kleiboeker et al., 1999; Nuttall, 2009; Lejal et al., 2019). When an unfed tick takes an infected blood meal, the pathogen is carried in the blood through the pharynx and esophagus into the midgut lumen, thence to the branched midgut diverticula where digestion takes place. As a consequence, elucidating infection in the digestive tract will yield information on acquisition and subsequent dissemination in the tick.

In the current study, we examined LGTV infection of I. scapularis midgut cultures. The midgut cultures in our experiments remained viable and intact for at least 8 d, confirming previous work in our lab (Grabowski et al., 2017). There was no difference in overall viability between infected and uninfected cultures. There was clear de novo production of approximately 1.5 log10 virus and peak levels were noted between 72 and 96 hpi (Fig. 1). Light microscopy with standard histological stains did not reveal any obvious difference between infected and uninfected cultures.

Based on these initial observations, we have extended our use of ex vivo midgut cultures from unfed adult I. scapularis to define the basic biology and cytoarchitecture of I. scapularis midguts infected with LGTV, a representative TBFV, in a controlled setting. Examination by light, confocal and electron microscopy confirmed a single layer of cuboidal epithelial generative cells lining the midgut lumen (Fig. 2). Separated from the generative cell epithelium by a basement membrane, elongated smooth muscle cells form an annular network around the midgut diverticula. The smooth muscle cells were readily identified by the pattern of polymerized actin filaments, which stained with phalloidin and had characteristic ultrastructure (Fig. 3).

Using a combination of immunofluorescence and confocal microscopy, we concluded that the generative cells and smooth muscle cells of the midgut epithelium are the primary target cells for LGTV infection. These cells stained positive for viral E and NS3 proteins as well as for dsRNA, an additional marker for viral replication (Fig. 4). Furthermore, infected cells had increased staining for PDI reflecting an increase in cellular membranes derived from the endoplasmic reticulum, an observation consistent with LGTV infected cells (Offerdahl et al., 2012; Mitzel et al., 2007).

When we studied thin sections of infected midgut cultures, we observed collections of generative cells with virus particles in membrane bound compartments and in the basolateral spaces between generative cells (Fig. 8). These cells also showed an increase in small membranous structures reminiscent of other cell types infected with TBFV, which likely accounts for the increased PDI staining (Offerdahl et al., 2012).

In our electron microscopy studies, we were unable to locate virus particles or viral membrane expression in characteristic smooth muscle cells, suggesting that they were not targets for LGTV infection. In summary, light microscopy and electron microscopy data strongly suggest generative cells as the primary target for LGTV with smooth muscle cells likely to be infected as a secondary target resulting from infection procedure used in ex vivo cultures of unfed adult female I. scapularis midgut.

A tick feeding on a TBFV infected animal may acquire the virus as it is transported to the midgut as part of the blood meal. It is not completely surprising that the virus targets the generative cells of the midgut for initial replication. We did not observe input virus particles binding to luminal surface of these cells, but with suitable techniques, it would be interesting to look for a TBFV receptor on these cells (Rodrigues et al., 2019). We also observed that over the course of infection, there was an increase in the number of infected cells adjacent to each other, suggesting virus spread to adjacent cells.

Virus particles were identified in the basolateral region between generative cells, suggesting a route for virus dissemination. Based on current concepts of virus dissemination within the ticks, we can speculate that virus replicates in the generative cells and is shed from these cells into the basolateral spaces. From here, the virus could traverse the basement membrane and enter the hemocoel, where it might spread to other organs including the salivary glands. However, we did not observe virus particles on the exterior of the basement membrane and additional investigation will be required to substantiate this hypothesis.

Our findings address infection of the midgut in the absence of a bloodmeal in a very controlled, artificial system. Although we have identified generative cells as the primary target for virus infection, we have not precisely modeled the in vivo situation, as the virus was delivered to the external surface of the cultured midguts. It is still uncertain how infection proceeds during feeding because the generative cells enlarge and differentiate to become digestive cells, detaching into the lumen. Will infection be limited to the resting generative cells or will virus continue to replicate in these cells as they proliferate into actual digestive cells and actively process the blood meal? This study used a bathing technique to infect the organ cultures, in which the medium bathes the external surface of the midgut, but entry of virus from the medium into the midgut lumen via the ends of the midgut excised from the esophagus and hindgut could also be possible. It will be interesting to investigate whether the muscle cells will be infected and the region infected when the virus is introduced by the natural route through the esophagus and whether there will be ultrastructural evidence of virus spreading from the midgut into the hemocoel. Therefore, the obvious next steps are to examine infection of the midgut during the dynamic process of feeding and taking in a bloodmeal.

One way to emulate this process under controlled conditions would be to feed ticks on blood inoculated with a known amount of virus using an artificial membrane chamber system. Feeding ticks can then be removed at various stages of repletion and prepared for study. We have recently used an artificial membrane feeding system with a tick-borne bacterial pathogen (Stewart et al., 2022), and plan to use this technique with TBFV as well.

In conclusion, these studies of TBFV infection in ex vivo midgut cultures of unfed adult I. scapularis have identified generative cells as probable target cells for initial virus replication in the tick midgut. The work provides a foundation to examine infection of intact ticks and to investigate how infection proceeds in other life stages of the tick, including during the molt. The results will provide significant information about tick-borne pathogens and may inform antiviral countermeasures.

Supplementary Material

1

Movie 1. Identification of specific Ixodes scapularis midgut cell types infected with Langat virus at 144 h post infection using confocal z-series of sections. In the movie, the nuclei are stained blue with Hoechst stain, dsRNA is stained red and phalloidin is stained green; phalloidin staining primarily indicates smooth muscle cells. The infected cells, indicated by presence of dsRNA, appear to be localizing to both the phalloidin signal (smooth muscle cell layers, green) as well as to the generative cells located deeper and away from the numerous muscle cell nuclei on the surface of the midgut.

Download video file (6.4MB, avi)

Acknowledgements

We acknowledge the support by Intramural Research Program of Intramural Research Program of National Institute of Allergy and Infectious Diseases (NIAID). The authors thank Lisa Coburn of the Oklahoma State University (OSU), tick rearing facility for continuously supplying I. scapularis ticks that we needed in this study. We thank Dr. Connie Schmaljohn, NIAID, who had provided LGTV monoclonal antibodies. We thank the Kenya Medical Research Institute (KEMRI), specifically Dr. James Kimotho and Prof. Sam Kariuki for their support. Other members of Rocky Mountain Laboratory (RML) histopathology group participated in sectioning and providing histological images used in this research.

This work was funded and supported by the NIAID of the National Institutes of Health (NIH).

Footnotes

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Conflict of interest: None

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Associated Data

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Supplementary Materials

1

Movie 1. Identification of specific Ixodes scapularis midgut cell types infected with Langat virus at 144 h post infection using confocal z-series of sections. In the movie, the nuclei are stained blue with Hoechst stain, dsRNA is stained red and phalloidin is stained green; phalloidin staining primarily indicates smooth muscle cells. The infected cells, indicated by presence of dsRNA, appear to be localizing to both the phalloidin signal (smooth muscle cell layers, green) as well as to the generative cells located deeper and away from the numerous muscle cell nuclei on the surface of the midgut.

Download video file (6.4MB, avi)

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