Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

<div><p>Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.</p></div

Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons.

<p>LGTV (4 MOI) was used to infect 1 x 10⁵ murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10⁷ cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10⁵ naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed <i>t</i> test is shown. Representative data is shown from at least three independent experiments.</p

Transmission of LGTV through infectious exosome to naïve cells is clathrin dependent.

<p>(A) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. TMTC indicates “too many to count”. (B) QRT-PCR analysis for the LGTV loads in fresh N2a (1 x 10 ⁵) cells at 24 h p.i., infected with exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24, 48 and 72 h p.i. (C) Mouse N2a cells showing infection kinetics with WNV (MOI 5) at different times (24, 48, 72 h) p.i., WNV <i>E</i> gene transcript levels were normalized to mouse beta-actin. (D) Relative loads of WNV in infected N2a cell-derived exosomes collected at 24, 48, 72 h p.i. LGTV loads (72 h p.i.) in N2a cells treated with either 4G2 antibody (5 μg for 4 h) (E) or Pitstop-2 clathrin inhibitor (F) (treated 30 μM for 15 min at 37°C) and infected with LGTV-containing (72 h p.i.) N2a cell-derived exosomes is shown. Infected but untreated samples serve as control in (E). Infected but DMSO-treated serve as control in (F). LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail <i>t</i> test is shown. Representative data is shown from at least three independent experiments.</p

Breeding preference ratio of the vector larvae of Chitwan district.

<p>Breeding preference ratio of the vector larvae of Chitwan district.</p

Endothelial cell-derived exosomes mediate transmission of LGTV RNA from bEnd.3 cells to N2a neuronal cells.

<p>The bEnd.3 cells (1 x 10⁵) were infected with 6 MOI of LGTV. QRT-PCR analysis showing total LGTV loads (A) or levels of LGTV positive-sense strand or negative-sense strand (B) in exosomes isolated from bEnd.3 cells at different time points (24, 48, 72, 96 and 120 h p.i.). (C) Viral loads were determined after 24 h p.i. by the presence of LGTV in N2a cells (1 x 10 ⁵) treated with LGTV containing exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24 or 48 h p.i. of bEnd.3 cells. (D) Viral loads at 48 h p.i. of N2a cells (plated in lower chamber) as determined by the presence of LGTV in a transwell assay performed with 1x 10⁵ of bEnd.3 cells treated with 20 μl brain endothelial cell-derived exosomes (in upper chamber) for 4 h in the presence or absence of exosome inhibitor (5 μM) (in upper chamber) or infected with LGTV laboratory virus stocks is shown. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail <i>t</i> test is shown. Representative data is shown from three independent experiments.</p

Clinical features of seropositive and seronegative cases.

<p>Clinical features of seropositive and seronegative cases.</p

Age wise distribution of seropositive dengue cases.

<p>Age wise distribution of seropositive dengue cases.</p

Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line.

<p>(A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10⁷). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10⁵) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10⁶ uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.</p

Arthropod exosomes mediate transmission of infectious LGTV RNA and proteins from tick to human cells.

<p>(A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 1; 72 h p.i.), ISE6 tick cells. Scale bar indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) tick cell-derived exosomes. Number of exosomes analyzed were n = 138 (uninfected) and n = 200 (infected) groups. (D) Comparison of exosome numbers per cryo-EM image from uninfected (n = 27) and infected (n = 14) groups is shown. (E) DG-Exos showing presence of LGTV envelope [E]-protein in fractions 1–6. QRT-PCR analysis showing total LGTV loads (F) and levels of LGTV positive-sense strand or negative-sense strand (G) in exosomes isolated from tick cells at 72 h (p.i.), Uninfected cells serve as control. LGTV transcript levels were normalized to tick beta-actin. (H) Immunoblotting analysis showing detection of LGTV E- glycoprotein in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) tick cells at 72 h (p.i.). Immunoblot detecting NS1 in both tick-cell derived exosomes and total cell lysates using monoclonal anti-Langat virus NS1 protein from LGTV-infected (MOI 1; 72 h p.i.) is also shown. HSP70 levels indicate enrichment of arthropod exosomal marker in exosome fractions. Uninfected samples and total protein profiles serve as controls. Tick cells (5 x 10⁶) were infected with 1 MOI of LGTV in both QRT-PCR and immunoblotting analysis. I) Native-PAGE followed by immunoblotting analysis showing presence of E-protein from LGTV-infected (MOI 1; 72 h p.i.) or uninfected tick cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated samples held on ice. Coomassie stained gel showing total protein profiles serve as loading control (I). (J) Antibody-beads binding assay performed on LGTV infected (MOI 1) tick cell-derived exosomes (collected from tick cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in Langat-E protein loads between 4G2 or isotype and untreated samples. (K) Quantitative assessment of number of plaques in exosomal and supernatant fractions is shown. TMTC indicates “too many to count”. (L) Viral re-infection kinetics as determined by the presence of total LGTV loads in HaCaT cells (1 x 10⁵ cells) at different time points treated with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 72 h p.i. LGTV-infected tick cells are shown. (M) Viral loads at 48 h p.i. was determined by the presence of LGTV in a transwell assay performed with 1 x 10⁵ tick cells (in upper chamber) or 1 x 10⁵ HaCaT cells (in lower chamber) treated with tick exosome fraction (20 μl, in upper chamber) for 4 h in the presence or absence of 5 μM exosome inhibitor GW4869. Tick cells infected with LGTV laboratory stocks were used as controls. LGTV transcript levels were normalized to human beta-actin in (L) and (M). P value determined by Student’s two-tail <i>t</i> test is shown. Representative data is shown from three independent experiments.</p

Treatment of primary cultures of cortical neurons with exosome inhibitor affects LGTV infection.

<p>(A) 1 x 10⁵ cortical neuronal cells was infected with LGTV (4 MOI). QRT-PCR analysis showing levels of LGTV in exosomes isolated from cortical neuronal cells at 48 h p.i. in the presence of exosome-inhibitor at different concentrations (1, 10, 20 μM). Cortical neuronal cells were pre-treated with exosomes inhibitor for 4 h followed by infection with LGTV. Exosomes isolated from cortical neuronal cells treated with DMSO served as control. (B) Levels of LGTV in fresh cortical neuronal cells at 48 h p.i., infected by treatment with exosomes (20 μl) isolated from control or inhibitor-treated mouse cortical neuronal cells is shown. UI indicates uninfected cells that serve as control. LGTV transcript levels were normalized to mouse beta-actin. (C) Plaque assays performed with different dilutions (1:10, 1:100, 1:1000) of exosomes fractions isolated from control DMSO-treated or inhibitor-treated cortical neuronal cells are shown. Ruler at the top determines the scale for the plaque assay. Representative images are shown from two independent experiments. (D) Quantitative assessment of the number of plaques from (C) is shown. TMTC indicates “too many to count”. P value determined by Student’s t test is shown. Representative data in A and B is shown from three independent experiments.</p