Robust dengue virus infection in bat cells and limited innate immune responses coupled with positive serology from bats in IndoMalaya and Australasia

Abstract

Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.

Keywords: Bats; Disease; Flavivirus; Immunity; Pteropus; Zoonosis.

Fig. 1

Productive DENV infection in bat cells. a Single confocal z-plane images of Dengue virus (NGC) entry into PakiT03 cells after 2 h at a MOI of 10 (washed, then fixed). Cells were stained with biotinylated 3H5 antibody (PreM-E) followed by TSA-Alexa594 amplification, Alexa-488 conjugated 4G2 antibody (Env) (green) and Hoechst3342 (blue). Membrane stain (Phalloidin-647) was visualised to ensure internalisation of virus particles but not included in the image. b J2 (dsRNA) replication after 24 h was visualised at various MOI’s (as indicated) with the J2-dsRNA antibody-alexa 594 conjugated (red), Alexa-488 conjugated 4G2 antibody (Env) (green) and counterstained with Hoechst 3342 (blue), as previously. c Productive infection was measured by titrating viral supernatant from PakiT03 cells after 48 h of infection with an MOI of 1 for either DENV2 (NGC) or DENV2 (ST2), infected cells were labelled with 4G2 (green), anti-βactin (blue), J2 (dsRNA) (red) or membrane DiO label (magenta)—right panel, as previously. Supernatant was titrated onto BHK cells for 5 days in 1% methyl-cellulose overlays. Example images are shown for MOI of 0.1, 1 and n = 3, d qPCR validation of viral replication at various MOI’s after 48 h of infection in PakiT03 cells using primers for NS2a, normalised to housekeeping gene expression (SNRPD3). One representative experiment is shown for n = 3

Fig. 2

High production of Dengue virus and infectivity with multiple strains. a Viral supernatant from PakiT03 cells post-infection (48 h) with DENV2 NGC as per Fig. 1c titrated onto BHK cells for 4 days with a methyl-cellulose overlay showing infective particles being produced from PakiT03 cells at 24 h with an MOI of 0.1 or 1. b Supernatant from DENV1,3,4 infected PakiT03 cells after 72 h with an MOI of 1, titrated onto BHK cells and left for 5 days in 1% methyl-cellulose as described previously. c DENV2 NGC and ST strains produced in PakiT03 cells for 96 h (Fig. 1c) after an initial MOI of 0.1 and then titrated onto both BHK cells and PakiT03 cells showing Paki cells are highly productive for DENV

Fig. 3

Infectivity of multiple cell types, species and immune cells by Dengue virus. a Single-plane confocal images of PaLu (Pteropus alecto lung epithelial cells, left panel) and EsLu (Eonycteris spelaea lung epithelial cells) cells after 48 h infection with an MOI of 1 and labelled with Actin (blue), dsRNA (magenta), 3H5-594 (red) and 4G2 (green), as previously. b Primary splenocytes infected with DENV2 (NGC) at an MOI of 0.1 overnight for ~ 16 h, fixed and stained as previous (antibodies as indicated) for Cynopterus brachyotis (left panel) and Pteropus alecto (right panel). c Bone-marrow-derived dendritic cells from Pteropus alecto (FLT3-ligand, as previously published, left) infected with DENV2 (NGC) for 24 h with an MOI of 0.1 and labelled as per Fig. 2 (direct labelling without TSA amplification). Image includes DiC brightfield images and merged image as last picture. Similarly, for primary Pteropus alecto lung (mixed) cell suspension (right). d as per c for 48 h in PaSPT.01 (SV40T-immortalised spleen-derived fibroblast cells). Example images for one experiment are shown for n = 3 in a–d

Fig. 4

Limited immune response triggered by Dengue virus. a RT-qPCR of Paki cells infected with DENV2 NGC at an MOI of 0.1 or 1 (as indicated), NBV MOI of 1 or transfected with polyI:C (1 µg/ml) for 24 h. Relative expression is shown for IFIT1 and IFNβ relative to uninfected and normalised against SNRPD3 housekeeping gene. b RT-qPCR of several genes for DENV2 NGC infection at 24 h with an MOI of 0.1, as per 1D. Relative expression to control, normalised against SNRPD3 is shown for IFIT1, BST2, IFNα3, MX1, MX2, IL-1β, TNF and CCL4. c Relative expression as per a but at 48 h for IFNβ, IFNα3 and IFIT1 with DENV2 NGC at MOI of 0.1 or 1 and NBV MOI of 1. A representative experiment is shown for each, n = 3. d NanoString expression data from 200 ng RNA on a custom bat panel (expressed as average fold change relative to uninfected PakiT03 control cells) is shown as averages (n = 3) for a 24 h infection at an MOI of 1 for DENV2 (NGC), DENV2 (ST), or Melaka virus (MelV). Scale is from 0–10-fold change, as indicated. N = 4 each. e As for d from E. spelaea primary splenocytes infected with DENV2 NGC for 16 h or treated with ODN2216 (TLR9 agonist) or polyIC (TLR3/RIG-I) for 5 h. Data expressed as fold change (Log₁₀) with 1.5× cutoff (red line)

Fig. 5

Quantitative proteomics of dengue infection reveals limited immune activation. a Pathway analysis (via reactome.org) of normalised LFQ protein counts from PakiT03 cells treated with polyI:C for 24 h (transfected) in biological triplicate. Graph overlay shows the percentage of genes from the pathway detected, the Z score p value as Log₁₀ and the p = 0.05 threshold line (as indicated in the legend). b As per a for treatment with NBV at an MOI of 0.1 for 48 h. c As per a with DENV2 NGC at 48 h with an MOI of 0.1 d Transcription factor enrichment for pathways in a and e Transcription factors enriched for pathways in b

Fig. 6

Serological results for Dengue Virus in Singaporean bats. a DENV2 luciferase immunoprecipitation system (LIPS) results for DENV2 NS1 antigen on 106 bat sera from Singapore (Supplemental Table S3). Score is colour coded as indicated, colour is recorded for all hits above the background threshold on each run (mean + 3 SD). Positive control sera were human and macacque positive sera (protein A/G capture). b Peptide mapping results of peptides identified by phage-display library screening (VirScan) with a Z score > 10 from sera used in a displaying DENV1 unique peptide results mapped to DENV1 amino acid sequence. Colour indicates individual bat number. c as per b displaying DENV2 unique peptide results mapped to DENV2 amino acid sequence. d as per b displaying DENV4 unique peptide results mapped to DENV4 amino acid sequence. Each colour represents an individual bat (indicated in legend). Arrows represent mapped peptides. Asterisk represents peptides partially overlapping with ZIKV (not in Virscan 2.0). Protein name as indicated

Fig. 7

Serology results of DENV2 positive bat sera from Australia. a Peptide Z scores for DENV2-unique peptides that have a Z scores > 10 in any of the Pteropus alecto bats. The cumulative Z scores of each peptide (individual Z scores for overlapping strain-specific peptides are added) is displayed per bat, bat number as indicated (different colour for each bat). Each dot represents one peptide. The Z scores cut-off line of 10 is displayed. Graph is in two segments as certain peptides have a very significant Z scores (multiple counts for the same peptide). Too many peptides were detected to map all. b Mapping of unique peptides (colored arrows) for one example individual bat (PA23) displaying all unique DE–NV2 peptides relevant to the amino acid sequence for DENV2