Fig 1: EGFR treatment enhances viral infection and dampens the interferon response to ZIKV and HSV-1.a Primary immortalised astrocyte cells were treated with 2 µM AG-1478 (EGFR inhibitor) for 16 h prior to infection with ZIKV and HSV-1. Cells were stained with Bodipy (493/503) to visualise LDs (green) and DAPI to visualise the cell nuclei (blue). ZIKV RNA was detected using anti-3G1.1 and 2G4 dsRNA antibodies and HSV-1 was detected using the anti-HSV-1 antibody (Abcam, ab9533), both viral proteins shown with red staining. Images are a representation of n = 3 independent experiments. b, c RT-qPCR was utilised to evaluate IFN-ß, IFN-? and viperin mRNA expression at 8, 24 and 48 hpi for both ZIKV at MOI 0.1 or HSV-1 at MOI 0.01. RT-PCRs were performed to detect viral nucleic acid levels of (d) ZIKV and (e) HSV-1. In (b–e) error bars, mean values ± SEM, P values were determined by unpaired two-tailed Student’s t test with a Holm-Sidak correction for multiple comparisons for 2 or more groups (greater than 300 cells; n = 3 biological replicates). Stimulated cells were statistically compared with their respective mock controls, ns = not significant. f IFN protein levels from these experiments were analysed via ELISA for IFN- ß and IFN-? protein at 16 h post infection. Error bars, mean values ± SEM, P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n = 3 biological replicates), nd = not detected. Source data are provided as a Source Data file.
Fig 2: Lipid Droplets accumulate in response to IAV, ZIKV and HSV-1 infections.a Human THP-1 monocytes were infected with two different strains of influenza- PR8 and X-31 at an MOI 5 for 8 h, Scale bars, 50 µm. b Primary immortalised astrocyte cells were infected with either the ZIKV (MR766 strain), DENV (DENV2) or HSV-1 (KOS strain) at MOI 5 for 8 h. All cells were stained with Bodipy (493/503) to visualise LDs (green) and DAPI to visualise the cell nuclei (blue). IAV was detected with a aNS2 antibody (1:1000), ZIKV and DENV RNA was detected using an anti-3G1.1 and 2G4 dsRNA antibodies (in combination, used neat) and HSV-1 was detected using the anti-HSV-1 antibody (Abcam, ab9533), all shown in red staining. Scale bars, 50 µm. c LD numbers were analysed using ImageJ analysis software. Error bars, mean values ± SEM, P values were determined by unpaired two-tailed Student’s t test with a Holm-Sidak correction for multiple comparisons (n = 2 biological replicates). Stimulated cells were statistically compared with their respective mock controls. d C57BL/6 mice were either mock infected or infected with 104 PFU of IAV for 24 or 72 h prior to removal of both lung lobes for immunofluorescence analysis of LDs via Bodipy (493/503) staining (green). DAPI was utilised to visualise the cell nuclei (blue), scale bars, 500 µm. e 1-day old BALB/c pups were either mock infected or infected with 800 PFU of DENV-2 (MON601) for 2 or 4 days prior to removal of pup heads for immunofluorescence analysis of LDs via Bodipy (493/503) staining (green) in the brain and eye. DAPI was utilised to visualise the cell nuclei (blue) and DENV RNA was detected using anti-3G1.1 and 2G4 dsRNA antibodies (in combination). Scale bars, 500 µm, n = 3 mice. Source data are provided as a Source Data file.
Fig 3: Increasing cellular LD numbers enhances IFN responses to restrict ZIKV and HSV-1 viral replication.a Primary immortalised astrocyte cells were treated with 500 µM oleic acid (OA) for 16 h prior to infection with ZIKV (MR766 strain) at MOI 0.1 or HSV-1 (KOS strain) at MOI 0.01 for 8 h. Cells were stained with Bodipy (493/503) to visualise LDs (green) and DAPI to visualise the cell nuclei (blue). ZIKV RNA was detected using an anti-3G1.1 and 2G4 dsRNA antibodies and HSV-1 was detected using the anti-HSV-1 antibody (Abcam, ab9533), both viral proteins shown with red staining. Images are a representation of n = 3 independent experiments. RT-qPCR was utilised to evaluate IFN-ß, IFN-? and viperin mRNA expression at 8, 24 and 48 hpi post (b) ZIKV or (c) HSV-1 infection (MOI 0.1). Primary immortalised astrocyte cells were treated with 500 µM oleic acid for 16 h prior to infection with (d) ZIKV at a MOI 0.1 or (e) HSV-1 at an MOI 0.1, and RT-qPCR was utilised to evaluate viral replication at 6, 24 and 48 hpi. In (b–e) error bars, mean values ± SEM, P values were determined by unpaired two-tailed Student’s t test with a Holm-Sidak correction for multiple comparisons for 2 or more groups (greater than 300 cells; n = 3 biological replicates). Stimulated cells were statistically compared with their respective mock controls, ns = not significant. f At 16 h post-infection secreted IFN protein levels from these experiments were analysed with ELISA plates for IFN- ß and IFN-? protein. Error bars, mean values ± SEM, P values were determined by two-way ANOVA post-hoc pairwise comparisons with Bonferroni correction (n = 3 biological replicates). Source data are provided as a Source Data file.
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