Fig 1.
Human primary cardiac cells are susceptible to infection by diverse arboviruses.
Human primary (A) cardiac fibroblasts, (B) aortic smooth muscle cells, (C) cardiac myocytes, and (D) cardiac microvascular endothelial cells were infected with MAYV, ZIKV, RVFV MP-12, or LACV at a MOI of 0.1. Supernatant was collected at 0, 6, 24, 48, and 72 hpi, and viral titers were quantified by plaque assay. Data points represent the mean of two independent experiments (n=4), with error bars representing the SEM. The limit of detection is indicated by the gray shaded area.
Fig 2.
Differential growth of alphaviruses in endothelial cells.
(A) hCMECs were infected with CHIKV-IOL, CHIKV-181/25, MAYV, SINV, or RRV at a MOI of 0.1. Viral supernatants were collected at 48 hpi and infectious virus was quantified by plaque assay. 5-8 independent experiments (n = 14-23) (B) hPMECs or (C) hBMECs were infected with each virus at a MOI of 0.1. Supernatant was collected 48 hpi and viral titers were quantified via plaque assay. 3-6 independent experiments (n = 6-15) (D) At 48 hpi, cells were fixed and stained with an anti-CHIKV-capsid antibody and DAPI. All data represent the mean with error bars representing the SD. The limit of detection (LOD) is indicated by the gray shaded area.
Fig 3.
CHIKV IOL restriction occurs at viral entry and egress in cardiac endothelial cells.
A fusion-from-without-assay was performed in hCMECs. CHIKV-IOL-ZsGreen or MAYV was bound to hCMECs at a MOI of 5 at 4°C for one hour. After incubation, buffer at a pH of 5.2 or 7.5 was added and incubated at 37°C for 3 mins, replaced with complete media and incubated for 2 days. Supernatant was collected at 0, 1, and 2 dpi, and cells were fixed and visualized by high-content microscopy 2 dpi. (A) CHIKV infection quantified as the percent of CHIKV-IOL-ZsGreen positive cells as compared to the total number of cell nuclei as indicated by DAPI staining by high-content microscopy. (B) Representative microscopy images showing uninfected (DMEM) and CHIKV-IOL-ZsGreen (green channel) infected cells at pH 5.2 and 7.5 with DAPI-stained cell nuclei (blue channel). (C) Viral titers from supernatant quantified via plaque assay. Data represents the mean of at least n = 3 independent trials in technical duplicate or triplicate, with error bars showing the SEM and the limit of detection indicated by the gray shaded area. Statistical significance was found via a Kruskal-Wallis test with Dunn’s multiple comparisons test (A) with p-values representing *p<0.05.
Fig 4.
Primary cardiac endothelial cells are susceptible and permissive to CHIKV IOL in absence of JAK/STAT signaling.
(A, B) HCMECs were pre-treated with 5 µM ruxolitinib or mock-treated for 2 days, then infected with CHIKV IOL ZsGreen at a MOI of 0.1 for 1 hour. Post-infection, cells were incubated with media supplemented with or without 5 µM ruxolitinib and fixed for high-content microscopy at 5 dpi. (A) Quantification of percent infected cells as measured by CHIKV-IOL-ZsGreen positive cells relative to number of total cells measured by DAPI staining. (B) Representative images from a CX7 high-content microscope showing cell nuclei stained with DAPI (blue channel) and CHIKV-IOL-ZsGreen infected cells (green channel). (C) Dot plot showing normalized expression levels of IFITM1, IFITM2, IFITM3, BST2, and CD74 in cardiac smooth muscle cells (CSMC), cardiac fibroblasts (CF), cardiac myocytes (CM), cardiac microvascular endothelial cells (CMEC), and pulmonary endothelial cells (PMEC) using the Tabula Sapiens dataset. (D and E) Representative images of western blots visualizing actin, IFITM2, and IFITM3 protein levels. (D) HCMECs were treated with 5 µM ruxolitinib for 2, 4, or 6 days or mock-treated. Cells were collected in laemmli buffer, proteins were separated by SDS-PAGE and visualized by immunoblotting. (E, F) HCMECs, hBMECs, hPMECs, and hCFs were treated with IFNβ or mock-treated for 24 hours. Cells were harvested and proteins analyzed as described above. (E) Values indicate relative intensity of expression as compared to hCMEC basal expression, with SD. (F) Quantified IFITM2 and IFITM3 expression after 24 hours IFNβ treatment. Data represents the mean of n = 3 independent trials in technical triplicate (A, B) with error bars showing the SEM and the limit of detection indicated by the gray shaded area. Western blots represent at least n = 2 independent trials (D-F). Statistical significance (A) was found by a Kruskal-Wallis test with multiple comparisons, with p-values representing *p<0.05.
Fig 5.
MAYV infection attenuates the type-I IFN response in cardiac endothelial cells.
(A-C) hCMECs were either pre-treated with Poly(I:C) or mock-transfected for 2 hours and then mock-infected with DMEM, or infected with CHIKV-IOL, CHIKV-181/25, MAYV, corresponding heat-inactivated controls (HI), or mock-infected (DMEM) at a MOI of 1 for 1 hour. Supernatants and cell monolayers were collected at 0 and 24 hpi. (A) Viral titers were quantified by plaque assay and (B) intracellular viral genomes were quantified by RT-qPCR. (C) ISG15 gene expression quantified relative to DMEM for each timepoint by RT-qPCR. Data represents the mean of n = 3 independent trials in technical duplicate with error bars representing the SEM and the limit of detection indicated by the gray shaded area. Statistical significance was found by (A - C) two-way ANOVA with multiple comparisons, with statistics in (A) representing comparisons to CHIKV-IOL at 24 hpi. P-values represent ****p<0.0001.