https://doi.org/10.5061/dryad.n2z34tn48

This dataset includes the data results from: (i)In vivo, using DBA/2J mice for infection with Coxsackievirus B3 and viral RNA transfection, and (ii) In vitro analysis, using human cardiomyocytes cells. The dataset includes the data results obtained by : RT-qPCR, ELISA, histological scoring, plaque forming units count, and quantification of 5'non-coding Coxsackievirus B3 RNA (by microelectrophoresis with 2100 Bioanalyzer Instrument). 

Description of the data and file structure

Our data raw are structured as follows:

Figure 1_Data.xlsx: Data representing the results summarized in Figure 1 (see Supplemental information figures): these data describe the dynamics of 5’terminally deleted Coxsackievirus B3 RNA population’s emergence in heart mice (DBA/2J) and their correlation with type I interferon beta level. The figure 1a represent the data of viral load (copies per µg of total nucleic acid extracted) quantified by qRT-PCR (quantitative Reverse Transcriptase-Polymerase Chain reaction). Figure 1b, the data of viral titer (number of PFU/mg of total protein) in heart tissue of mice at different time post-infected measured by plaque forming units assay (PFU). Data of Figure 1d show the viral load (copies per µg of total nucleic acid extracted) of different 5’ terminally deleted Coxsackievirus B3 RNA forms measured by microelectrophoresis with 2100 Bioanalyzer Instrument. n/a in fig1d means “no data”. Data of figure 1 e and f represent the results of measured cytokine (ng or pg per total protein) (IL-10: Interleukin-10, MCP-1: Monocyte chemoattractant protein-1, TNF-alpha: Tumor necrosis Factor alpha, IFN-beta: Interferon beta) by ELISA (enzyme-linked immunosorbent assay). To evaluate the association between total viral RNA and/or the 5’ terminally deleted Coxsackievirus B3 RNA forms and interferon beta, GraphPad Prism 8 (Prism) was used to measure the Linear regression curves and Spearman R coefficient of correlation.

Figure 2_Data.xlsx: To evaluate the implication of these 5’ terminally deleted Coxsackievirus B3 RNA forms in induction of acute and chronic myocarditis in mice, homogenized heart from mice infected by CVB3 at different time post infection were inoculated in DBA/2J mice. Heart were than harvested at different time post-inoculation and viral loads (copies per µg of total nucleic acid extracted) quantified by qRT-PCR (quantitative Reverse Transcriptase-Polymerase Chain reaction) (Fig 2b) and viral titers (number of PFU/mg of total protein) measured by plaque forming units assay (PFU) (Fig2c). Interferon beta (protein (pg/mg of total protein) and mRNA (fold increase)) was measured at different time post-inoculation by ELISA (enzyme-linked immunosorbent assay) or RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chain reaction) (Figure 2 e and f).

Figure 3_Data.xlsx: in order to investigate the direct impact of 5’ terminally deleted Coxsackievirus B3 RNA forms on interferon beta production and induction of acute myocarditis, we transfected mice with Coxsackievirus B3 deleted in 5’non-coding region by 8, 21 or 50 nucleotides. After 8 hours post-transfection, hearts were harvested, and we measured viral loads (copies per µg of total nucleic acid extracted ) by qRT-PCR (quantitative Reverse Transcriptase-Polymerase Chain reaction) (Figure 3a) and interferon beta and cytokines (ng og pg per total protein) (MCP-1: Monocyte chemoattractant protein-1, TNF-alpha: Tumor necrosis Factor alpha) production by ELISA (enzyme-linked immunosorbent assay) (figure 3b and c). Using a specific ELISA (enzyme-linked immunosorbent assay) for double strand RNA (dsRNA), we measured the dynamic of 5’ terminally deleted Coxsackievirus B3 RNA forms replication. Data from figure 3e summarize the histological scoring in the heart tissue of mice transfected by different 5’ terminally deleted Coxsackievirus B3 RNA forms at 7 days post-transfection (figure 3f:  Histology (HES) and immunohistochemistry for viral protein 1 of mouse hearts at 7 DPI). n/a in fig3e means “no data”

Figure 4_Data.xlsx: then, to investigate the direct impact of  5’ terminally deleted Coxsackievirus B3 RNA forms on interferon beta pathway activation in human cardiomyocytes, cultured human cardiomyocytes were transfected by synthetic Coxsackievirus RNA forms, full length or 5’terminally deleted RNA (5’TD: deleted in 5’non-coding region from 8, 15, 21 or 50 nucleotides) and then we quantified viral RNA load (cp/µg of nucleic acids extracted) in cells at 8hours post-transfection (figure 4b) and interferon beta (IFN-beta) production (pg/ml) in the supernatant of cells at 8 hours post-transfection by  ELISA (enzyme-linked immunosorbent assay). By Western blot assay we evaluated the impact of the transfection of 5’terminally deleted RNA forms on cell functionality (measuring eIF4G cleavage: Eukaryotic translation initiation factor 4 G) and innate immune activation by measuring the phosphorylation of IRF3 (Interferon regulatory factor 3).

Figure 5_Data.xlsx: finally, since 5’TD CVB3 RNA forms proportions can impair IFN-β signaling pathway activation in our mouse myocarditis model, we postulated that the in vivo induction of high IFN-β production levels could significantly decrease CVB3-5’TD RNA forms loads in hearts of CVB3/28-infected mice. To this, mice were infected or not with coxsackievirus B3 and then treated or not with Poly(I:C) (known to induce type I IFN response in vitro and in vivo through innate immune sensors activation pathway). The data represented in figure 5a show the survival (0) and death (1) curve and follow-up of body weight (% to basal weight) of CVB3/28-infected mice compared to infected Poly(I:C) HMW-treated mice. GraphPad Prism 8 (Prism) was used to draw the curves. HMW: High Molecular Weight. Figure 5b show the semi-quantification of inflammation and necrosis (from histology results) in treated group and untreated group of mice at 14 DPI (days post-infection). We then measured the impact of the treatment on interferon beta production (by RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chain reaction) expressed by relative fold-change of Ifn-b mRNA and ELISA (enzyme-linked immunosorbent assay) by pg/mg of total protein (figure 5 c and d), viral titer measured by plaque forming units (PFU) number of  PFU/mg of total protein, and finally on 5’terminally deleted RNA forms emergence and maintenance in the heart tissues, measured by qRT-PCR (quantitative Reverse Transcriptase-Polymerase Chain reaction), cp/µg of total nucleic acids extracted (figure 5 f and g).

Supplementary Figure S1-Data.xlsx: these data represent the supplement information of figure 1 in which we described the percentage of 5’terminally deleted RNA forms according to 5’terminal deletion position on 5’non-coding Coxsackievirus B3 genome (figure S1a) detected by RACE-PCR (Rapid amplification of cDNA ends-PCR) in the heart of infected mice at indicated time post-infection. data from figure S1 b represent interferon beta level (by ng/mg of total protein) in the heart tissue of mice infected by different viruses (Mengovirus and Coxsackievirus B3) or stimulated by Poly(I:C).UI: Uninfected. n/a means "no data".

Supplementary Figure S2-Data.xlsx: data show the level of cytokines (interleukin 10, MCP-1: Monocyte chemoattractant protein-1 and TNF-alpha: Tumor necrosis Factor alpha) by ng or pg per mg of total protein extracted from heart tissue of mice infected with Coxsackievirus B3 at indicated time (2, 3- and 7-days post-infection) and from uninfected mice (figure S2a). we then assessed the association between the level of these cytokines with the percentage of deleted viral RNA forms; GraphPad Prism 8 (Prism) was used to measure the Linear regression curves and Spearman R coefficient of correlation (figure S2b).

Supplementary Figure S3-Data.xlsx: this dataset present the result of viral load (copies per µg of total nucleic acid extracted) in the heat of mice inoculated with homogenized heart of mice infected with coxsackievirus B3 at different time post-infection, and the detection of 5’terminal deletion position on 5’non-coding Coxsackievirus B3 genome (copies per µg of total nucleic acid extracted) (figure S1a) detected by RACE-PCR (Rapid amplification of cDNA ends-PCR) (figure S3 a and b). figure S3d represent the data of histological scoring (numbers) of inflammatory and necrosis infiltrates in the heart tissues of mice inoculated with homogenized heart tissue of mice first infected with Coxsackievirus B3 at different time post-infection.

Table 1_Data.xlsx: dataset from table 1 represent Viral properties of detected CVB3 populations at 3, 14 and 28 post-infection.  Data represent cp/µg of nucleic acids extracted of (+)/(-) or single Strand/double strand viral RNA Ratio: positive/negative or single/double-strands viral RNA Ratio. Viral infectivity (PFU/ml) PFU: Plaque forming unit. Encapsidation of viral RNA, demonstrated by a viral entry (cp/µg of nucleic acids extracted) challenge with or without proteinase K, was maintained at all times post infection. ND: non-detectable.

Analyses

We built different graphs for figures using GraphPad Prism 8 (Prism).