Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis

Figure 1. Structural characteristics of VP1 771–790 of CVB3 showing their relationship to the binding region between viral canyon and CAR, and an approach to create mutant viruses. (a) Ribbon and surface models of CVB3 VPs. Using the published pentamer structure of CVB3 (PDB: 1COV)16,17, all four structural proteins of CVB3 (VP1 [red], VP2 [blue], VP3 [turquoise], and VP4 [purple]) were localized in the protomer, as illustrated in the ribbon (left panel) and surface (right panel) models. (b) Localization of VP1 771–790 in the VP1 region. The amino acid residues of VP1 771–790, depicted in balls between two protomers (green and red) are shown in the top panel, and their positions are indicated in yellow. The surface model (bottom panel) shows the exposed amino acid residues that may potentially interact with the CAR; their positions are shown in yellow in the inset. The interactions of VP1 771–790 with CAR were analyzed using the BIOVIA Discovery Studio v4.0 (https://www.discngine.com/discovery-studio). (c) Depiction of putative amino acid residues that may interact with CAR. After localizing the residues within the VP1 region in relation to CAR as described above, the putative residues…

Figure 2. Recovery of infectious viruses from mutant clones. (a) Detection of infectivity by mutant viruses. Vero cells grown in monolayers on coverslips in 12-well plates were cultured in medium alone or infected with EMEM containing wt CVB3, pBRCVB3, Mt 2, Mt 3, Mt 4, Mt 8, or Mt 10, then incubated for 12 h at 37 °C. Cells were fixed and incubated with anti-CVB3 serum, and a secondary FITC-conjugated IgG was added to detect viral antigens. The coverslips were washed and mounted, then examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60×. (b) Comparison of viral titers. Monolayers of Vero cells were grown to 80–90% confluence in 12-well plates. Triplicate wells were infected at an MOI of 0.1 or 3.0 with passage 2 viruses of wt CVB3, pBRCVB3, Mt 2, Mt 3, Mt 4, Mt 8, or Mt 10. After noting the CPE, the respective viral supernatants were harvested, and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3). Mann–Whitney test and Kruskal–Wallis test were used to determine significance between wt CVB3/pBRCVB3 and other mutant viruses at different…

Figure 3. Disease phenotypes induced by various mutant viruses. (a) Experimental design. A/J mice were infected with wt CVB3, pBRCVB3, Mt 2, Mt 3, Mt 4, Mt 8, and Mt 10 viruses, with saline recipients as controls. At termination on day 21, heart and pancreas were collected for histology. (b) Clinical phenotypes. Body weights were taken every one to two days until termination and compared between groups (left panel); mortalities, if any, were noted to calculate survival rates (right panel). (c) Histopathology. Hearts and pancreata collected at termination on day 21 were processed for standard histology to evaluate inflammatory changes. Representative sections of hearts (top panel) and pancreata (bottom panel) from saline (negative control), wt CVB3 and pBRCVB3 (positive controls), and Mt 10 virus groups are shown. Solid and empty arrows represent infiltrations of MNCs in the heart and pancreatic sections, respectively. Circles indicate necrotic areas in the pancreatic sections. Magnification, 20×; scale bars, 20 µm. Data sets obtained from two individual experiments, each involving n = 4 to 5 mice, are shown. Two-way ANOVA with a Sidak’s post-test was used to compare body weight changes in…

Figure 4. Mt 10 virus offers protection against wt CVB3 infection in challenge studies. (a) Experimental design. Two groups of mice each were administered saline or Mt 10 virus on day 0 after serum was collected. After 14 days, serum was collected, and one group from each was challenged with CVB3. Experiments were terminated 21 days later, and serum and tissues were collected for in vitro experimentation. (b) Clinical phenotypes. Body weights (left panel) and survival rates (right panel) between different groups are shown. (c) Histopathology. Hearts and pancreata collected from the indicated groups and processed by H and E staining to evaluate inflammatory changes. Representative heart (top panel) and pancreatic (bottom panel) sections are shown. Heart sections from wt CVB3 group shows infiltrations (solid arrow) and mineralization, necrosis, and fibrosis (empty arrow). Pancreatic sections from wt CVB3 group had infiltrations (solid arrow) as opposed to normal sections in the saline, Mt 10 virus, and Mt 10 virus/wt CVB3-challenged groups. Magnification, 20×; scale bars, 20 µm. Data sets obtained from three individual experiments, each involving n = 3 to 8 mice, are shown. Two-way ANOVA…

Figure 5. Mt 10 virus induces neutralizing antibodies of IgG isotypes. (a) Virus neutralization assay. Sera were obtained from saline and Mt 10 virus groups on days 14 and 35, and after diluting serially, samples were incubated with wt CVB3. The mixtures were transferred to plates containing Vero cells, and incubation was continued for four days to calculate the percentage neutralization based on CPE. Data from n = 6 samples, each representing a pool of sera from 3 to 5 mice, are shown. (b) Detection of VP1-reactive antibodies. Serum samples collected from the above groups were diluted (1:150) and added in duplicates to high-binding plates previously coated with CVB3-VP1 or KLH (control). After adding HRP-conjugated goat anti-mouse total Ig, IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, and IgE as detection antibodies, reactions were stopped. Plates were read at 450 nm to obtain the OD values. Mean ± SEM values obtained from n = 6 samples, each representing 3 to 5 mice, are shown. Mann–Whitney test was used to determine significance between groups. **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 6. Mt 10 virus induces the generation of antigen-specific, T cells capable of producing predominantly, IFN-γ. (a) Antigen-specificity. Groups of mice were injected with saline or Mt 10 virus, and 35 days later, lymphocytes were prepared. Cells were stimulated with VP1 681–700 or 721–740 and maintained in IL-2 medium. Cells harvested between days, 7–10 post-stimulation were stained with the indicated IAk/tetramers and dextramers, anti-CD4 and 7-AAD. After acquiring the cells by flow cytometry, tetramer and dextramer + cells were analyzed in the live (7-AAD¯) CD4 subset using FlowJo software. RNase 43–56, control. Representative flow cytometric dot-plots and the mean ± SEM values from three individual experiments, each involving n = 3 to 8 mice are indicated. (b) Cytokine analysis. Supernatants harvested from the above cultures on day 3 post-stimulation were analyzed for cytokines by LEGENDplex cytokine bead array as described in the methods section. Mean ± SEM values obtained from three individual experiments, each involving n = 3 to 8 mice are indicated. Unpaired Student’s t-test (two-tailed) was used to determine significance between groups [for panels (a,b)]. *p ≤ 0.05, **p ≤…

Figure 7. Analysis of anti-viral cytokines and chemokines in response to Mt 10 virus. Groups of mice were administered with saline or Mt 10 virus, and sera were collected periodically from day 0 to day 35. Samples were processed for a panel of indicated anti-viral cytokines and chemokines by LEGENDplex cytokine bead array as described in the methods section, and their levels were compared. Mean ± SEM values obtained from three individual experiments, each involving n = 3 to 8 mice are indicated. Unpaired Student’s t-test (two-tailed) was used to determine significance between groups. *p ≤ 0.05, ***p ≤ 0.001, and ****p ≤ 0.0001.

Figure 8. Mice injected with Mt 10 virus did not reveal autoimmunity or cardiac injury. (a) Proliferative response to Myhc-α 334–352. Groups of mice were administered saline or Mt 10 virus, and after 14 days, they were challenged with or without CVB3. Three weeks later, lymphocytes were prepared, and cells were stimulated with or without Myhc-α 334–352 or control (RNase 43–56) for 2 days. After pulsing with 3[H] thymidine for 16 h, proliferative responses were measured as cpm. Mean ± SEM values obtained from three individual experiments, each involving n = 3 to 8 mice, are shown. (b) cTnI detection. Undiluted sera obtained from the above groups were added in duplicates to pre-coated, cTnI ELISA strip plates. After incubation and a series of washes, HRP-conjugated rabbit anti-mouse antibody was added followed by TMB substrate and stop solution. Plates were read at 450 nm to obtain OD values, and concentrations of unknown samples were determined using the standard curve. Mean ± SEM values representing 6 samples per group, each containing n = 3 to 5 mice, are shown. Unpaired Student’s t-test (two-tailed) was used to determine significance between groups [for panels (a,b)]. *p ≤ 0.05, and ***p…

Figure 9. Recipients of Mt 10 virus challenged with the wt CVB3 virus did not reveal viral RNA. Groups of mice were administered saline or Mt 10 virus, and 14 days later, they were challenged with or without wt CVB3. Three weeks post-challenge, hearts and pancreata were collected, total RNA was extracted, and CVB3 RNA was estimated by quantitative PCR targeting VP1 sequences, as described in the methods section. After normalizing the expression levels viral RNA relative to GAPDH, 2−(∆∆Ct) values were calculated. Mean ± SEM values representing 4 samples per group, each containing n = 3 to 5 mice, are shown. Mann–Whitney test was used to determine significance between groups. **p ≤ 0.01.