Refining a preclinical model of viral myocarditis in accordance with biotech standards

FIGURE 1. Experimental design and pilot study characterizing the activity of heart‐passaged CVB3. (A) Scheme outlining experimental workflow. (B) Timeline of pilot study indicating treatment and assessment timepoints. (C) Plot of body temperature change relative to day 0, showing no significant differences across all groups. (D) Quantitation of body weight change compared to day 0. Virus (1×)‐treated mice show progressive reduction in body weight. (E) Decreased blood glucose levels in virus (1×)‐treated animals. Graph shows non‐fasting blood glucose levels at day −7 (basal) and day 9 (final). (F) Validation of inflammation upon virus injection. Graphs show histopathologic scores of inflammation derived from H&E and MT staining, and quantitation of inflamed tissue area using VisioPharm's AI‐based analysis. Virus (1×)‐treated animals show highest degree of inflammation. (G, H) Analysis of systolic heart function, showing LVEF derived from 4D‐imaging (G) and endocardial longitudinal strain analysis (H). Groups included vehicle, heart homogenate, virus 0.1× (8.8 ± 2.1 PFU), and virus 1× (88 ± 21 PFU) (n = 4–5 per group). Data are presented as mean ± SD. Statistical significance is indicated as…

FIGURE 2. Diet refinement improves the weight loss burden of CVB3‐infected mice. (A) Scheme outlining timeline and assessment points of the characterization study. Four cohorts were monitored with different final timepoints (day 7, 14, 35, and 56). (B) Graphs showing body weight change of all cohorts over time. Purified amino acid diet led to attenuated body weight loss in virus treated animals. (C) Blood glucose analysis at baseline and final timepoints across all cohorts. Diet refinement improved blood glucose levels. Groups included vehicle, heart homogenate, and virus 0.5× (44 ± 10.5 PFU), 1× (88 ± 21 PFU), and 2× (176 ± 42 PFU) (n = 4–6 per group and timepoint). Data are presented as mean ± SD. d, day; SD, standard deviation.

FIGURE 3. Histopathological characterization of the CVB3‐induced myocarditis model. (A) Histopathologic scoring of inflammation (H&E) and fibrosis (PSR) across all cohorts. Inflammation severity correlated with viral dose and peaked at day 7. Fibrosis remained mild across groups and peaked at days 35 and 56. (B) Representative H&E‐stained heart sections from day 7, comparing vehicle and virus (2×)‐treated animals. Insets highlight inflammatory foci in virus‐treated animals. (C) Representative PSR‐stained heart sections from day 56, comparing vehicle and virus (2×)‐treated animals. Insets of virus‐treated animals show reactive interstitial fibrosis. (D) Confirmation of pathology findings using VisioPharm's AI‐based analysis. Graphs show total inflammation foci area, total nuclei count, and fibrotic area. Groups included vehicle, heart homogenate, virus 0.5× (44 ± 10.5 PFU), 1× (88 ± 21 PFU), and 2× (176 ± 42 PFU) (n = 4–6 per group and timepoint). Data are presented as mean ± SD or as boxplots showing the median, interquartile range, and whiskers extending to the minimum and maximum values. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001, based on…

FIGURE 4. Comparison of imaging modalities for assessing left ventricular systolic function. (A, B) Representative echocardiographic images of SAX M‐mode (A) and PLAX B‐mode (B) from vehicle‐ and virus (2×)‐treated animals at day 56. Virus‐treated animals show reduced ventricular contraction. Dashed lines indicate endocardial tracings. (C) Surface renderings of reconstructions from 4D imaging. Panels show superimposed end‐diastolic and end‐systolic volumes from anterior and posterior views of vehicle‐ and virus (2×)‐treated animals at day 56. (D–F) Quantitation of LVEF using M‐mode (D), B‐mode (E), and 4D imaging (F). 4D imaging yields the most consistent measurements and shows reduced LVEF in virus‐treated animals at day 56. (G) Quantitation of cardiac output derived from 4D imaging, shows reduction in virus‐treated groups. Groups included vehicle and virus 0.5× (44 ± 10.5 PFU), 1× (88 ± 21 PFU), and 2× (176 ± 42 PFU) (n = 4–6 per group and timepoint). Data are presented as boxplots showing the median, interquartile range, and whiskers extending to the minimum and maximum values. Statistical significance is indicated as *p < 0.05, and **p < 0.01 based on comparisons against the vehicle…

FIGURE 5. Global and segmental analysis of longitudinal, circumferential, and radial strain. (A, B) Representative echocardiographic images from vehicle‐ and virus (2×)‐treated animals at day 56 showing peak systolic and diastolic strain. (A) Longitudinal strain in PLAX B‐mode images. (B) Circumferential and radial strain in SAX B‐mode images at basal, mid, and apical levels. Arrow length corresponds to the magnitude of strain. (C) Quantitation of global endocardial longitudinal, circumferential, and radial strain. Global longitudinal strain is severely reduced in all virus‐treated groups at days 35 and 56. (D) Bullseye maps showing segmental analysis of longitudinal, circumferential, and radial strain of vehicle‐ and virus (2×)‐treated animals at day 56. Apical segments show localized dysfunction in circumferential and radial strain in virus‐treated animals. Groups included vehicle and virus 0.5× (44 ± 10.5 PFU), 1× (88 ± 21 PFU), and 2× (176 ± 42 PFU) (n = 4–6 per group and timepoint). Data are presented as mean ± SD in bullseye maps or as boxplots showing the median, interquartile range, and whiskers extending to the minimum and maximum values. Statistical significance is indicated as…