Cellular drivers of long-term CVB3 persistence in chronic myocarditis

Abstract Background Myocarditis exhibits a highly variable clinical course, ranging from full recovery to the development of chronic heart failure. The Coxsackievirus B3 (CVB3)-infected mouse model is a well-established system for studying viral myocarditis. Elimination of the virus is crucial for the regeneration of the heart with minimal consequences; persistent infections, however, are associated with increased fibrosis and long-term functional impairment. Furthermore, current findings suggest that the cellular localisation of viral persistence significantly influences the course of the disease. However, the specific cell types that harbour CVB3 during the chronic stage and thereby maintain viral persistence remain insufficiently defined. Purpose This study aims to clarify which cardiac cell types are capable of generating functional viral particles, thus driving acute infection, and to identify those that act as reservoirs driving long-term viral persistence. Methods To investigate the ability of different cell types to replicate CVB3, primary B cells, T cells and macrophages were infected for 6 and 24 h. Virus load and virus replication was determined using qPCR. Furthermore, cell culture supernatants were used to determine the production of infectious virus particles via TCID50 assays. To determine in which cell types the virus persists, viral RNA was stained together with cell type specific markers on tissue sections from mice with virus persistence (16 days post-infection). Results Quantification of viral RNA from 6 to 24 h p.i. revealed marked cell type-specific difference in CVB3 permissiveness: cardiomyocytes showed the strongest increase (300-fold), followed by B cells (2.24-fold; p=0.0027) and a modest rise in T cells (1.17-fold), whereas macrophages showed a decline (0.24-fold; p=0.0223), indicating a non-permissive phenotype. Strand-specific PCR confirmed low-level CVB3 negative-strand synthesis in B cells (2.14-fold) and minimal levels in T cells (1-fold), while macrophages showed no detectable replication intermediates. Despite intracellular viral RNA and detection of CVB3 negative-strand, no infectious virus particles were detected in the supernatants of any immune cell. First results of in situ hybridization of heart tissue showed that CVB3 was mainly localized in immune cell infiltrates. Summery Our findings show that, although viral RNA slightly increases from 6 to 24 hours in B and T cells, these immune cells support only minimal or even non-productive CVB3 replication, while macrophages do not replicate the virus entirely. In contrast, cardiomyocytes exhibit robust virus amplification, confirming them as the dominant permissive cell type. In situ hybridization at 16 days p.i. further revealed that persistent CVB3 is localized within immune cell infiltrates rather than cardiomyocytes. Ongoing analyses are now characterizing these infiltrates in detail to define the cellular reservoirs sustaining long-term viral persistence.