The generation of a novel viral expression system for the in-vitro modelling of viral myocarditis

For a number of RNA viral diseases, including SARS-CoV-2, Coxsackie and other RNA viral infections, there are still no effective vaccines and/or treatments available. Therefore, improving the diagnosis and treatment of viral infections is of great importance for medical research. In order to conduct efficient research, valid disease models that closely mimic human pathology are essential. To date, three approaches have been used to study viral infections: Via human samples, animal models and human cell lines infected in vitro with virus particles. The possibility of obtaining samples from humans is often limited, as tissue samples can only be taken from patients as biopsies or from deceased persons. Therefore, a model system with human tissue that is more easily accessible is highly desirable and would solve this problem. With the development of (induced) pluripotent stem cells, which can proliferate virtually indefinitely and potentially differentiate into any type of tissue, a large number of organ-specific cells can be obtained. However, to date, in vitro experiments have been performed using infectious viral particles, which require an appropriately equipped laboratory (often biosafety level 2 or higher) and are potentially harmful to the experimenters and limit the capabilities of standard laboratories authorised to perform drug screening. To counteract this limitation and enable research in normally equipped biosafety level 1 laboratories, we have developed a human induced pluripotent stem cell line (hiPSC) that can express viral genes but does not produce infectious viral particles. As a model system for RNA viruses, we used the well-studied Coxsackie virus B3 (CVB3) from the group of RNA enteroviruses as an infection reagent. CVB3 has been shown to cause myocarditis, meningoencephalitis, insulitis, diarrhoea and insulin-dependent type 1 diabetes. We stably transfected hiPSCs with a construct of the CVB3 viral genome carrying two mutations in the part of the viral genome encoding the viral capsid proteins. The CVB3 genome, including the two mutations hereafter referred to as CVB3ΔVP0, prevents competent viral capsid formation, rendering the system non-infectious and thus allowing downgrading from biosafety level 2 to 1. This approach is a huge advantage for experimenters and allows most laboratories to work with RNA viruses in a very controlled manner. In addition, the construct has a doxycycline-dependent Tet-on promoter and a fluorescent Venus reporter that allow the duration and extent of viral infection to be controlled and monitored by adjusting the timing of application and concentration of doxycycline administration. Differentiation of the generated CVB3-expressing hiPSC line into cardiomyocytes with subsequent CVB3 induction resulted in reduced contractility, prolonged QT intervals, increased mitochondrial ROS production and loss of cellular membrane integrity. This new disease model provides the scientific community with a new tool to study enteroviral infections under controlled conditions.