Mechanisms of Coxsackievirus B3 dissemination and persistence in the Host : consequences for neurodegenerative disease
- Author(s): Robinson, Scott Michael
- Robinson, Scott Michael
- et al.
Enterovirus infections are quite common in the United States, causing approximately 10-15 million symptomatic infections every year. Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, has been shown to be responsible for viral myocarditis, aseptic meningitis, and pancreatitis in humans. The mechanism responsible for CVB3 persistence in the host (chronic / sporadic viral replication or viral latency) remains unclear. Therefore, we utilized the inherent long-term instability of our recombinant coxsackievirus expressing the enhanced green fluorescent protein (eGFP-CVB3) in two mouse models (BALB/c and B cell knockout mice) to determine the mechanism of virus persistence in the heart. These studies may help to determine if antiviral treatment during persistent infection may be productive in patients suffering from chronic viral myocarditis. Also, we genetically engineered a unique molecular marker - "Fluorescent timer" protein - within our infectious viral clone and isolated a high-titer viral stock (Timer-CVB3) following transfection in HeLa cells. "Fluorescent timer" protein changes its fluorescence from green to red over time. Timer-CVB3-infected HeLa cells, neural progenitor and stem cells (NPSCs), or C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours, as determined by fluorescence microscopy or flow cytometric analysis. The slow change in fluorescence for Timer-CVB3- infected HeLa cells could be interrupted by fixation, suggesting that the fluorophore associated with "fluorescent timer" protein was stabilized by formalin cross-linking reactions. Induction of a type I interferon response reduced the progression of cell-to-cell infection in both HeLa cells and NPSCs. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discreet virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. Unexpectedly, Timer-CVB3 infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of numerous extracellular microvesicles containing viral material which may represent a novel route of dissemination. Through the use of this novel recombinant virus, we hope to gain a better understanding of CVB3 tropism, alterations in host cell organization, and virus dissemination within the host. Finally, we wished to determine if a previous CVB3 infection during the neonatal period may provide a "fertile field" for the acceleration or exacerbation of subsequent neurodegenerative and autoimmune disease in the aging host. We hypothesized that a previous neurotropic infection which persists in the host may aggravate a subsequent neurological disease in a mouse model of Alzheimer's disease, and separately in a mouse model of multiple sclerosis. The induction of key inflammatory and cytokine genes during persistent infection, and the nature of the neurological disease may be important determinants for enhancement or reduction of disease following neonatal CVB3 infection