New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Abstract Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A pro and 3C pro ), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A pro cause direct damage to the infected heart and tools to investigate 2A pro and 3C pro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A pro and 3C pro ; Two monoclonal 2A pro antibodies and one 3C pro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C pro antibody detects all of the EV species B (EV-B) viruses tested and that the 2A pro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A pro and 3C pro , and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.