Expression of coxsackievirus B3 capsid proteins in Escherichia coli and generation of virus-specific antisera.

Subgenomic fragments of cloned infectious coxsackievirus B3 (CVB3) cDNA up to the size of the complete coding sequence of the viral polyprotein were inserted into the prokaryotic expression vector pPLc24 and expressed in Escherichia coli. Fusion proteins, containing 54 amino acids of MS2 replicase at their amino terminus followed by different parts of the CVB3 structural proteins, were expressed from several constructs. The expression product of a plasmid encoding the capsid proteins VP4, VP2, and the amino-terminal part of VP3 was obtained in high amounts. However, primary expression products containing the complete viral capsid precursor VP4-VP1 were completely degraded, indicating the presence of domains downstream from VP3 that are accessible to E. coli proteases. This finding is consistent with the observation that the structural intact expression product of the separately subcloned VP1 gene is also extremely unstable and consequently obtained only in low amounts. Two fusion proteins of non-overlapping parts of the viral structural proteins containing VP4, VP2, and VP3 or VP1, respectively, were isolated and used for the generation of antisera in rabbits. The antisera obtained recognize distinct CVB3 structural proteins in infected cell cultures as well as from purified CVB3 preparations. In addition, significant cross-reactivity of the described antisera with the corresponding structural proteins of other enteroviruses was observed, indicating that these antisera provide a valuable tool for an improved broad spectrum diagnosis of enteroviral infections.

A search for the presence of the enteroviral capsid protein VP1 in pancreases of patients with type 1 (insulin-dependent) diabetes and pancreases and hearts of infants who died of coxsackieviral myocarditis.

Using an antiserum raised to a recombinant coxsackie virus B 3 capsid protein, VP1, an immunocytochemical technique was developed which was capable of detecting the presence of all coxsackie B viruses in formalin fixed paraffin embedded infected tissue culture cells. This technique was tested on autopsy heart and pancreas from 21 patients who were thought to have died of acute coxsackievirus B myocarditis. Cardiac myocytes were positive for the VP1 protein in 12 of 20 cases where the heart was available for study. Insulitis was present in the pancreas in seven of these cases and in all seven islet endocrine cells containing VP1 were found. VP1 was only rarely found in exocrine pancreas. In heart and pancreas, cells shown to contain VP1 usually showed signs of necrosis. Autopsy pancreases from 88 patients who had died at clinical presentation of Type 1 (insulin-dependent) diabetes mellitus showed no evidence of the presence of VP1. The continuing destruction of insulin-secreting B cells seen at the time of death in the diabetic pancreas is unlikely to be due to a direct cytopathic effect of a coxsackie B virus. However, this study does not exclude the possibility that a persistent infection of B cells by a defective enterovirus may result in their destruction by an autoimmune mechanism.