In vitro studies with primary human pancreatic islets suggest that several enterovirus serotypes are able to infect and replicate in beta cells. Some enterovirus strains are highly cytolytic in vitro whereas others show virus replication with no apparent islet destruction. The capability to induce islet destruction is determined only partially by the virus serotype, since strain specific differences have been detected within some serotypes including echovirus 9 (E-9). In this study, the viral genetic factors determining the outcome of islet infection (i.e., destructive vs. benign) were investigated by constructing parallel infectious clones of lytic E-9-DM strain that was isolated from a small child at the clinical onset of type 1 diabetes. The capabilities of these clone-derived viruses to induce islet destruction were monitored and the lytic potential of clones was modified by site-directed mutagenesis. The lytic capabilities of these clone-derived viruses in human pancreatic islets were modified by a single amino acid substitution (T81A) in the capsid protein VP1. The data presented outline the importance of amino acid point mutations in the pathogenetic process leading to islet necrosis. However, although the amino acid substitution (T81A) modifies the lytic capabilities of E-9-DM strain-derived microvariant strains, it is likely that additional viral genetic determinants of pancreatic islet pathogenicity exist in other E-9 strains.