Materials and Methods

Virus. The virus used in this study was isolated in rhesus renal cell cultures from myocardial tissue of an infant with encephalohepatomyocarditis and focal necrosis and inflammation of the pancreas (5). It was identified as Coxsackie virus group B, type 4 (CB4; Edwards strain) by neutralization tests with specific antiserum in monke~ renal cell cultures. The isolate was passed once intraperitoneally in the suckling mouse, then inoculated into monkey renal cell cultures. This culture fluid was stored in 1958 in sealed ampuls at-55~ C until recently, when it was passed once in HeLa cells to check its infectivity. Virus from the HeLa cell cultures was then passed three times sequentially by intraperitoneal inoculation into outbred adult male and female CD-1 mice (Charles River Breeding Laboratories, Wilmington, Mass.). Each successive inoculum consisted of a cell-free suspension of homogenized pancreatic tissue taken 3 days after the previous inoculation. A virus pool was then prepared in HeLa cells and stored at-70cC as a cell-free suspension. This constituted the virus inoculum for the present study. The titer of this working pool was 2 x 10 H plaque-forming units (PFU}/ml for HeLa cells. Preparation of tissues for viral assay and techniques for viral quantitation have previously been described (6). Animals. Three genetic variants of the inbred murine strain C57BL/Ks (The Jackson Laboratory, Bar Harbor, Maine) were used. These differed only in the presence of the unit autosomal recessive gene for diabetes, db, located on chromosome 4 (linkage group VIII). They consisted of (a) the parental background strain, C57BL/Ks+/+, which has no reported predisposition to diabetes;(b) mice heterozygous for the diabetic gene, C57BL/Ks db/+, which are phenotypically and physiologically similar to the+/+ variant; and (c) mice homozygous for the diabetic gene,