VIRUS-LIKE, DOUBLE-STRANDED RNAS IN THE PARASITIC PROTOZOAN CRYPTOSPORIDIUM-PARVUM

We have discovered and analysed two novel, linear extrachromosomal dou ble-stranded RNAs (dsRNAs) within oocysts of major north Amercian isol ates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. The se dsRNAs were found to reside within the cytoplasm of sporozoites, an d were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and ea ch encoded one large open reading frame (ORF). The deduced protein seq uence of the larger dsRNA (L-dsRNA) had homology with viral RNA-depend ent RNA polymerases (RDRP), with more similarity to polymerases from f ungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activ ated c-June NH2 terminal protein kinases (JNK) from mammalian cells. A ttempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activit y was identified in crude extracts from C. parvum sporozoites and prod ucts of RNA polymerase activity derived in vitro were similar to the d sRNAs purified directly from the parasites.