Identification And Characterization Of DsRNA Virus In Crytposporidium Parvum

Identification and Characterization of virus-liked dsRNA in C~yptosprodium parvumPostgraduate student:Chen Jian BaoDirected by Prof.Zhang Xi ChenIn this paper we provide evidence for the presence of two unusually dsRNA in sporozoites of C.parvum isolated from China,and describe the cloning and sequencing of both dsRNAs elements. RNA-dependent RNA polymerase activity in the cytoplasm of sporozoites are shown and each segment may exist as an unencapsidated dsRNA that has the coding capacity for protein.Agarose gel electrophoretic analysis of RNase A (0.01 ~i g/mI.0. hi g/ml.1.0 i?glml.l0 ii g/ml) treated nucleic acid from 2.0 X ~ Cparvuin revealed two distinct bands with estimated sizes of 1.7 X 1 O3nt and 1.3 X 1 0~nt. and with approximately identical intensities after staining with ethidiurn hromide.The two pairs of primer were designed and synthesized according to the sequence of virus-like double-stranded RNAs in the parasitic protozoan C.parvuni discovered by Nikolai V . Khramtsov (1997). By RT-PCR amplified the nucleic acid samples from Cryptosporidia. the result showed that virus-like dsRNAs were contained in the nucleic acid from C.pcwvum. neither bands were found in nucleic acid extracted from (.~. niuri~ C. Baileyi, C. ine/cugridis .The products of RT-PCR wre purified and ligated into pMDI8-T vector ,then cloned and sequenced .The sequences of cDNAs representing both dsRNAs genome were compared with the sequences published in GenBank. homology with the sequences having been piihlisheJ was 98% and 96% respectively . L-dsRNA and S-dsRNA each CDdCd one large open reading frame (ORF). L-dsRNA has an ORF encoding \irUs-IikJ RDRP. Cytoslic from Cparvztn? were assayed for R\i\ p01 ?ier~sc act1v~ty by following the incorporation [32P]-UTP in the presence of ~I té‹—picin.RDRP activity was shown. and was DNA independent.Vira]pariclec were not detected h~ TEM, suggesting that the virus is not ~i~xi with protein.