| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 93 === 英文摘要
Defective interfering (DI) RNAs are deletion mutants of viral genomes that are known in many cases to contribute to persistent infection and modification of viral pathogenesis. Previous studies in our laboratory demonstrated the generation of DI RNAs in mosquito (C6/36) cells infected with Japanese encephalitis virus (JEV) that are associated with persistent infection. In this study, I confirm and extend the observation by sequencing more of DI RNAs from different cell passages and virion in both mosquito cells (C6/36) and mammalian cells (BHK-21). Sequence analyses of deleted region from 40 clones revealed 22 kinds of different deletions. These DI RNAs all contained in-frame deletions ranging from 2,256 to 2,775 nucleotides (nts) covering the region of the E gene and some flanking C, prM and NS1 gene sequences. One of the DI RNA clones with 8703 nts identified from persistently-infected BHK-21 cells was completely sequenced and shown deletions at nt 610-2283, 14 point mutations (3 silent mutations and 11 missense mutations), and one base insertion at the 3’-untranslated region. When synthetic capped DI RNA transcripts of this clone were transfected into BHK-21 cells, replication was observed, as demonstrated by RT-PCR showing increasing of plus-strand and minus-strand RNA. Furthermore, DI RNAs identified from 35th passages of persistently-infected mosquito cells all contained deletion of nt 541-3015 and also showed replicability by transfecting of in vitro transcripts into mosquito cells. Taken together, these results indicated that the identified DI RNAs all contained in-frame deletions and at least two of these DI RNAs are self-replicating molecules.
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