Viral replication analysis of Japanese encephalitis virus and discovery of RT-primer-independent amplification of viral cDNA
| Autor: | Chia-Chen Weng, 翁嘉珍 |
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| Rok vydání: | 2007 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 95 Japanese encephalitis virus (JEV) possesses a single positive- stranded RNA genome of 10,976 nucleotides in length. Previous studies in our laboratory have reported the abundant accumulation of a 3'-terminal 521- to 523-nucleotides genome fragment named small RNA, representing the highly conserved region of the 3'-untranslated region, in JEV-infected cells. To address possible functions of the small RNA during viral replication, unit-length (i.e. 523-nt) sense- and antisense-small RNA were separately transfected into JEV-infected cells and the effects on plus- and minus-strand accumulation were measured by strand-specific Northern hybridization. Transfecting of small RNA appeared not to affect plus- or minus-strand viral RNA accumulation, while transfecting of anti-sense small RNA caused an inhibition of plus-strand RNA but not minus-strand RNA accumulation. The high molecular weight viral RNAs (relative 2X genome size) observed earlier and in this study were demonstrated not a dimer or a multimer of viral genome by serial RT-PCR analyses. Through these analyses, an interesting phenomenon that JEV specific cDNA could be amplified by RT-PCR without primer added during RT step was found. These results suggested that JEV genome, presumably the 3'-long stable hairpin, might serve as a primer during RT step and JEV specific cDNA subsequently were amplified through PCR. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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