登革二型病毒PL046感染性質體之建構

Abstract

登革病毒為全長10.7 kb的正單股形RNA病毒，屬黃質病毒科（Flaviviridae）黃質病毒屬（Genus Flavivirus）。目前登革病毒感染性質體（infectious cDNA clones or infectious clone）的研究，皆是利用原核生物啟動子調控病毒基因表現，需再經過體外轉錄（in vitro transcription）及接上5’端cap的加工過程，用加工後的病毒RNA轉染至宿主細胞後產生病毒顆粒。根據同屬的西尼羅病毒（West Nile virus）研究指出，以巨細胞病毒（cytomegalovirus；CMV）啟動子替代原核生物啟動子後，可以直接由轉染感染性質體的方式獲得病毒。本研究亦致力以此新穎的模式，建構本土登革二型病毒PL046之感染性質體。 先前由實驗室前人賴建□，將登革二型病毒PL046全長基因組建構於pcDNA3質體後，成功建構出由巨細胞病毒啟動子所調控之感染性質體 pcDNA3/DV2F。但進一步將其轉染至宿主細胞（BHK-21）後，未能獲得病毒顆粒。為了找出可能的原因，將病毒基因序列進行全長定序分析，發現其中產生了數個無義突變（nonsense mutation）與轉譯閱讀框架位移突變（frame shift mutation）。此外，在建構感染性質體的時候，5’端及3’端非轉譯區（3’-untranslated region；3’-UTR）外側所殘留的多餘序列，亦可能影響病毒複製。因此，本研究嘗試將突變的序列修正，再以聚合□鏈反應（polymerase chain reaction；PCR）及核醣□（ribozyme）等方法，修飾出完整的病毒基因5’端與3’端非轉譯區。期望修正後的感染性質體能產出病毒顆粒，為本土登革病毒後續研究提供幫助。 結果顯示，修正後的登革二型病毒PL046感染性質體轉染至宿主細胞後，可以偵測到病毒RNA的表現，但仍然無法檢測出病毒顆粒的產生。

Dengue virus (genus Flavivirus, family Flaviviridae) is a single-stranded and positive-sense RNA virus with a 10.7 kb genome. Generally, most of the dengue virus infectious clones (or infectious cDNA clones) are under the control of prokaryotic promoter. To generate the virus RNA, the process requires in vitro transcription and addition of 5’-cap. According to the study of West Nile virus (Flavivirus), the virus could be recovered from the plasmid DNA-transfected cells directly while prokaryotic promoter was replaced by cytomegalovirus (CMV) promoter. Based on this concept, the objective of the present study was to employ this strategy to construct the DNA-launched dengue virus type 2 PL046 strain (Taiwan local isolated) infectious clones. Previously, the whole genome of dengue virus type 2 PL046 strain was successfully constructed in pcDNA3 plasmid by laboratory predecessors. This infectious clone was named pcDNA3/DV2F and the viral genome was placed under the control of CMV promoter. However, no virus particles were produced in transfected host cells (BHK-21). After full-length sequencing of the clones, several mutations such as nonsense mutation and frame shift mutation were discovered in this study. In addition, there remained certain superfluous nucleotide at the 5’- and 3’- end of viral genome. These non-viral sequence may affect viral replication. Restriction fragment replacement, polymerase chain reaction (PCR), and introduction of ribozyme were applied to correct the mutation and remove excess sequence from the 5’- and 3’- untranslated region (UTR) of viral genome. The new infectious clone was transfected into host cells for the production of viruses. However, no virus particle was detected, although viral RNA was detected.