第五型鸚鵡玻那病毒在不同細胞之複製特性分析與受感染細胞之轉錄體表現探討

Replication studies of parrot bornavirus type 5 in different cell types and analysis of cellular transcriptomic profiles after infection

吳孟錡(Meng-Chi Wu)

指導教授 ： 陳慧文

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101769

摘要

鸚鵡玻那病毒感染造成鸚鵡發生高致死性的前胃擴張症，共有八種基因型。其中，最為盛行的是第四型病毒，而第五型病毒則是較罕見的型別，單獨分類在第二群演化支，和水鳥或雀科玻那病毒關係較近，先前研究顯示水鳥或雀科之玻那病毒具有感染哺乳類細胞能力，然而，第五型玻那病毒的病例稀少，其感染性、病原性及宿主與病原之交互作用等目前皆為未知。本實驗室先前自臨床檢體中分別分離到第四型與第五型玻那病毒，而本研究則進一步建立持續性感染玻那病毒的鵪鶉細胞株，用以瞭解病毒感染細胞之生長曲線，並利用RNA定序法初步探究病毒感染後宿主細胞之轉錄體調控。結果顯示，不論是第四型或第五型病毒，皆高度依賴細胞的生長而增殖，而第五型病毒在培養的第五天過後，病毒核酸有非常明顯的增加。轉錄體基因表現結果顯示，兩種型別病毒感染對於宿主細胞之轉錄調控有顯著不同，兩者的差異表現基因數量高達111個；而第五型病毒持續感染細胞與未感染細胞比較之差異表現基因數量有67個，其中的兩個與神經調控相關之基因皆顯著表現增加：VIPR2及LPAR4。另外，將病毒接種至多種禽類及哺乳類來源細胞，持續培養五代或十代，以定量PCR檢驗病毒核酸，並透過免疫化學染色法偵測病毒蛋白質，進而分析對第五型鸚鵡玻那病毒在不同細胞的之複製特性。結果顯示，第五型病毒具有在Vero細胞複製的能力 ，而對於禽類細胞的感受性依序為：QM7 > DF-1和QT6 > DEF，其中DEF只適合初代分離。我們亦將第五型病毒接種於鴨胚胎蛋之尿囊絨毛膜，再檢驗組織內的病毒核酸，結果顯示大部分為陰性，推測此模式不適用於增殖病毒。總結來說，第五型鸚鵡玻那病毒相對於第四型病毒具有不同的生長曲線與基因轉錄體調控，且第五型病毒具有廣泛的宿主細胞感染能力，包含多種禽類細胞和哺乳類Vero細胞，此結果將有利於深入解析鸚鵡玻那病毒對宿主之致病與免疫機制，也將有助於未來動物感染模式之建立。

關鍵字

第五型鸚鵡玻那病毒 ； 感受性 ； 複製特性 ； 轉錄體調控

並列摘要

Parrot bornavirus (PaBV) is the causative agent of proventricular dilatation disease. Eight genotypes, PaBV-1 to PaBV-8, has been described. Among them, PaBV-4 is the most dominant infection; PaBV-5 is rare and uniquely classified into the clade II virus, in which includes waterfowl and canary bornaviruses. Based on the distinct classification, PaBV-5 has been hypothesized to possess a cross-species infectivity. Nevertheless, due to the limited virus resources, infectivity, pathogenicity and host ranges of PaBV-5 are largely unknown. Previously, our lab has isolated the PaBV-4 and PaBV-5 from diseased parrots and established persistent infection in the quail QM7 cell line. Here, we aim to characterize the replication kinetics of PaBV persistently-infected cells, to investigate the transcriptomic profiles, and to conduct replication studies in avian and mammalian cells. In this study, growth kinetics of uninfected, PaBV-4 and -5 persistently infected QM7 cells were determined. PaBV replication highly relied on cell growth, and PaBV-5 showed a significant increase on day 5 of culture. In the comparisons of transcriptomic profiles of PaBV-4 or PaBV-5 persistently-infected cells, a total of 111 differential expression genes (DEGs) was noted. Furthermore, a total of 67 DEGs was identified between PaBV-5 persistently-infected cells and mock-infected cells, including two up-regulated neuro-associated genes, VIPR2 and LPAR4. For replication studies of PaBV-5, different inoculation doses were inoculated on avian cell lines (QM7, QT6, and DF-1), mammalian Vero cells, and primary duck embryo fibroblasts. Infected cells were serially passaged for 5 to 10 times, viral RNA was evaluated by RT-qPCR and viral proteins were detected by immunocytochemistry staining. The results showed that all the tested avian-origin cell lines as well as Vero cells were susceptible and permissive for PaBV-5. The QM7 sustains the viral persistence the most, followed by DF-1 and QT-6, while DEF only supported for primary isolation. Moreover, the chorioallantoic membrane of duck embryos did not seem to allow for viral growth. Collectively, this study demonstrates that PaBV-5 possesses distinct growth kinetics and transcriptomic signaling compared to PaBV-4, and PaBV-5 exhibited a broad range of infectivity in different cells. These findings are beneficial in understanding PaBV's pathogenesis and host responses, and provide insights into animal model development.

並列關鍵字

Parrot bornavirus type 5 ； susceptibility ； replication studies ； transcriptomic profiles

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