- 學位論文
評估益生菌和干擾素α在鴿子環狀病毒感染模型中的抗病毒特性
Antiviral properties of probiotics and interferon alpha in pigeon circovirus infection model
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摘要
自1980年代發現以來，鴿子環狀病毒 (PiCV)一直是鴿子(Columba livia)中研究和報導最為廣泛的病毒。由於病毒會影響鴿子的免疫系統，尤其是華氏囊，PiCV的感染會導致免疫抑制。鴿子感染PiCV後會引起淋巴球凋亡及免疫器官的萎縮，這可能導致隨後的細菌、病毒或寄生蟲的二次感染。幼鴿疾病症候群（YPDS）是一種複雜的疾病，PiCV被認為是造成此疾病的因素之一。PiCV可以感染幼鴿和成鴿，但更常見於四到六個月大的鴿子。為了防止PiCV在鴿子中傳播，需要有效的治療方案。 本研究的目的是在分離與研究貝萊斯芽孢桿菌(Bacillus velezensis)作為益生菌的功效，以及它對感染PiCV的鴿子作用。另一方面，本研究合成並生產了重組鴿子干擾素α（PiIFN-α），並在體內檢測了其對PiCV的抗病毒活性。 B. velezensis 從鴿子糞便中分離出來，給予鴿子口服60天，隨後以PiCV攻毒。檢測鴿子脾臟組織和糞便中PiCV病毒量和細胞激素的表現水平。攻毒後5天，服用益生菌組顯著降低了鴿子糞便和脾臟中的PiCV病毒量。在服用益生菌的鴿子中，Toll樣受體2 (TLR2)和4 (TLR4)顯著升高(P<0·05)。此外，在益生菌處理的鴿子中，干擾素-γ (IFN-γ)、myxovirus resistance 1 (Mx1)以及信號轉導和轉錄激活蛋白1 (STAT1)基因表現均增加，但白血球介素4 (IL-4)的表現沒有被檢測到。 PiIFN-α的基因克隆到pET24a載體中，並利用大腸桿菌表達系統產生蛋白質。純化後，以西方點墨法證實其抗原性，將蛋白質皮下注射於自然感染PiCV的鴿子。使用西方點墨法檢測到36.1 kDa的明顯條帶。經過PiIFN-α治療後，自然感染PiCV的鴿子的PiCV病毒量較低。 這項研究表明，無論是使用益生菌菌株B. velezensis或 PiIFN-α，都可以用作預防PiCV在鴿子中傳播的方法。
並列摘要
Since its discovery in the 1980s, pigeon circovirus (PiCV) has been the most widely studied and reported virus in pigeons (Columba livia). Because the virus affects the pigeons' immune system, mainly the bursa, PiCV infections can result in immunosuppression. PiCV infection in pigeons can cause lymphocyte apoptosis and immunological organ atrophy. This could lead to subsequent bacterial, viral, or protozoan infections. Young pigeon disease syndrome (YPDS) is a complicated condition in which PiCV is suspected to have a role. PiCV can infect both young and old pigeons, however it is more commonly found in 4 to 6-month-old pigeons. Effective treatment strategies are required to prevent the spread of PiCV in pigeons. The goal of this study is twofold: first, to identify and examine the efficacy of Bacillus velezensis as a probiotic, and second, to assess its effect on pigeons infected with PiCV. Recombinant pigeon interferon α (PiIFN-α) on the other hand, was cloned and generated utilizing a prokaryotic expression system, and its antiviral efficacy against PiCV was examined in vivo. B. velezensis was isolated from the feces of young pigeons and given to the birds orally for 60 days. Following the PiCV challenge, the pigeons' spleen tissues and feces were collected, and the viral load of PiCV and the expression level of innate immunity indicator genes were determined. 5 days after challenge (dpc), PiCV viral load was considerably reduced in the feces and spleens of pigeons treated with B. velezensis. Toll-like receptor 2 (TLR2) and 4 (TLR4) expression was considerably higher in treated pigeons than in controls (P < 0.05). Furthermore, the interferon-γ (IFN-γ) gene was upregulated in probiotic-treated pigeons, but not the interleukin-4 (IL-4) gene (no detected). The genes for myxovirus resistance 1 (Mx1) and signal transducer and activator of transcription 1 (STAT1) were also upregulated. The PiIFN-α gene was cloned into the pET24a vector and protein was produced utilizing the Escherichia coli expression system. Following purification, antigenicity was confirmed by western blot analysis. After that, the protein was delivered subcutaneously to pigeons, which naturally infected PiCV. A substantial band of 36.1 kDa was detected using the Western blot test. After therapy with PiIFN-α, pigeons with naturally infected PiCV had lower levels of PiCV viral titers. This study indicates that either a probiotic strain (B. velezensis) or PiIFN-α could be utilized as a therapy to inhibit PiCV spread in pigeons.
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