- 學位論文
台灣地區非人靈長類動物B型肝炎和瘧疾之盛行率及親緣關係
Prevalence and phylogentic analyses of hepatitis B and malaria among nonhuman primates in Taiwan
摘要
非人靈長類動物（nonhuman primates, NHPs）由於其基因、生理學及行為上與人類極為相似，因此是人畜共通傳染病之重要感染源。儘管非人靈長類之B型肝炎及瘧疾是重要之疾病且對人類健康具有潛在性危險，但在台灣地區其相關研究卻極為有限。本論文將分為B型肝炎及瘧疾等兩部份，探討其於台灣地區非人靈長類之盛行率、病原性、分子生物學特徵及親緣關係。B型肝炎為一種全球性公共衛生問題，除可感染人類外，也被證實可感染非人靈長類動物。本研究收集台灣各地區之圈飼動物共286例血清樣本，其中包含32隻巨猿科動物（Pan troglodytes及Pongo pygmaeus）、42隻長臂猿科動物（Hylobates sp.及Nomascus sp.）及212隻獼猴科動物，以B型肝炎特異性血清標誌進行篩檢。結果發現所有獼猴科動物之血清樣本其B型肝炎特異性血清標誌檢查皆呈現陰性。然而21.9%（n=7）巨猿科及40.5%（n=17）長臂猿科動物其受檢血清對一種或以上之B型肝炎特異性血清標誌呈現陽性。所有B型肝炎陽性動物之D型肝炎抗體檢測皆呈陰性。經親緣分析完整B型肝炎病毒基因序列，發現此次自長臂猿分離之B型肝炎病毒與東南亞所分離之長臂猿與巨猿類動物之B型肝炎病毒之親緣關係極為親近，而與人類之B型肝炎病毒之親緣關係則相當疏遠。根據上述結果證明台灣地區之圈飼巨猿類及長臂猿動物其具有高B型肝炎盛行率，且此B型肝炎病毒可能是過去自東南亞引進已感染B型肝炎動物而進入台灣。於猴瘧疾（simian malaria）方面，台灣獼猴為被認為具有人畜共通傳染性猴瘧原蟲Plasmodium knowlesi、P. cynomolgi 及P. inui之自然宿主。台灣為人類瘧疾根除地區，而自1970後並無猴瘧疾之相關研究報告。為更新猴子瘧疾盛行率資料，本研究以分子生物學調查方法進行相關研究。針對台灣北、南及東部等地區之圈飼及野生台灣獼猴（Macaca cyclpois）共計採集286例血液樣本，進行鏡檢及巢式聚合酶鏈鎖反應（nested-PCR）檢查。結果2.4％ (7/286) 血液樣本其顯微鏡檢及聚合酶鏈鎖反應皆呈現陽性，這些陽性分離株皆採集於南部地區，而北部及東部地區血液樣本均未發現有感染。經增幅無性期之核糖體小亞單位（asexual stage small subunit ribosomal RNA, SSU rRNA）部分基因片段進行親緣分析，確定這些血液樣本皆為單一種P. inui感染。為究明此次分離P. inui之分子生物學特徵，增幅裂殖子表面蛋白（42 kDa fragment of the merozoite surface protein 1；MSP-142）基因片段並與其他分離自不同地理位置及宿主之P. inui瘧原蟲株進行親緣分析。結果發現此7株野外株與1963自台灣獼猴所分離之Taiwan I與Taiwan II株親緣關係最為接近，而與其他地理位置所分離P. inui不同。根據上述結果證明P. inui為台灣猴瘧疾之唯一感染種類，且主要分布於南台灣。為更進一步區別抗原性及病原性不同之Taiwan I與Taiwan II株，增幅環孢子蛋白（circumsporozoite protein; CSP）基因並進行親緣關係分析。結果全部7株野外分離株與Taiwan I及Taiwan II株非常相似，呈現單一親緣分枝（monoclade）。此結果顯示尚無法以CSP基因進行親緣關係分析來區別Taiwan I及II株。
並列摘要
Based on the genetic, physiological, and behavioral similarities with humans, nonhuman primates (NHPs) are the natural reservoirs of zoonotic diseases. Despite the importance and potential hazard to human health, the related studies about hepatitis B virus (HBV) and malaria infection in NHPs in Taiwan are limited. The objectives of this thesis are to understand the prevalence and pathogenicity of HBV and simian malaria, and further investigate their molecular characterization and phylogenetic analyses in Taiwan. HBV is a public health problem worldwide and apart from infecting humans, HBV has been identified in NHPs. Serum samples from captive NHPs (n=286), including great apes (Pan troglodytes and Pongo pygmaeus) (n=32), gibbons (Hylobates sp. and Nomascus sp.) (n=42) and Cercopithecidae monkeys (n=212), were collected and tested for the presence of HBV-specific serological markers. None of the Cercopithecidae monkeys were reactive against serological markers of HBV. In contrast, 21.9% (n=7) of great apes and 40.5% (n=17) of gibbons were positive for at least one serological marker of HBV. None of the HBV-infected animals were reactive against hepatitis D virus (HDV). Phylogenetic analysis of the complete HBV genome revealed that gibbon isolates clustered with other HBV of great apes and gibbons from Southeast Asia and separated from human-specific HBV. These findings indicate that the high prevalence of HBV among captive gibbons and apes in Taiwan, and HBV might have been introduced into Taiwan via the direct import of infected animals from Southeast Asia in the past. To update the prevalence of simian malaria, a molecular-based survey was performed. In this study, 286 blood samples collected from captive and wild-caught Formosan macaques (Macaca cyclopis) the northern, southern, and eastern Taiwan were tested for Plasmodium species by microscopy and polymerase chain reaction (PCR). Of these, 2.4% (7/286) were positive by both microscopy and nested-PCR. All the positive isolates were collected from southern Taiwan, whereas no evidence of malarial parasites was observed among monkeys from eastern and northern Taiwan. Molecular and phylogenetic analyses based on the asexual stage small subunit ribosomal RNA (SSU rRNA) gene clearly identified these samples as a single infection with P. inui. For further characterization of P. inui obtained in this study and compared with other P. inui isolates from different geographical areas and different hosts, a phylogenetic tree was constructed according to the 42 kDa fragment of the merozoite surface protein 1 (MSP-142) gene sequence. Phylogenetic analysis showed that 7 field isolates were closely related to Taiwan strains I and II, which were isolated from Formosan macaques in 1963, and distinct from the other geographical isolates of P. inui. Furthermore, in order to further discriminate between strains Taiwan I and II, which have been shown to have significant antigenic and pathologic differences, the circumsporozoite protein (CSP) gene sequences were amplified and analyzed. The results showed that the 7 isolates were clustered together with strains Taiwan I and II, allowing for the construction of a monoclade. This indicate that phylogenetic analysis based on CSP genes still did not enable us to discriminate between strains Taiwan I and II.