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  • 學位論文

臺灣禽類副黏液病毒分離、鑑定與親緣分析

Isolation, Identification, and Phylogenetic Analyses of Avian Paramyxoviruses in Taiwan

指導教授 : 蔡向榮
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摘要


禽類副黏液病毒為Paramyxoviridae科的Avulavirinae亞科下病毒群,目前已知共22型,且廣泛分布於世界眾多鳥種中。禽類副黏液病毒一型(亦稱為新城病病毒)為家禽新城病的致病原,已廣佈於全球,能夠感染兩百種以上的鳥類。目前已有許多即時反轉錄聚合酶反應針對新城病病毒不同標的基因偵測被研發出,但檢測的敏感性與效力仍待改進。在本研究中,於2009年至2020年期間由臺灣家禽、候鳥、輸入鳥類及本地留鳥檢體中分離禽類副黏液病毒,並進行病毒基因體或融合蛋白基因全長定序及分析。同時建立核鞘蛋白即時反轉錄聚合酶反應技術以檢測具高度變異性的新城病病毒核酸。本研究共分離100株禽類副黏液病毒株,分屬於一型(52株)、二型(1株)、四型(20株)、六型(7株)、十二型(2株)、二十一型(2株)以及二十二型(16株)等7型。核酸序列分析顯示除了10株第一類禽類副黏液病毒一型及2株四型病毒屬於北美株外,其餘所有病毒均歸屬於歐亞株。禽類副黏液病毒一型的融合蛋白親緣分析顯示52株分離株中,50株屬於第一類的第一基因型或第二類的第一、六及七基因型病毒、1株為第一類新增的第二基因型及1株為無法分類之特異病毒;臺灣近十年的鴿子副黏液病毒流行株為第二類第六基因型的2.1.1.2.1與2.1.1.2.2亞型;造成臺灣家禽新城病疫情的病毒分離株均歸屬於第七基因型的1.1亞型。本研究在2009年的禽流感主動監測檢體中分離到一株特殊的副黏液病毒株(APMV/dove/Taiwan/AHRI33/2009),經基因體核酸序列比對,AHRI33相近於第七型禽類副黏液病毒,但相似度僅62.8%。依據RNA依賴性RNA聚合酶基因親緣性分析,AHRI33病毒株應新成立為禽類副黏液病毒二十二型。本研究建立之核鞘蛋白即時反轉錄聚合酶反應檢測技術在不同型別新城病病毒株的測試下,全部23株均成功檢出,而經美國農業部認證之基質蛋白即時反轉錄聚合酶反應則未能檢出8株第一類及2株鴿子副黏液病毒株;新檢測方法對於35種非新城病病毒均無陽性反應,顯示高度之特異性;實際應用於臨床檢體時,與黃金標準的病毒分離檢測結果比對,其敏感性與特異性分別為100%與96.61%。綜合上述,本篇研究證實臺灣禽鳥至少帶有7種不同型別之禽類副黏液病毒,而候鳥對於歐亞及北美間的跨洲際病毒傳播扮演重要之角色。AHRI33病毒應於Avulavirinae亞科Metaavulavirus屬中設立為Avian metaavulavirus 22新病毒種;臺灣家禽與野鳥至少帶有5種不同基因型之新城病病毒;其中AHRI67病毒株應於第一類下新成立第二基因型;而新建立的核鞘蛋白即時反轉錄聚合酶反應技術具高敏感性、特異性,可檢出高度演化變異的新城病病毒。總結本篇研究結果可更深入瞭解臺灣禽類副黏液病毒之天然宿主、演化及遺傳多樣性。

並列摘要


Avian paramyxoviruses (APMVs) belonging to the subfamily Avulavirinae within the family Paramyxoviridae consist of twenty-two known species, and are constantly isolated from a wide variety of avian species around the world. Avian paramyxovirus 1, synonymous with Newcastle disease virus (NDV), is a worldwide viral agent that infects over 200 species of birds and responsible for outbreaks of Newcastle disease. Although a number of real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been developed for detecting different genes of NDV, diagnostic sensitivity and efficiency could be improved. In this study, the APMV isolates collected from poultry, migratory, imported and resident birds during 2009-2020 in Taiwan were genetically characterized by sequence analysis of the complete fusion protein gene or full-length genome. Furthermore, this study describes a nucleocapsid protein gene rRT-PCR screening assay based on TaqMan technology for the detection of divergent NDV strains and isolates. One hundred isolates belonging to seven species were identified as APMV-1 (n=52), APMV-2 (n=1), APMV-4 (n=20), APMV-6 (n=7), APMV-12 (n=2), APMV-21 (n=2) and APMV-22 (n=16). Genetic studies showed that the recovered APMVs isolates had highest homology with Eurasian isolates, except ten class I APMV-1 and two APMV-4 isolates related to North America strains. Our phylogenetic analysis of complete fusion protein gene of the APMV-1 isolates revealed that 50 of the 52 Taiwanese isolates were closely related to APMV-1 of class I genotype 1 or class II genotypes I, VI or VII, one isolate belonged to a group that can be classified as a novel genotype 2 within class I, and one isolate was grouped within class I viruses but formed a monophyletic lineage, with a genetic distance of 5.6% between them. Viruses placed in class II sub-genotype VI.2.1.1.2.1 and sub-genotype VI.2.1.1.2.2 were the dominant pigeon paramyxovirus 1 circulating in the last decade in Taiwan. All the Newcastle disease outbreak-associated isolates belonged to class II sub-genotype VII.1.1, which was mainly responsible for the present epizootic of Newcastle disease in Taiwan. In 2009, the isolate APMV/dove/Taiwan/AHRI33/2009 was isolated from swabs of red collared doves during active surveillance of avian influenza in resident birds in Taiwan. Nucleotide sequence comparisons of the genome between each prototype of APMVs had shown AHRI33 to be more closely related to APMV-7 than to the others, with a sequence identity of 62.8%. Based on topology of the phylogenetic tree of RdRp genes and the branch length between the nearest node and the tip of the branch, AHRI33 met the criteria for designation as distinct species. Using the NP-gene rRT-PCR assay, all 23 representative NDV strains of class I and II in the tested panel were detected, whereas eight class I and two class II NDV isolates cannot be detected by the USDA-validated matrix-gene assay. The new assay also has a high degree of specificity with no false-positive results of 35 non–NDV viruses. A total of 146 clinical specimens were also tested and the NP-gene assay gave high relative sensitivity (100%) and specificity (96.61%) when compared with virus isolation. We conclude that at least seven species of APMVs were obtained from multiple avian host species in Taiwan. Migratory birds may play an important role in intercontinental spread of APMVs between Eurasia and North America. The data suggest that the isolate APMV/dove/Taiwan/AHRI33/2009 should be considered as the prototype strain of the new species Avian metaavulavirus 22 in the genus Metaavulavirus in the subfamily Avulavirinae. The current data confirm that at least five genotypes of APMV-1 circulate in both domestic and wild birds throughout Taiwan. One genetically divergent group of APMV-1 should be considered as a novel genotype within class I. Furthermore, the developed NP-gene rRT-PCR assay offers a sensitive, specific and rapid assay for detecting both class I and II NDV and could be used as part of a panel of diagnostic assays for this notifiable disease agent. Together, this study contributes to the knowledge of the distribution, evolution, and genetic diversity of APMVs in Taiwan.

參考文獻


1. Aldous, E.W., Fuller, C.M., Ridgeon, J.H., Irvine, R.M., Alexander, D.J., Brown, I.H., 2014. The evolution of pigeon paramyxovirus Type 1 (PPMV-1) in Great Britain: A molecular epidemiological study. Transbound. Emerg. Dis. 61, 134–139.
2. Alexander, D.J., 2000. Newcastle disease and other avian paramyxoviruses. Rev. Sci. Tech. 19, 443–462.
3. Alexander, D.J., 1987. Taxonomy and nomenclature of avian paramyxoviruses. Avian Pathol. 16, 547–552.
4. Alexander, D.J., Hinshaw, V.S., Collins, M.S., Yamane, N., 1983. Characterization of viruses which represent further distinct serotypes (PMV-8 and PMV-9) of avian paramyxoviruses. Arch. Virol. 78, 29–36.
5. Alexander, D.J., Manvell, R.J., Collins, M.S., Brockman, S.J., Westbury, H.A., Morgan, I., Austin, F.J., 1989. Characterization of paramyxoviruses isolated from penguins in Antarctica and sub-Antarctica during 1976-1979. Arch. Virol. 109, 135–143.

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