台灣土雞與蛋雞家禽白血病之病毒分離、序列分析及流行病學調查

Virus isolation, sequence analysis and epidemiological survey for avian leukosis virus from Taiwan Country chickens and egg-type chickens in Taiwan

張書維(Shu-Wei Chang)

指導教授 ： 王金和

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02283

摘要

家禽白血病病毒（avian leukosis virus, ALV）為雞場中常見可引起腫瘤性疾病及造成生產問題的病原，根據其封套醣蛋白質的不同，可將ALV區分為A、B、C、D、E、J共六個亞群，其中亞群A（ALV-A）、B、C、D、J屬於外源性病毒，而E亞群屬於內源性病毒。由於台灣的土雞並未進行任何外源性ALV的清除撲滅計畫，因此本研究之目的，是藉由病毒株分離、序列分析、分子生物學及血清學的調查，建立外源性ALV於台灣土雞的感染概況。本研究一共自五個未發生腫瘤疾病的土雞場分離到了五株重組家禽白血病病毒及兩株ALV-J，並定序分離株ALV TW-3577與ALV TW-3593之全proviral DNA基因體，及與國際間ALV代表毒株之序列進行分析比較。ALV TW-3577擁有了與myeloblastosis-associated virus 1高度相似的gp85序列及屬於ALV-J的三端非轉譯區（3’ untranslated region, 3’UTR）及長端點重複片段（long terminal repeat, LTR）；而ALV TW-3593的gag基因、pol基因、gp37序列及3’UTR則與內源性病毒的序列高度相似，並擁有一不同於各亞群病毒的gp85序列，與親源關係最接近的ALV-C的核苷酸相似度僅88.1%。本研究設計了一個聚合酶鏈鎖反應（polymerase chain reaction, PCR）之引子（TWA），其可用來檢測除了ALV-J之外的外源性ALVs。我們一共針對了61個取自於屠宰場的土雞場、2個蛋種雞場及一個種土雞場，以PCR來偵測外源性ALVs，及使用可檢測ALV-A/B抗體、ALV-J抗體及群特異性抗原（group-specific antigen, GSA）的商品化之酵素連結免疫吸附法（enzyme-linked immunosorbent assay, ELISA）診斷套組，進行ALV感染之調查。在61個土雞場中，分別可在60場及43場檢測到ALV TW-3593-like viruses及ALV-J的感染；此外，在種土雞場亦可偵測到ALV TW-3593-like viruses的感染。在抗體的調查方面，在28個土雞場的血漿樣本中可檢測出ALV-J抗體，其陽性率介於10-60%；但在ALV-A/B抗體的檢測部分，僅在6個土雞場及種土雞場有約10-20%的低抗體陽性率。本研究中所調查的兩個蛋種雞場，無論在偵測外源性ALVs的PCR或是檢測ALV-A/B及ALV-J抗體的ELISA中，都是陰性反應。在使用ELISA偵測GSA的部分，我們發現在同一批雞隻的血清及血漿樣本其GSA的檢測結果有明顯地差異，血清樣本的陽性率極顯著地高於血漿樣本(P < 0.01)。本研究指出，在台灣的雞場中，目前至少存在了兩種重組的家禽白血病病毒；此外，ALV TW-3593-like viruses及ALV-J的雙重感染在台灣的土雞場非常普遍，也顯示了ALV TW-3593-like viruses為目前台灣土雞中最盛行的家禽白血病病毒。

關鍵字

家禽白血病病毒 ； 病毒分離 ； 序列分析 ； 重組 ； 外源性病毒 ； 聚合酶鏈鎖反應 ； 台灣土雞 ； 流行病學調查

並列摘要

Avian leukosis viruses (ALVs) are common in many poultry flocks and can cause neoplastic diseases and other reproductive problems in chickens. Based on differences in their envelope glycoproteins, ALVs are divided into six subgroups (A, B, C, D, E, and J). Subgroup A (ALV-A), B, C, D and J (ALV-J) are considered exogenous, whereas subgroup E encompasses the endogenous viruses. Because the eradication of ALV has not carried out in Taiwan Country chicken industry, the purpose of this study is to investigate the profile of exogenous ALV infection in Taiwan Country chickens by virus isolation, sequence analysis, and molecular and serologic surveillance. 5 recombinant ALVs and 2 subgroup J ALVs were isolated from 5 Taiwan Country chicken farms with no tumor occurrence, and the full proviral DNA genomes of isolates ALV TW-3577 and ALV TW-3593 were sequenced, analyzed and compared with representative strains of ALV. Isolate ALV TW-3577 possessed a myeloblastosis-associated virus 1 gp85 coding region and an ALV-J 3’ untranslated region (3’UTR) and long terminal repeat (LTR); the LTR, gag, pol, gp37 coding region, and the3’UTR of ALV TW-3593 displayed high identity to endogenous counterpart sequence, and the gp85 coding region of ALV TW-3593 was different from all the subgroups, with a moderate nucleotide sequence identity to ALV-C (88.1%). In this study, we designed a primer (TWA) for molecular detection of exogenous ALVs except ALV-J. 61 Taiwan Country chicken farms from slaughter house, 2 layer farms, and one Taiwan Country broiler breeder farm were screened for exogenous ALVs by PCR, for ALV-A/B and ALV-J antibody by enzyme-linked immunosorbent assay (ELISA), and for group-specific antigen (GSA) by ELISA. ALV TW-3593-like viruses and ALV-J were detected in 60 and 43 farms of 61 Taiwan Country chicken farms, respectively. The broiler breeder farm was also positive for ALV TW-3593-like viruses. ALV-J antibodies was detected in the plasma samples of 28 Taiwan Country chicken farms with moderate positive rates (10-60%), while only 6 Taiwan Country chicken farms and the broiler breeder farm had ALV-A/B antibodies, and the positive rates of these farms were quite low (10-20%). These two layer farms were negative for exogenous ALVs by PCR and ALV-A/B, ALV-J antibodies by ELISA. The GSA tested by ELISA showed significantly difference between serum and plasma samples collected from the same flocks, the positive rate of serum samples was extremely higher than plasma samples (P < 0.01). Our results reveal that there are at least two recombinant ALVs circulating in Taiwan flocks. In addition, dual infection with ALV TW-3593-like viruses and ALV-J are common in this study, indicating that ALV TW-3593-like viruses are the most prevalent ALV in Taiwan Country chicken population.

並列關鍵字

avian leukosis virus ； virus isolation ； sequence analysis ； recombinant ； exogenous virus ； PCR ； Taiwan Country chicken ； epidemiological survey

參考文獻

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