家禽傳染性支氣管炎病毒之基因序列分析及減毒疫苗開發

Sequence analysis and development of attenuated vaccines from local infectious bronchitis virus strains

黃元品(Yuan-Pin Huang)

指導教授 ： 王金和

國立臺灣大學/生物資源暨農學院/獸醫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.10054

摘要

家禽傳染性支氣管炎病毒 (Avian infectious bronchitis virus, IBV) 引起的傳染性支氣管炎 (Infectious bronchitis, IB) 是雞的一種急性、高傳染力的呼吸道疾病。由於呼吸道疾病 (包括IB) 對於家禽產業有很大的影響，因此基於經濟與應用在臨床上區別診斷的考量，本實驗室發展多引子反轉錄聚合酶鏈鎖反應，一共使用四組針對傳染性喉頭氣管炎病毒 (ILTV)、傳染性支氣管炎病毒、新城病病毒 (NDV)、以及家禽流行性感冒病毒 (AIV) 的特異性引子來進行測試。結果顯示在單管反轉錄聚合酶鏈鎖反應或是多引子反轉錄聚合酶鏈鎖反應皆可得到相同的預期產物。在敏感性測定方面，多引子反轉錄聚合酶鏈鎖反應針對ILTV、IBV、NDV及AIV，分別可測得3.5 ng、0.14 ng、0.4 ng、0.36 ng的病毒核酸。在所有測試的樣本中，共有22個樣本為病毒分離陽性，其中有77%的樣本是呈現病毒核酸偵測陽性的結果；而在64個病毒分離陰性的樣本中，有91%的樣本是呈現病毒核酸偵測陰性的結果。因此，此多引子反轉錄聚合酶鏈鎖反應可使用在家禽病毒性呼吸道疾病的快速鑑別診斷上。除了臨床診斷外，此項技術還應用在兩座屠宰場雞隻樣本的IBV檢測上，結果顯示在總共82個雞群中，一共分離出1個疫苗毒株，以及其他12個IBV分離株，其中包含11個台灣一型分離株，及一個未確定基因型毒株，但無台灣二型分離株。而估計之雞隻分離率為1.6%至3.1%之間。由欲被屠宰雞隻之IBV高分離率，可以得知從屠宰場採樣是一個方便又有效的監測IBV方法。
同時我們想要探討台灣之傳染性支氣管炎病毒的分子流行病學，因而挑選一株1964年所分離的病毒以及31株從1991年至2003年間分離的病毒來進行S1基因的N端序列分析，並參考親緣樹狀圖後挑選13株來針對整段S1基因及部分N基因進行定序。分析結果顯示這些台灣的傳染性支氣管炎病毒株主要仍可分成兩個族群，即台灣一型與台灣二型，以及一株麻州型毒株2994/02和一株似中國毒株2992/02。結果顯示多數分離毒株屬於台灣基因型。此外，在S1基因中並沒有發現台灣毒株與疫苗株H120之間有重組的現象，且台灣毒株的基因體3’端區域的其他基因與Beaudette CK毒株間亦沒有發現重組的現象。由S1基因以及N基因的親緣樹狀圖顯示毒株2992/02及2994/02分別發生基因間重組，且S1基因明顯的比N基因出現更高的差異程度。
由前述研究結果顯示多數台灣分離毒株仍然屬於台灣基因型，而且進口的IBV疫苗並沒有良好的保護能力，因此為了想要有效控制IBV感染，本實驗室選取台灣本土分離株，以雞胚胎連續繼代減毒後進行疫苗檢定相關測試，結果顯示經過74 - 76代繼代後，所選取之台灣一型減毒病毒株2575/98對一週齡小雞不具有致病性，中和指數為4.4，合乎法規規定值，並且有90%的雞隻在免疫後可以抵抗攻毒毒株的侵害，並且在雞體內繼代5代之後並沒有發現毒力回歸的現象。台灣二型減毒病毒株2296/95亦有類似的結果。因此將此台灣一型及台灣二型毒株交付廠商製成冷凍乾燥疫苗後進行更進一步的測試，包括病毒力價測定、安全試驗、抗體力價試驗及攻毒試驗，結果顯示前者102.3、103.3、104.3 EID50/隻的免疫劑量對3至10日齡小雞皆不具有致病性。以102.3、103.3、104.3 EID50/隻的劑量免疫試驗組後的中和指數皆大於法規規定值，而對照組中和指數皆小於規定值，並且在製成的冷凍乾燥疫苗中並無檢測到活菌，亦無測到新城病及家禽流行性感冒的病毒核酸，後者亦有類似的結果。在田間試驗中，接種減毒病毒株2575/98或者是2296/95的雞隻在中和指數、育成率或者是出售體重方面，皆比接種疫苗H120的雞隻表現要好。綜合上述結果，此繼代減毒病毒株2575/98及2296/95有潛力可以作為一良好減毒疫苗毒株。
更進一步，我們針對三株減毒前與減毒後的傳染性支氣管炎病毒，使用反轉錄聚合酶鏈鎖反應增幅病毒基因體3端 (約7.3 kb)，並將其序列資料與基因庫上的其他毒株資料進行比對，藉此探討病毒毒力與基因序列的關係。結果顯示，經過74至76代的無特定病原雞胚胎蛋繼代後，在S1基因上有2至6個胺基酸改變，在S2基因上有2至3個胺基酸改變。在E基因上僅有1個或是沒有胺基酸改變，而在M基因上則有1個或是3個胺基酸改變。在N基因上則沒有發現胺基酸改變，表示此減毒效果可能與N基因無關。在N基因3端的未轉譯區域則是發現有一株病毒在減毒後出現部分序列的缺失，此變異也許與毒力的改變有關。此部分研究是第一篇比較傳染性支氣管炎病毒之基因體3端在減毒後的序列改變情形，而上述結果也許對未來關於病毒毒力的研究上會很有幫助。
本研究探討台灣家禽傳染性支氣管炎病毒的分子流行病學情形，以及發展出兩株疫苗株可以用來控制疾病的發生。

關鍵字

減毒疫苗 ； 基因分析 ； 傳染性支氣管炎病毒 ； 多引子反轉錄聚合酶鏈鎖反應 ； 屠宰場 ； 台灣一型 ； 台灣二型

並列摘要

Infectious bronchitis (IB) is an acute, highly contagious disease of chickens caused by infectious bronchitis virus (IBV). Since respiratory tract infections, including IB, are critical in the poultry industry, a multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects avian viral respiratory pathogens in the same reaction was developed for the economic and clinical needs. Four sets of specific oligonucleotide primers for infectious laryngotracheitis virus (ILTV), IBV, Newcastle disease virus (NDV), and avian influenza virus (AIV) were used in this study. Expected products were generated from both multiplex RT-PCR and single RT-PCR. The detection limits of this multiplex RT-PCR were 3.5 ng, 0.14 ng, 0.4 ng, and 0.36 ng for ILTV, IBV, NDV, and AIV, respectively. Among the tested samples, seventy-seven percent of the 22 virus isolation-positive samples showed positive by genome detection, and ninety-one percent of the 64 virus isolation-negative samples showed negative by genome detection. This multiplex RT-PCR could be used for rapid screening and differential diagnosis of poultry viral respiratory infections. In addition to clinical diagnosis, this technique was also applied to the IBV detection in samples from two slaughter houses. Besides one vaccine isolate, twelve flocks showed positive IBV isolation from 82 chicken flocks (14.6%), including eleven TW I isolates and one novel genotypic isolate, but no TW II isolate. The estimated total chicken isolation rate was 1.6% to 3.1%. The high IBV isolation rate from healthy chickens indicates that sampling in the slaughter houses is a convenient and effective way for IBV monitoring.
To investigate the molecular epidemiology of IBV in Taiwan, an old IBV strain isolated in 1964 and other 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on the results of phylogenetic analysis, 13 strains were selected for sequencing the entire S1 and partial N genes. The results indicated that most Taiwan IBV strains could be divided into two distinct lineages, Taiwan Group I (TW I) and Taiwan Group II (TW II). Only one Massachusetts strain, 2994/02, and one China-related strain, 2992/02, were detected. No recombination was found between H120 and the Taiwan strains in the S1 gene, and neither was between Beaudette CK and Taiwan strains at the 3’ genome. However, the S1 gene showed a noticeable higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination of strain 2992/02 and 2994/02 has occurred.
Since most Taiwan isolates are unique, and vaccination of chicks with imported vaccines fails to protect chicks from IBV infections in Taiwan. In order to control IBV infection, selected Taiwan strains were attenuated through SPF chicken embryonated eggs. A TW I strain 2575/98 was passaged 74 times through SPF embryonated eggs, and then tested in SPF chickens. The attenuated vaccine was not pathogenic in 1-week-old chicks, had a neutralization index (NI) of greater than 4.4 and showed a protective rate of 90% when inoculated birds were challenged with a field IBV strain. Similar results were obtained for a vaccine made from a TW II IBV strain 2296/95. Additionally, the TW I attenuated vaccine strain had no reversion to virulence after five back passages in chicks. Therefore, these two IBV strains were transferred to a manufacturer for the vaccine production and evaluation. The safety and the efficacy of this attenuated strain were also tested in SPF chickens. The results of evaluation indicated that the attenuated strain 2575/98 at 102.3, 103.3, or 104.3 embryo infectious dose (EID50)/dose was not pathogenic to 3-to-10-day-old chicks. After vaccination in SPF chickens, the NI in vaccinated with 102.3, 103.3, or 104.3 EID50/dose were greater than 2, and in control groups were lower than 1. No bacteria, Newcastle disease virus, or avian influenza virus contamination was found in this attenuated vaccine preparation. Similar results were obtained for attenuated strain 2296/95. In field tests, the groups vaccinated with the attenuated strain 2575/98 or 2296/95 showed better performance in NI, livability and weight comparing to whom vaccinated with vaccine H120. In conclusion, the attenuated IBV 2575/98 and 2296/95 have potential for controlling IBV infection.
Furthermore, the sequences in the 3’ 7.3 kb of the genome amplified using RT-PCR before and after attenuation were compared to study the relationship between virulence and the sequences of three IBV strains, 1171/92, 2575/98 and 2296/95. After attenuation, two to six amino acid substitutions were found in the S1 subunit, and two or three amino acid substitutions were found in the S2 subunit. No or one amino acid substitution was found in the small membrane protein, and one or three amino acid substitutions were found in the membrane protein. However, no amino acid substitution was found in the N protein, indicating that the N protein might not be related to this attenuation. The un-translated nucleotide sequence after the N of one strain was partially deleted after attenuation, and might be correlated with virulence. This study is the first demonstration comparing sequence changes in the 3’ 7.3 kb of the genome of IBV after attenuation. The above mentioned information might be useful in future virulence studies.
This study describes the IBV molecular epidemiology, and develops two vaccine strains for controlling IBV infection in Taiwan.

並列關鍵字

Attenuated vaccine ； Gene analysis ； Infectious bronchitis virus ； Multiplex RT-PCR ； Slaughter house ； Taiwan Group I ； Taiwan Group II

參考文獻

55. Chang PC, Kuo TY, Chueh LL, Wang CH. Sequencing and analysis of S1 gene of infectious bronchitis viruses isolated in Taiwan. J Chin Soc Vet Sci 24: 179-187, 1998.

117. Huang YP, Chang WC, Wang CH. Evaluation of an attenuated TW I infectious bronchitis vaccine. Taiwan Vet J 31: 148-154, 2005.

118. Huang YP, Chang WC, Wang CH. Evaluation of an attenuated TW II infectious bronchitis vaccine. Taiwan Vet J 32: 126-131, 2006.

165. Lee HC, Hsu CN, Wang CH. Molecular epidemiology of avian influenza virus H6N1 in Taiwan. Taiwan Vet J 31: 230-239, 2005.

246. Tseng ZM. Analysis of nucleotide and preparation of inactivated vaccine of infectious bronchitis virus isolated in Taiwan. In: Master thesis, National Chung-Hsing University, Taichung, Taiwan. pp. 13, 1999.