家禽傳染性支氣管炎病毒之序列分析、血球凝集特性及診斷方法的開發

Sequence analyses, hemagglutination activity and development of diagnostic assays of avian infectious bronchitis viruses

陳慧文(Hui-Wen Chen)

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/博士(2010年)

https://doi.org/10.6342/NTU.2010.02157

摘要

在論文的前三分之一部份中 (第三及第四單元)，研究著重在台灣地區家禽傳染性支氣管炎病毒(IBV)之序列分析，並藉由親源樹狀圖以及simplot分析法來探討1992至2007年之間病毒分離株的重組情形。針對8株台灣分離株進行7.3kb的定序並用來分析，結果顯示在台灣病毒株與中國CK/CH/LDL/97I-type病毒株之間有著許多重組事件，而推測的重組位置則落於S1、S2、3b、M基因以及M基因與5a基因間的區域。此外，亦於2005-2006年間至家禽屠宰場採樣進行病毒監測，結果自47場中的8場分離到IBV共13株，並由S1基因的親源樹狀圖得知其中的11株屬於台灣一型。更進一步發現其中一株與中國病毒株在S基因有重組事件，另外一株則是分別和中國病毒株與疫苗株H120在S2基因及M基因有著基因重組。由單向中和試驗可以發現到部份抗原性的改變，但是並沒有發現到正向天擇壓力。這些結果顯示台灣田間分離株存在著重組事件及抗原性多樣性。
論文的中間三分之一部份 (第五及第六單元)，則著重於研發IBV的分子生物學及血清學的診斷工具。由於世界上普遍使用麻州型病毒株當作疫苗，開發具型別特異性的病毒與抗體檢測方法便成為一個重要的課題。第五單元中描述一種新開發之快速且精確的多引子反轉錄酶聚合酶連鎖反應來區別IBV的基因型，先以已知的本土病毒株及商業疫苗株用於本方法的開發，另外使用眾多田間分離株以進行實際應用的評估。結果顯示所有用來檢測的IBV皆可正確地經由辨識預期片段而被區分其基因型別，本方法的偵測極限對於台灣一型、台灣二型及麻州型病毒分別為103, 105 及103個病毒S1 RNA數目，本方法確實可以用來區分野外病毒株以及疫苗毒株。另一方面，為了進行具有型別特異性的抗體檢測，本研究首先篩選出適合的單株抗體，開發一種阻斷型酵素連結免疫吸附法，並以血球凝集抑制試驗當作抗體診斷的金標準，採集共390個田間血清或血漿來樣本評估此阻斷型酵素連結免疫吸附法的臨界值、敏感性以及特異性。評估結果顯示抑制百分比之臨界值為29.08%，診斷之敏感性為97.96%，特異性為97.16%，而新開發的阻斷型酵素連結免疫吸附法與金標準之間有高度的一致性，且無顯著差異 (Kappa = 0.95, McNemar p = 0.72)，顯示本阻斷型酵素連結免疫吸附法為一種具有型別特異性的IBV抗體檢測方法，可避免疫苗接種後的抗體干擾。以上兩種診斷工具皆有潛力作為快速且精確的野外病毒檢測及血清陽性率調查。
最後三分之一的部份 (第七及第八單元) 則是探討台灣分離株之血球凝集特性以及發現桿狀病毒來源的S1蛋白具有細胞相關的吸附雞紅血球特性。本研究一共挑選13株台灣型IBV依照標準流程以及最佳化流程操作，製備HA抗原進行分析。結果顯示有9株台灣型IBV經過最佳化流程製作之HA抗原仍不具有血球凝集特性，但是這樣的差異性與病毒之基因型無關，而且S1基因總數與血球凝集力價無絕對相關性，我們也以西方墨點法確認這些HA抗原中的確有S1蛋白的存在。本研究發現台灣型IBV在血球凝集特性方面具有毒株之間的差異性，並嘗試探討造成這些差異的原因。目前雖未發現確切的血球凝集抗原決定位或決定因子，但這些研究成果仍有助於未來研究台灣病毒株的生物特性。另外，為了研究由S1蛋白所調節的血球凝集特性，因而利用桿狀病毒表現系統將台灣病毒株2575/98的S1 基因加以選殖並在Sf9細胞中表現，針對S1基因在不同感染倍數及不同時間下的表現情況作更進一步的探討。結果顯示重組S1蛋白在細胞打破後的細胞上清液或團塊中皆可被偵測到，而重組S1蛋白以及重組桿狀病毒皆可與抗IBV高免血清有良好的反應。此外，桿狀病毒感染之Sf9細胞在未經神經胺酸酶處理之前，就具有吸附雞紅血球的特性，而這個現象能夠被IBV雞隻抗血清所抑制，足見此血球吸附現象是為IBV相關蛋白所引起；但是其餘所有材料皆無血球凝集力價，即使是經過神經胺酸酶處理之後。這是第一篇關於細胞-血球吸附的IBV研究，而這些成果可能可以作為探討血球凝集特性與細胞接觸、融合的一種便利模式，亦可更進一步經由基因工程的方式，可以製造特定的突變S1蛋白，藉之更深入的探究其生物學或免疫學特性。

關鍵字

阻斷型酵素連結免疫吸附法 ； 血球吸附特性 ； 血球凝集特性 ； 傳染性支氣管炎病毒 ； 多引子反轉錄酶聚合酶連鎖反應 ； 重組S1 蛋白 ； 序列分析

並列摘要

In the beginning one-third part of this dissertation (Unit 3 and 4), research has focused on the sequence analyses of IBVs isolates in Taiwan. The putative recombinant events of Taiwan IBVs isolated from 1992 to 2007 were investigated by phylogenetic analysis and simplot analysis. The 3’ 7.3 kb structural protein genome of eight Taiwan strains was directly sequenced. Frequent recombination events were identified among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. In addition, this study reports on a viral surveillance program in Taiwan from 2005 to 2006 with sampling conducted in poultry slaughter houses. Eight out of 47 flocks (17%) were IBV-infected, from which 13 IBV isolates were recovered. Eleven of 13 isolates (84.6%) clustered with Taiwan group I based on the S1 gene. One IBV isolate showed evidence of frequent recombination events with China-like IBVs in the S gene. Another isolate demonstrated the incorporation of China-like and H120-like genome fragments within the S2 gene and the M gene region, respectively. Some antigenic changes were found in the one-directional neutralization test. However, no positive selection pressures were related to those variations in the S1 genes among Taiwan IBVs. Field IBVs in Taiwan revealed intertypic genetic recombination and antigenic diversity.
For the second one-third part of this dissertation (Unit 5 and 6), molecular and serological diagnosis of IBV were the research topics. Since a heterologous Mass-serotype vaccine has been used in Taiwan for a decade, group-specific identification on virus and antibody has been a difficult problem. The Unit 5 reports on a rapid and reliable multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay for the genotyping of IBVs. Local IBV strains and commercially available vaccines were used for evaluating the viral genotyping assay. A number of field isolates and were examined for clinical application. The results showed that all of the examined IBVs were accurately genotyped by identifying the corresponding bands on agarose gels. The mRT-PCR assay was able to detect as low as 103, 105 and 103 viral RNA copies of the TW-I, TW-II and Mass-type strains, respectively. The mRT-PCR assay accurately detected and differentiated vaccine viruses from wild-type strains in the field. Another aspect, in order to understand the status of field infection, a monoclonal antibody (mAb) blocking ELISA (b-ELISA) against local IBVs was developed. The selected mAb showed specificity to Taiwan IBV strains but no cross reactivity against the vaccine strain H120. By using the hemagglutination inhibition (HI) test as a gold standard, the cut-off value, sensitivity and specificity of the b-ELISA were evaluated with 390 field samples. The type-specificity of detection was validated with a panel of chicken hyperimmune sera. The results showed that the b-ELISA demonstrated high sensitivity (97.96%) and specificity (97.16%) of detection. The agreement between the results of b-ELISA and HI test was statistically significant (Kappa = 0.95) and no significant difference between these two methods (McNemar p = 0.72). The b-ELISA specifically detected Taiwan IBV serotypes rather than other three IBV serotypes and sera against other avian pathogens. This b-ELISA provides type-specific antibody detection to the local IBV strains. It has the potential to serve as a rapid and reliable diagnostic method of IBV clinical infections in the field of Taiwan.
In the last one-third part of this dissertation (Unit 7 and 8), research started with the characterization of the hemagglutination activity (HA) in Taiwan IBV strains and ended up with discovering the cell-associated hemadsorption activity of baculovirus-derived S1 protein. The HA activity of 13 Taiwan IBV strains were investigated. The results showed 9 of 13 Taiwan IBV strains failed to show any HA activity after the neuraminidase treatment under standard or optimized protocols. This difference is not genotype-dependent. The amount of S1 gene did not equivalently correlate to the obtained HA titers. The presence of S1 protein in the prepared HA antigens was verified by western blot. The present study provides the information on the diversity of HA activity that Taiwan IBVs display and primary investigation on the factors that may influence the HA activity. Furthermore, to investigate the hemagglutination activity mediated by the S1 protein, the full S1 gene of the Taiwan IBV 2575/98 was cloned and expressed in Sf9 cells with the baculovirus expression vector system. Both of the recombinant S1 protein and the recombinant baculovirus possessed good reactivity with the chicken hyperimmune sera against several IBVs. Most notably, the baculovirus infected Sf9 cells acquired the ability to directly hemadsorb the chicken erythrocytes without neuraminidase treatment. The phenomenon of hemadsorption was inhibited by the IBV chicken antiserum, indicating the hemadsorption activity was specific induced by IBV-related proteins. However, no HA titer was obtained from any materials, even after additional neuraminidase treatment. This is the first observation of cell-associated hemadsorption activity in IBV research. This finding may provide a simple model for investigating the mechanism of virus-mediated hemagglutination activity, cell attachment and fusion.

並列關鍵字

Blocking ELISA ； Hemadsorption activity ； Hemagglutination activity ； Infectious bronchitis virus ； Multiplex RT-PCR ； Recombinant S1 protein ； Sequence

參考文獻

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