傳染性支氣管炎病毒減毒株在雞胚胎增殖之分析

Abstract

家禽傳染性支氣管炎(Avian infectious bronchitis; IB)是一種侵害世界各國雞隻的病毒性急性高度傳染病。家禽傳染性支氣管炎病毒(IB virus)可經過連續接種雞胚胎蛋培養進行減毒程序降低對雞隻之病害而獲得疫苗株，但減毒疫苗株對雞胚胎的致病性卻增加。為探討家禽傳染性支氣管炎病毒減毒株之雞胚胎致病性及毒力相關的作用機制與可能的分子決定位，以TW2575/98野外株(TW2575/98w)與減毒疫苗株(TW2575/98vac)接種雞胚胎蛋，證實減毒疫苗株對雞胚胎的致病程度高於野外株，胚胎死亡率較高、早期死亡較多與胚胎矮化現象較少，且病毒增殖效率較高與產量較多。而雞胚胎蛋分別接種TW2575/98病毒之野外株或減毒疫苗株後的病理學變化無明顯差異。通過免疫組織化學檢測，病毒抗原主要存在於絨毛膜上皮、肺旁支氣管、腎小管上皮細胞及一些在脾臟和心臟漿膜層間皮細胞，顯示早期的胚胎死亡和矮化與疫苗株的細胞/組織親和性的變化無關，但與早期感染的建立和病毒量有關。進而對兩毒株的病毒基因體完全定序後，與國內外數個家禽傳染性支氣管炎病毒野外株及減毒疫苗株比對分析，可在TW2575/98vac病毒基因體中發現44個突變核苷(造成30個變異胺基酸)，且均未在國內外其他病毒株(TW2296/95、Ark/Ark-DPI/81、麻薩諸塞州株、GA98/CWL0470/98和CK/CH等)中看到同樣變化，反之亦然。經過三個方面的進一步比較和評估：(1)對特定的同源毒株繼代中出現變異時間進行縱向分析；(2)對其他5個病毒株之野外株和減毒疫苗株間的胺基酸變化進行水平評估；(3)檢查病毒蛋白中變異的胺基酸化學特性變化，四個發生取代變異的胺基酸位點[P87蛋白之V342D、HD1蛋白之S1493P及P2025S與HD1(P41)蛋白之F2308Y]被選為具高度可能性之分子決定位，與TW2575/98病毒之減毒作用有關。可知不同病毒株成功減毒相關的胺基酸變異位點及型態，其實是多樣性的，並不僅限於特定病毒蛋白的改變或發生取代的胺基酸。再者，為克服減毒疫苗株對雞胚胎具有更高的毒力而降低孵化率，推測可能藉由調低疫苗株於卵內接種之最佳劑量而達成。

Avian infectious bronchitis (IB) caused by the IB virus (IBV) is an acute, highly contagious infectious disease in chickens around the world. Live IB vaccine strain could be acquired because its pathogencity for chickens has lowered after attenuation by continuous serial passages in chicken embryos (CEs). However, its pathogencity for CEs was increased. To investigate the mechanism for pathogenicity of the attenuated IB virus in CEs and virulence related possible molecular determinants, wild strain (TW2575/98w) and vaccine strain (TW2575/98vac) were inoculated into CEs, respectively. It has proved that the attenuated vaccine strains are more pathogenic than the wild virus strains. TW2575/98vac has higher embryonic leathal rate and early death as well as lower dwarfing effect for CEs. Meanwhile, replication efficiency and yield of vaccine strain in CEs are higher. There were no significant differences in the pathological changes in CE infected by both wild and attenuated strains. Detected by immunohistochemistry, the viral antigens of both strains could be found mainly at the epithelium of the chorioallantoic membrane, lung parabronchi, renal tubules and some in the spleen and heart serosa. These findings indicated that the early embryonic death and dwarfing is not related to the change in cell/tissue tropism of the vaccine strain, rather on the early infection establishment and viral load. Full length of viral genomes from TW2575/98w and TW2575/98vac were determined and subject to comparisons with gene sequences of wild strains and vaccine strains from the other IB viruses in Taiwan and other countries. In TW2575/98vac, there are 30 varied amino acid residues resulting from 44 mutated nucleotides compared to TW2575/98w. However, all of the molecular variations lead to attenuation, found in TW2575/98, were not observed consistently in the other IBVs (TW2296/95, Ark/Ark-DPI/81, the Massachusetts strain, GA98/CWL0470/98, and CK/CH/LDL/97I) and vice versa. After further comparisons and evaluations from three aspects: (1) longitudinal analysis on the timing of variations appeared in specific homologous strain passages, (2) horizontal evaluations with the amino acid changes between wild and vaccine strains among the other 5 IBVs, and (3) inspection on alterations in the chemical characteristics of substituted amino acid residues in viral proteins, four amino acid substitutions [V342D in P87, S1493P and P2025S in HD1, as well as F2308Y in HD1(P41)] were selected as highly possible candidates for successful TW2575/98w attenuation. Furthermore, the results in the study imply that molecular variations, which contribute to the successful attenuation of different IBVs, are diverse and not restricted to a fixed pattern or specific amino acid substitutions in viral proteins. Finally, it suggests that the vaccine strain inoculated titer might be adjusted to an optimal low level for in ovo vaccination to overcome the poor hatching rate for its higher virulence to CEs.