Department of Infection Biology, Faculty of Health and Life Sciences, University of Liverpool, Leahurst Campus, Neston, Cheshire, CH64 7TE, UK.
Vaccine. 2013 May 24;31(22):2565-71. doi: 10.1016/j.vaccine.2013.03.055. Epub 2013 Apr 12.
The study investigates the ability of subtype A Avian metapneumovirus (AMPV) to accept foreign genes and be used as a vector for delivery of Infectious bronchitis virus (IBV) QX genes to chickens. Initially the GFP gene was added to AMPV at all gene junctions in conjunction with the development of cassetted full length DNA AMPV copies. After recombinant virus had been recovered by reverse genetics, GFP positions supporting gene expression while maintaining virus viability in vitro, were determined. Subsequently, either S1 or nucleocapsid (N) genes of IBV were positioned between AMPV M and F genes, while later a bivalent recombinant was prepared by inserting S1 and N at AMPV MF and GL junctions respectively. Immunofluorescent antibody staining showed that all recombinants expressed the inserted IBV genes in vitro and furthermore, all recombinant viruses were found to be highly stable during serial passage. Eyedrop inoculation of chickens with some AMPV-IBV recombinants at one-day-old induced protection against virulent IBV QX challenge 3 weeks later, as assessed by greater motility of tracheal cilia from chickens receiving the recombinants. Nonetheless evidence of AMPV/IBV seroconversion, or major recombinant tracheal replication, were largely absent.
本研究调查了禽 A 亚型副黏病毒（AMPV）接受外源基因的能力，并将其作为传染性支气管炎病毒（IBV）QX 基因传递给鸡的载体。最初，在 AMPV 的所有基因连接处添加 GFP 基因，同时开发盒式全长 DNA AMPV 拷贝。通过反向遗传学回收重组病毒后，确定了 GFP 位置在支持基因表达的同时保持病毒在体外的存活能力。随后，将 IBV 的 S1 或核衣壳（N）基因置于 AMPV M 和 F 基因之间，后来通过在 AMPV MF 和 GL 连接处分别插入 S1 和 N 制备了二价重组体。免疫荧光抗体染色显示，所有重组体在体外均表达插入的 IBV 基因，此外，所有重组病毒在连续传代过程中均高度稳定。在 1 日龄时用一些 AMPV-IBV 重组体滴眼接种鸡，3 周后用强毒 IBV QX 攻毒，结果显示接受重组体的鸡的气管纤毛运动性更强，从而诱导了对强毒 IBV QX 的保护。然而，AMPV/IBV 血清转化率或主要重组气管复制的证据基本上不存在。
