Study on development of a live attenuated vaccine for equine influenza infection using reverse genetics

马流感是马的一种重要的传染性呼吸道疾病。在兽医学领域，马流感被认为是一个严重的问题，因为它会造成经济损失。马流感灭活疫苗已用于控制感染；然而，其效果并不令人满意。本研究的目的是研制一种马流感减毒活疫苗。通过将编码靶蛋白的基因插入到流感病毒的基因组中，可以制备流感病毒减毒活疫苗。为此，我们建立了一种利用反向遗传学产生含有外源基因的重组流感病毒的策略。我们得到的结果如下。在基因组RNA片段中发现了有效整合病毒颗粒所需的区域。这些区域位于每个基因组片段的两端，包含蛋白质编码序列。因此，我们确定了甲型流感病毒基因组片段中的包装信号。对包装信号的识别使我们能够生产一种带有编码外源蛋白基因的重组病毒。通过将包装信号与外源基因融合，成功地合成了含有绿色荧光蛋白、流感B病毒血凝素和/或神经氨酸酶或水疱性口炎病毒糖蛋白编码基因的甲型流感病毒。乙型流感病毒糖蛋白编码基因感染甲型流感病毒诱导小鼠免疫应答。到目前为止，关于导致甲型流感病毒衰减的突变的知识已经积累起来。因此，引入衰减突变和产生重组甲型流感病毒的系统使我们能够建立一种不仅针对马流感而且针对其他感染的减毒活疫苗。

Equine influenza is an important contagious respiratory disease of horses. In the field of veterinary medicine, equine influenza is recognized as a serious problem because it causes economical loss. Inactivated vaccine for equine influenza has been used to control infection; however its efficacy is not satisfactory.The aim of this study is to develop a live attenuated vaccine for equine influenza infection. A live attenuated vaccine for influenza virus can be generated by insertion of a gene encoding target protein into genome of an attenuated virus. To this end we established a strategy to generate recombinant influenza virus that contained a foreign gene using reverse genetics.The results we obtained were as follows.1.Regions in the genome RNA segments required for their efficient incorporation into virus particle were found. The regions were located at the both ends of each genome segments and contained the protein encoding sequences. Thus we identified packaging signals in the genome segments of influenza A virus.2.Identification of the packaging signals allowed us to produce a recombinant virus with a gene encoding foreign protein. By fusion of the packaging signals to the foreign genes, influenza A virus with the gene encoding the green fluorescent protein, the hemagglutinin and/or neuraminidase of influenza B virus or the glycoprotein of vesicular stomatitis virus was successfully generated.3.Infection of the influenza A virus with the gene encoding influenza B virus glycoprotein induced immune response in mice.So far, knowledge of mutations that lead to attenuation of influenza A virus has been accumulated. Therefore introduction of attenuation mutations and the system to generate recombinant influenza A virus enable us to establish a live attenuated vaccine not only for equine influenza but also other infections.