Characterization of a neuraminidase-deficient influenza A virus as a potential gene delivery vector and a live vaccine
Characterization of a neuraminidase-deficient influenza A virus as a potential gene delivery vector and a live vaccine
复制标题
DOI：
10.1128/jvi.78.6.3083-3088.2004
复制
发表时间：
2004-03-01
影响因子：
5.4
通讯作者：
Kawaoka, Y
中科院分区：
文献类型：
--
作者：
Shinya, K;Fujii, Y;Kawaoka, Y
我们最近在甲型流感病毒的神经氨酸酶（NA）病毒RNA（vRNA）片段中鉴定了包装信号，使我们能够产生突变病毒[GFP（NA）-Flu]，该病毒缺乏大部分NA开放阅读框，但含有编码绿色荧光蛋白（GFP）的基因。为了利用vRNA包装信号的扩展知识来建立用于表达外源基因的流感病毒载体，我们研究了该病毒在细胞培养物和小鼠中的复制特性。与野生型病毒相比，GFP（NA）-Flu在正常培养的细胞中高度减毒，但在细胞表面上表达降低水平的唾液酸的突变细胞系中能够生长至> 10（6）PFU/ml的滴度。该病毒的GFP表达即使在后者细胞中传代5次后也是稳定的。在鼻内感染的小鼠中，感染后4天在鼻粘膜、细支气管和肺泡的上皮细胞中检测到GFP。我们将GFP（NA）-Flu的生长减弱归因于细支气管上皮细胞表面的病毒粒子聚集。在测试该突变体作为减毒活流感疫苗的潜力的研究中，当用致死剂量的亲本病毒攻击时，用大于或等于10（5）PFU的GFP（NA）-Flu接种的所有小鼠存活。这些结果表明，流感病毒可能是一个有用的载体表达外源基因和唾液酸酶缺陷的病毒可能提供一种替代活流感疫苗最近批准用于人类使用。
We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of > 10(6) PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with greater than or equal to 10(5) PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.