Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.
Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.
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DOI：
10.1371/journal.pone.0147723
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Martínez-Sobrido L
中科院分区：
文献类型：
--
作者：
Breen M;Nogales A;Baker SF;Perez DR;Martínez-Sobrido L
甲型和B型流感病毒（分别为IAV和IBV）每年都会引起季节性人类呼吸道疾病流行。此外，IAV还与偶尔发生的大流行病有关，造成了严重的健康和经济后果。在体外或体内研究流感病毒需要使用费力的二级方法来识别感染的细胞。为了规避这一要求，可以使用表达容易追踪的荧光报告蛋白的可复制的传染性流感病毒。Timer是一种荧光蛋白，其经历从绿色到红色的时间依赖性颜色发射转换。光谱变化率与Timer蛋白浓度无关，可用于按时间顺序测量其表达的持续时间。在这里，我们描述了复制能力的IAV和IBV的病毒非结构蛋白1（NS1）融合到荧光动态计时器蛋白的生成。表达定时器的IAV和IBV在组织培养中显示出与野生型病毒相似的空斑表型和生长动力学。在受感染的细胞内，Timer的光谱偏移可用于使用荧光显微镜、读板器或流式细胞术测量感染的速率和细胞间传播。还在小鼠模型中评估了表达Timer的IAV感染的进展，证明了表征体内IAV细胞间感染的可行性。通过提供按时间顺序追踪病毒传播的能力，表达Timer的流感病毒是评价病毒感染的体外和体内动力学的极好选择。
Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer’s spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.