Department of Microbiology and Immunology, University of Rochester, Rochester, New York, USA.
Center for Animal Health Research, INIA-CISA, Madrid, Spain.
J Virol. 2019 May 1;93(10). doi: 10.1128/JVI.00032-19. Print 2019 May 15.
Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. , BIRFLU effectively infected mice, and both reporter genes were detected using imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections. Influenza A virus (IAV) causes a human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable fluorescent or luciferase reporter protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we generated, for the first time, a recombinant replication-competent bireporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections and The combination of cutting-edge techniques from molecular biology, animal research, and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection.
研究甲型流感病毒（IAV）需要使用辅助方法来检测感染细胞中病毒的存在。为了克服这个问题，我们和其他人已经生成了表达荧光或荧光素酶报告基因的重组 IAV。这些外源报告基因可以作为追踪病毒存在的有效替代物。然而，流感病毒基因组中有限的容纳外源序列的能力迫使研究人员根据研究类型选择荧光或荧光素酶报告基因。为了规避这一限制，我们设计了一种新型的重组复制型双报告基因 IAV（BIRFLU），它可同时表达荧光和荧光素酶报告基因。在培养细胞中，BIRFLU 的生长动力学与野生型（WT）病毒相当，可用于筛选中和抗体或具有抗病毒活性的化合物。两种报告基因的表达可以通过荧光或生物发光来监测病毒的抑制，克服了仅使用一种报告基因作为读数的局限性。BIRFLU 有效地感染了小鼠，并使用成像系统（IVIS）检测到了两种报告基因。生成携带多个外源基因的重组 IAV 的能力为研究病毒-宿主相互作用以及将 IAV 用于高通量筛选（HTS）以鉴定可纳入治疗手段以控制 IAV 感染的新型抗病毒药物开辟了独特的可能性。此外，对病毒基因组进行遗传操作以表达两种外源基因的能力为开发新型流感疫苗提供了可能性，并为使用重组 IAV 作为疫苗载体来治疗其他病原体感染提供了可行性。甲型流感病毒（IAV）引起人类呼吸道疾病，与重大健康和经济后果相关。近年来，表达易于追踪的荧光或荧光素酶报告蛋白的复制型 IAV 的使用极大地促进了流感研究的进展。然而，由于流感病毒基因组中包含外源序列的能力有限，研究人员被迫选择荧光或荧光素酶报告基因。为了克服这一限制，我们首次生成了一种重组复制型双报告基因 IAV（BIRFLU），它稳定表达两种报告基因（一种荧光和一种荧光素酶）以追踪 IAV 感染。分子生物学、动物研究和成像技术的尖端技术的结合为研究人员带来了独特的机会，使他们能够使用这种新一代表达报告基因的 IAV 来研究培养细胞和动物模型中病毒感染的动力学。
