Department of Microbiology and Immunology, University of Rochester, Rochester, New York, USA.
Texas Biomedical Research Institutegrid.250889.e, San Antonio, Texas, USA.
Microbiol Spectr. 2021 Dec 22;9(3):e0160121. doi: 10.1128/Spectrum.01601-21. Epub 2021 Nov 24.
Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. , these bi-reporter-expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. , rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using imaging systems (IVIS) and in the lungs from infected mice. Importantly, we used these bi-reporter-expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections and , including host-pathogen interactions and dynamics or tropism of viral infections. Despite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for and studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection and . This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection.
表达报告基因的重组病毒可用于病毒感染的可视化和定量分析，可作为鉴定感染细胞和动物模型中病毒存在的有效替代物。然而，表达报告基因的重组病毒的一个局限性是使用荧光蛋白或荧光素酶蛋白，它们可根据不同目的交替使用。痘苗病毒（VV）被广泛用作病毒载体，包括单独表达荧光或荧光素酶报告基因的重组（r）VV，这些报告基因在特定用途中很有用。在本报告中，我们构建了两种新型 rVV，它们在病毒基因组的不同位置稳定表达荧光（Scarlet 或 GFP）和荧光素酶（Nluc）报告基因。与亲本 WR VV 分离株相比，这些双报告基因表达的 rVV 具有相似的生长动力学和蚀斑表型。rVV Nluc/Scarlet 和 rVV Nluc/GFP 可有效感染小鼠，并可使用活体成像系统（IVIS）和在感染小鼠的肺部中轻松检测到。重要的是，我们使用这些双报告基因表达的 rVV 来评估病毒发病机制、肺部免疫细胞浸润，并直接鉴定 VV 感染的不同细胞亚群，而无需抗体染色。总之，这些表达两种报告基因的 rVV 为研究病毒感染的生物学特性和病毒感染的动态或嗜性打开了可行性，包括宿主-病原体相互作用。尽管天花病毒（VARV）已被根除，但正痘病毒仍对人类健康构成重大威胁，因为它们可能被用作生物恐怖主义制剂，并且出现了人畜共患的正痘病毒病。表达易于追踪的荧光或荧光素酶报告基因的重组痘苗病毒（rVV）为痘病毒研究的进展做出了重大贡献。然而，表达一种标记基因的 rVV 对和研究有几个限制，因为荧光和荧光素酶蛋白对特定应用有一定的限制。为了克服这些限制，我们生成了稳定表达荧光（Scarlet 或 GFP）和荧光素酶（Nluc）报告基因的优化 rVV，以轻松跟踪病毒感染和。这种新一代双报告基因表达的 rVV 是研究感染细胞和感染动物模型中病毒感染动力学的绝佳选择。
