Bi-Reporter Vaccinia Virus for Tracking Viral Infections In Vitro and In Vivo

• Published in: Microbiology spectrum Vol. 9; no. 3; p. e0160121
• Main Authors: Chiem, Kevin, Lorenzo, Maria M, Rangel-Moreno, Javier, Garcia-Hernandez, Maria De La Luz, Park, Jun-Gyu, Nogales, Aitor, Blasco, Rafael, Martínez-Sobrido, Luis
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 22.12.2021
• Subjects: Animals; bioluminescence; Cell Line; Female; Fluorescence; Gene Expression; Genes, Reporter; Genome, Viral; GFP; Humans; In Vitro Techniques; Lung - diagnostic imaging; Lung - pathology; Mice; Mice, Inbred BALB C; reporter genes; Research Article; Scarlet; Staining and Labeling; Vaccines, Synthetic; vaccinia virus; Vaccinia virus - genetics; Virology; Virus Diseases - pathology; Virus Diseases - prevention & control; Virus Replication; GFP; in vivo imaging; bioluminescence; fluorescence; in vitro; luciferase; in vivo; NanoLuc; Scarlet; vaccinia virus; reporter genes
• ISSN: 2165-0497; 2165-0497
• DOI: 10.1128/Spectrum.01601-21

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.