A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Abstract

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

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

“…1 ). We previously demonstrated the feasibility and advantages of generating bi-reporter influenza A virus (IAV) ( 32 , 33 ) and the prototype arenavirus lymphocytic choriomeningitis virus (LCMV) ( 34 ). This new dual reporter-expressing rVV possess the advantages of both fluorescent and luciferase reporters, which could provide promising applications for the study of VV in vitro and in vivo .…”

Bi-reporter vaccinia virus for tracking viral infections in vitro and in vivo

“…The copyright holder for this preprint this version posted August 26, 2021. ; https://doi.org/10.1101/2021.08.24.457594 doi: bioRxiv preprint Here, we have described the generation of novel rVV stably expressing two different reporter fluorescent and luciferase genes to overcome the current limitations of using single reporter-expressing rVV (Figure 1). We previously demonstrated the feasibility and advantages of generating bi-reporter influenza A virus (IAV) (37,38) and the prototype arenavirus lymphocytic choriomeningitis virus (LCMV) (47). This new dual reporter-expressing rVV possess the advantages of both fluorescent and luciferase reporters, which could provide promising applications for the study of VV in vitro and in vivo.…”

“…Whole lungs were excised and washed with PBS. Scarlet or GFP expression were analyzed in the IVIS as previously described (15, 33, 37, 38). Living Image (v.4.5) software was used to acquire and analyze images to determine the radiant efficiency of the ROI.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…These luciferase-tagged viruses are applicable not only for studying the virus life cycle in vitro and in vivo (viral proliferation, replication, assembly/release and entry) and analyzing virus-host interactions but also for evaluating therapeutic strategies and developing new inhibitors and vaccines. NanoLuc-based reporter viruses were successfully designed to study such dangerous diseases as: influenza A virus [ 232 , 233 , 234 , 235 , 236 ], hepatitis B [ 236 , 237 , 238 , 239 ] and E viruses [ 240 ], HIV [ 241 ], rotavirus [ 242 ], flavivirus [ 243 , 244 , 245 ], and arboviruses [ 246 ] which indicates the great potential of NanoLuc for such application.…”