A paper published on October 2 in the Journal of Virology describes an exciting development in the world of influenza research—the construction of a luciferase reporter virus that does not affect virulence and can be used to track development and spread of infection in mice.
Insertion of luciferase reporter genes into viruses has been accomplished before for several viruses, but has not been successful for influenza. Construction of influenza reporter viruses is complicated because the viral genome is small and all the viral genes are critical for infection. Therefore, replacement of an existing gene with a reporter gene or insertion of additional reporter sequences without affecting the virus’s ability to replicate and cause infection has proven difficult. To be successful, a reporter gene needs to be small enough to insert into the viral genome without eliminating any other vital functionality.
The authors of the J. Virology paper, Tran et al from the Department of Medical Microbiology and Immunology at the University of Wisconsin, describe construction of an influenza virus containing NanoLuc luciferase—a new, small (19KDa) luciferase reporter distinguished by its small size and extremely bright luminescence. They were able to create a reporter virus that was stable and retained its pathogenicity by attaching the NanoLuc reporter gene to the c-terminal end of the gene for the viral polymerase subunit PA. Sequence encoding the self-cleaving 2A peptide from porcine teschovirus was placed between the PA and the NLuc genes so that separate PA and NLuc proteins could be generated from a single protein precursor.
The NanoLuc reporter virus displayed the same features as the wildtype virus in cell culture, was able to replicate in mice at a similar rate, and caused similar pathogenic effects. Bioluminescence imaging in live mice detected luminescence at two days post infection, a detection level that was more sensitive than plaque assays. Lethal dose was the same for both viruses, and calculation of viral load using the luciferase reporter assay gave the same results as TCID50 assays. Also, due to the brightness of the NanoLuc luminescence, imaging of the reporter virus was sensitive enough to detect viral clearance of a sublethal infection.
NanoLuc Luciferase was developed by scientists at Promega. It’s small size and bright luminescence make it possible to use this new luciferase in several applications where other luciferases may fail. This paper provides one example where both the small size and the brightness were exploited successfully. The bright signal allowed sensitive detection of the early stages of infection, and the small size allowed construction of a reporter in a situation where gene size was critical.
Here’s the Paper:
Tran V, Moser LA, Poole DS, & Mehle A (2013). Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. Journal of virology, 87 (24), 13321-9 PMID: 24089552