Luciferase Imaging of a Neurotropic Viral Infection in Intact Animals

Abstract

The identification of viral determinants of virulence and host determinants of susceptibility to virus-induced disease is essential for understanding the pathogenesis of infection. Obtaining this information requires infecting large numbers of animals to assay amounts of virus in a variety of organs and to observe the onset and progression of disease. As an alternative approach, we have used a murine model of viral encephalitis and an in vivo imaging system that can detect light generated by luciferase to monitor over time the extent and location of virus replication in intact, living mice. Sindbis virus causes encephalomyelitis in mice, and the outcome of infection is determined both by the strain of virus used for infection and by the strain of mouse infected. The mode of entry into the nervous system is not known. Virulent and avirulent strains of Sindbis virus were engineered to express firefly luciferase, and the Xenogen IVIS system was used to monitor the location and extent of virus replication in susceptible and resistant mice. The amount of light generated directly reflected the amount of infectious virus in the brain. This system could distinguish virulent and avirulent strains of virus and susceptible and resistant strains of mice and suggested that virus entry into the nervous system could occur by retrograde axonal transport either from neurons innervating the initial site of replication or from the olfactory epithelium after viremic spread.Understanding the pathogenesis of a viral infection requires a knowledge of the sites of viral replication and pathways of spread throughout the body. Traditionally, this knowledge has been obtained by infecting sufficient numbers of animals to be able to evaluate several animals per time point and assaying organs for infectious virus at many times after the initiation of infection. This process requires large numbers of animals, and free virus circulating in the blood can confound the interpretation of data on the amounts of virus in a given organ. Furthermore, important sites of virus replication may be missed because appropriate samples were not taken. In vivo imaging might provide an alternative method of assaying viral infection that would eliminate these concerns.In vivo imaging was first used to detect sites of bacterial replication in intact, living animals in 1995 (3). Both green fluorescent protein (26) and firefly luciferase (3) have been used as reporters. Luciferase offers the advantages of producing an inherently low background in animals and not accumulating (25). Luciferase can therefore be used to monitor realtime activity in living animals (6). Significant technological advances continue to be made in our ability to detect small amounts of light, and luciferase imaging has been used successfully to monitor tumors (4, 17, 21), bacterial infections (3, 19), herpes simplex virus expression (12), and viral gene expression (5, 25). However, its usefulness for studies of viral pathogenesis, host susceptibility, and virus strain differences is unclear....

Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: Assessment of an oral agent that stimulates erythropoietin production

“…injection with estimated tissue levels of 60-180 M, with somewhat lower levels in the brain, at the doses used here (26,27). These levels exceed the firefly luciferase luciferin K m (22), and luciferase activity has been successfully imaged in a wide variety of tissues (including brain) by using protocols similar to the one used by us (10,28,29). Nonetheless, the intracellular concentrations of luciferin achieved in different tissues after systemic administration are not known, and differences in luciferin availability might confound luciferase-based imaging in certain settings.…”

Plasmid DNA Vaccine-Elicited Cellular Immune Responses Limit In Vivo Vaccine Antigen Expression through Fas-Mediated Apoptosis

“…This imaging strategy exploits the ability of luciferase protein to catalyze the light-producing oxidation of the small molecule luciferin. Luciferin is inoculated into mice that have received luciferase-expressing immunogens, and the quantity of light emitted by this reaction is monitored in living mice (25). Inoculating mice with increasing quantities of luciferase protein by the i.m.…”

Inhibition of host translation by virus infection in vivo

“…By means of recombinant viruses expressing engineered reporter mRNAs, we present indirect but solid evidence that translation of nonviral mRNA is strongly inhibited in infected mouse-brain neurons. Virus-expressing reporter genes were used in earlier studies to track replication and spreading of the virus to different organs of infected animals (37,41,48). However, to date, the shut off phenomenon has not been addressed in vivo, probably because of the technical difficulties that such a study raises.…”