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.

FIG. 1.

Schematic representations of recombinant viruses used. The arrows indicate the subgenomic promoter. ns, nonstructural; GOI, gene of interest. Percentages of mortality of 4-week-old female albino B6 or BALB/c mice inoculated intracerebrally with 10³ PFU of recombinant viruses are shown. a, P was 0.0096 when values with NSV7-Luc were compared to those with TRNSV-Luc; b, P was 0.0054 when values for albino B6 mice were compared to those for BALB/c mice (Kaplan-Meier analysis).

FIG. 2.

Imaging of 4-week-old albino B6 mice after i.c. inoculation of NSV7-Luc. Virus is detectable in the brain (A) and spinal cord (B). Animals were subjected to imaging for 3 (A) or 5 (B) min. The animal in panel B had its head covered to prevent camera saturation by the stronger signal from the brain.

FIG. 3.

Correlation between light detected by the camera and in vitro plaque assays. Light was correlated with infectious virus as measured by plaque assay. Each point represents data from a single 4-week-old albino B6 mouse studied 1 to 3 days after infection with TRNSV-Luc (A) or NSV7-Luc (B).

FIG. 4.

Detection of differences in levels of virus virulence associated with the ability of virus to replicate in the CNS. Four-week-old BALB/c mice were infected i.c. with 10³ PFU TRNSV-Luc (closed symbols) or NSV7-Luc (open symbols) and subjected to imaging with the IVIS camera system on days 1, 3, and 5 after infection. The calculated number of PFU per brain was determined for each mouse at each time point from the appropriate correlation curve in Fig. 3.

FIG. 5.

Detection of differences in levels of virus replication in susceptible and resistant strains of mice. Four-week-old susceptible albino B6 or resistant BALB/c mice were infected with TRNSV-Luc (A) or NSV7-Luc (B) and subjected to imaging with the IVIS system. Values plotted are averages of results for four mice, and error bars are ± standard errors. *, P < 0.10; **, P < 0.05 (Student's t test).

FIG. 6.

Tracking the spread of virus from the periphery to the CNS in 11-day-old albino B6 mice inoculated s.c. with 10³ PFU of TRNSV-Luc in the right hind footpad. Representative images of viral replication at the site of inoculation 8 h postinfection (A), the nose on day 1 (B), the lower spinal cord on day 3 (C), and the brain on day 4 (D). All animals were subjected to imaging for 5 min.