Spread of HSV-1 Expressing the Enhanced Green Fluorescent Protein (EGFP) Following Anterior Chamber Inoculation in BALB/c Mice

Abstract

Abstract: : Purpose: To determine the pathway and time course of movement of enhanced green fluorescent protein (EGFP) HSV-1 from the anterior chamber into the central nervous system and to the contralateral eye following unilateral ocular infection. Methods: Male BALB/c mice were inoculated intracamerally in the anterior chamber of the right eye with 2 µl (2.6 x 10⁵ PFU/eye) of EGFP HSV-1. Eyes, optic nerves, trigeminal ganglia (TG), and brain were collected for six consecutive days beginning 1 day after inoculation, and infectious virus was quantified. Whole mounts of iris, ciliary body, and retina from the inoculated eyes and the contralateral eyes were examined microscopically for cells expressing EGFP. EGFP expression in inoculated eyes, contralateral eyes, and brain regions was also examined in tissue sections. Results: One day after inoculation, EGFP was found in the iris and ciliary body of inoculated eyes. EGFP was observed in the ipsilateral TG 1 day after inoculation and in the contralateral TG 4 days after inoculation. EGFP expression was seen in the right optic nerve 4 days after inoculation and in the left optic nerve 5 days after inoculation. The mice developed encephalitis as early as 4 days after inoculation, coincident with EGFP expression in numerous brain regions. In the retinas of the injected eyes, EGFP was seen adjacent to the ciliary body on day 4; thereafter, the area of EGFP expression expanded progressively until it included the entire retina. In the contralateral eyes, EGFP-expressing retinal ganglion cells were seen as scattered lesions, and small fluorescent spots were observed in the iris on days 5 and 6. Viral titers in the eyes were determined by plaque assay. High titers of HSV-1 were recovered from the infected eyes on days 1-6. In the contralateral eyes, virus was first detected on day 4 and high titers were detected on days 5 and 6. Conclusions: The results suggest that HSV-1 rapidly causes ocular and neurological damage in infected mice. The spread of HSV-1 in contralateral eyes differs from that in the inoculated eyes. The use of EGFP HSV–1 facilitates examination of the spread of infection in mice, permitting the identification of retinal lesions caused by the virus as well as the transneuronal pathways involved in its dissemination.

Keywords: herpes simplex virus • retinitis • microscopy: light/fluorescence/immunohistochem