The effects of cyclophosphamide and ultra-violet B on reactivation of herpes simplex virus-1 in mice

Stephanie Brosius, Dr. Tammie Keadle, Dr. Patrick Stuart, Ophthalmology Department, Washington University School of Medicine

Ocular herpes is a form of herpes simplex virus-1 (HSV-1) characterized by symptoms such as corneal opacity, neovascularization, and blepharitis.  A recurrent ocular herpes infection begins with an acute HSV-1 infection.  The virus then travels to the trigeminal ganglia where it enters the latent phase until a stimulus, typically ultra violet light (UV), triggers reactivation.  Symptoms associated with the virus are actually caused by the immune system’s reaction to the virus rather than the virus itself.  Two types of immune cells, CD4⁺ and CD8⁺, have been shown to cause further disease by fighting the virus and maintaining latency in the trigeminal ganglia respectively.  Since immune cells rapidly divide, it was hypothesized that an alkylating agent such as cyclophosphamide (CyP), which is toxic to these cells, would affect the corneal opacity, the number of days eyes shed virus, and the amount of HSV-1 shed.

To determine the effects of CyP on recurrent HSV-1 induced keratitis, we infected C57BL6 mice with HSV-1, then allowed the virus to establish a latent infection.  The virus was reactivated from latency by exposing the eyes to UVB light.  The day of exposure was referred to as day 0.  In most cages, both eyes were exposed so that the left (uninfected) eye could serve as a control.  On days 1, 3, and 5, half the mice received 4 mg of CyP and the other half received an equal volume of HBSS to account for the shock of injection.  Eyes were swabbed on days 0-7, 11, and 14 and then the swabs were plated on confluent 24 well Vero plates to determine if the eye was shedding virus.  Eyes were also examined on days 7 and 14 for corneal opacity and neovascularization.

Preliminary data from plating days 3-6 showed no significant difference in reactivation rate or in virus shedding.  While it was not significant, there was a trend towards increased mortality in the mice treated with CyP.  It was also observed that the mice treated with UVB only were much more likely to experience clouding of the cornea than the mice that received UVB and CyP.  The current hypothesis for this data is that the CyP killed the immune cells responsible for inducing corneal opacity by fighting the virus.  This would also explain the higher mortality rate as the virus could easily get out of control with nothing repressing it.

The next stage of this research involves finishing plating days 0-2, 7, 11, and 14 to determine if CyP induced prolonged shedding of HSV-1.  If it is determined that CyP had any effect the shedding of HSV-1, the swabs will be titered.  Immunohistology will also be performed to test for the presence of CD4⁺ and CD8⁺ cells in the eyes, blood, trigeminal ganglia, and spleens in both groups.