Vol. 15, No. 3 Printed in U.S.A.

INECTION AND IMMUNITY, Mar. 1977, P. 772-775 Copyright e 1977 American Society for Microbiology

Reliable In Vivo Model for Latent Herpes Simplex Virus Reactivation with Peripheral Virus Shedding ANTHONY B. NESBURN,* MARY T. GREEN, MARIA RADNOTI, AND BRUCE WALKER Estelle Doheny Eye Foundation* and Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles, California 90033

Received for publication 25 June 1976

A long-sought laboratory model, a reliable system for in vivo reactivization of latent herpes simplex infection with peripheral shedding of active virus, is presented. Results demonstrate that surgical stimulation of latently infected rabbit trigeminal ganglia induces peripheral release of infectious herpes simplex virus. Virus could be cultured from the tear film in 83% of the stimulated animals' eyes within 48 h. A pattern of latency, followed by reactivation, is characteristic of many herpes viruses (8). In humans, the propensity for recurrence of disease is of particular importance. Repeated bouts of dermal and oral herpes appear to heal without great consequence. However, ocular infection and its recurrences are a major cause of corneal blindness and a less important, but significant, cause of iritus and secondary glaucoma. In this country, it is the most frequent, serious viral infection of the eye. Although the mechanism is not understood, the nervous system acts as a reservoir for herpes simplex virus (HSV) in its dormant state (1, 2, 4, 10, 12, 18). The key to the latency-reactivation cycle lies in the sequence by which latent infection of the nervous system is translated into active disease in the peripheral tissues. The neuronal theory states that after primary infection, the neuron cell bodies become reservoirs for latent HSV, the axons functioning as conduits transporting the virus to and from peripheral tissues (18). Although we and others have conducted experiments that provide indirect proof of the neuronal theory, the lack of a reliable animal model has handicapped researchers seeking to elucidate this process. Previous attempts to reactivate peripheral infection in either the skin or the eye have been disappointing, for the results were neither rapid nor reproducible (5, 9, 17, 19). Using the method reported here, we have succeeded in overcoming these limitations. By direct mechanical stimulation of latently infected rabbit trigeminal ganglia, we can induce peripheral ocular shedding of infectious HSV on command in over 80% of animals within 48 h. This system should facilitate investigation of the herpes virus latency-reactivation cycle. For many reasons, including its close resem-

blance to human disease, the rabbit spontaneous recurrence model has been used extensively in HSV research. Our earlier work has shown that, in rabbits, ocular HSV infections simulate the human syndrome. Like humans (6), the latently infected rabbit continues to shed virus in the tears and sporadically displays clinical lesions (15). The interval of natural shedding was documented in an earlier study in which 10 rabbits infected with McKrae strain HSV were cultured daily for 6 months. More than 2 months after the signs of primary infection had cleared completely, 13 of 20 eyes (65%) spontaneously released HSV, yielding positive cultures for 1 to 11 days (average, 7 days). Six to 10% of the eyes were positive at any one time (11, 15, 16). The relevance ofthese animal data to human disease has been strengthened by the recent demonstration of latent HSV infection of human trigeminal (2) and sacral ganglia (1). Neurosurgical observations in humans have demonstrated that stimulation of trigeminal ganglia by transaction normally leads to clinical outbreak of herpes (8). Interestingly, direct mechanical stimulation by blunt probing of latently infected trigeminal ganglia proved to be the most effective means of precipitating peripheral HSV shedding in rabbits. MATERIALS AND METHODS Infection. New Zealand albino rabbits (2 to 3 kg) were bilaterally infected without scarifying the cornea. A total of 106 primary rabbit kidney plaqueforming units of McKrae strain HSV, type 1, were applied to the eye, as previously described (12, 16). Daily conjunctival swab cultures (6 days/week) were initiated 6 weeks after infection to document the rate of spontaneous HSV shedding into the tear film. Animals selected for the study were known ocular shedders of HSV but had negative cultures 772

IN VIVO HSV REACTIVATION

VOL. 15, 1977 for a minimum of 2 weeks, as well as a negative clinical ocular examination immediately prior to stimulation. Uninfected animals served as negative controls. Procedure for mechanical stimulation. Preoperative intramuscular acepromazine (15 mg), ketamine (50 mg), and scopolamine (0.25 mg), combined with intravenous Nembutal (20 to 25 mg), provided excellent anesthesia with no anesthetic deaths. Mannitol (1 g/kg) was given intravenously to dehydrate the brain and thus facilitate surgical manipulation. Aseptic precautions were used for all surgical procedures. On the side to be stimulated, a 1- by 0.7-cm parietal craniectomy and removal of meninges were performed. The brain was gently retracted toward the opposite side until the tentorium, covering the trigeminal ganglion, could be located. At times, some cerebral cortex was removed. In the 31 animals (16 infected and 15 noninfected) subjected to mechanical stimulation, a sterile, blunt, right-angle tipped probe, 5 mm long and 0.75 mm in diameter, was inserted through the soft center part of the tentorium covering the trigeminal ganglion. The probe was run posteriorly in the canal containing the ganglion three times for 1 s each. Blood loss was minimal. The cranial cavity was irrigated with 10 ml of gentamicin (0.8 mg/ml). The removed calvarium was replaced with a Gelfoam skull cap. The skin was closed with wound clips. The procedure for the 10 sham-operated animals was identical, except that the trigeminal ganglion and the tentorium covering it were avoided. Postoperative monitoring for HSV recurrence. Beginning 4 h after surgery, conjunctival swab cultures were taken bilaterally every 2 h through h 72 and then daily on days 4, 5, and 6. The swabs were inoculated directly into the medium on monolayer cultures of primary rabbit kidney cells. Cell cultures. Monolayer cell cultures of primary rabbit kidney were prepared by overnight trypsinization at 40C of minced kidney from 2- to 5-week-old animals. Cells were grown to confluency and maintained for 1 to 3 weeks in 32-ounce (ca. 1-liter) prescription bottles in our standard medium (16). Cells from these bottles were trypsinized, spun, resuspended in medium, and used to seed 2-dram (ca. 2.4g) rectangular perfume bottles. Cultures were taken

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using a sterile dacron swab, which was rotated in the upper fornix, gently swept across the cornea, and rotated in the lower fornix. The swab was immediately inoculated into the medium over the monolayer culture of primary rabbit kidney cells. (The swab was swirled in the medium over the monolayer for approximately 5 s.) Cultures were incubated at 350C in a stationary position and were examined daily for typical herpes virus cytopathic effect. Cultures were held for 2 weeks before being considered negative. All degenerating, suspicious, and positive cultures were passed in primary rabbit kidney cells. Identification of isolates. Cultures exhibiting cytopathic changes were frozen at -70'C for later serological identification. In these tests, equal volumes of appropriately diluted normal or anti-HSV (McKrae) rabbit serum and virus were incubated for 1 h at 370C..Two--t .nths milliliter of reaction mixture containing 100 50% tissue culture infective doses of virus as inoculated into each of four wells of a microtesting plate containing monolayers of primary rabbit kidney cells. Plates were incubated at 370C in a 5% carbon dioxide atmosphere and read at 72 h, when the virus control well showed 3 to 4+ cytopathic effect.

RESULTS

Four of the 16 infected animals and 6 of the 15 noninfected animals died within 16 h of trigeminal manipulation. We did not count them in our results, since the first positive culture was obtained at h 18. In the stimulated infected group, two ipsilateral eyes did not respond at all. However, the remaining 10 of 12 eyes (83%) on the side of the stimulated ganglion shed virus within 48 h. Two of the 10 eyes were positive at 18 h. By h 24, 7 of 10 had shed virus, with the other three shedding at 18, 30, and 48 h, respectively. In the same time period (48 h), none of the ipsilateral eyes of the 10 sham-operated animals showed evidence of virus shedding. By the end of the experiment (day 6), a single sham-operated animal had shed HSV (at h 64). At both 48 h and 6

TABLE 1. Effect of unilateral mechanical stimulation of trigeminal ganglia latently infected with HSV Determination

Stimulated

Sham-operated

group'

No. of ipsilateral eyes shedding HSV by 48 h after stimula10/12 tion/no. of eyes No. of ipsilateral eyes shedding HSV by end of expt (day 10/12 6)/no. of eyes No. of HSV isolations through day 6 from ipsilateral eyes/ 101/382 no. of cultures No. of HSV isolations from contralateral eyes/no. of cul16/382c tures a Stimulated group consisted of 12 infected animals, and 9 animals served as positive cultures were obtained from the noninfected animals. b Ten animals served as sham-operated controls. " These 16 positive cultures occurred in 4 of 12 animals.

groupb 0/10

2Probability xPrbilt

Reliable in vivo model for latent herpes simplex virus reactivation with peripheral virus shedding.

Vol. 15, No. 3 Printed in U.S.A. INECTION AND IMMUNITY, Mar. 1977, P. 772-775 Copyright e 1977 American Society for Microbiology Reliable In Vivo Mo...
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