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Recurrence of herpes simplex virus in rabbit eyes: Results of a three-year study Peter R. Laibson and Sidney Kibrick Spontaneous reactivation of herpes simplex virus in rabbit ocular tissue was found on 112 occasions in 50 rabbits for up to 3 years after the primary eye infection. To detect these spontaneous reappearances of virus, cultures for herpes simplex virus were obtained 5 or 6 clays a week from the cul-de-sac of rabbit eyes for various periods up to 3 years. Keratitis was also intermittently observed but 72 per cent of the viral reactivations occurred without coincidental corneal disease. Once the rabbit eye is infected with herpes simplex virus a chronic inapparent infection ensues which probably persists for the life of the animal. Key words: chronic ocular herpes simplex infection, herpes simplex virus, virus reactivation, herpes simplex keratitis, epinephrine, ophthalmic ointment, time factors, virus isolation, cornea, corneal ulcer, corneal vascularization, fluorescein stain. D In 1961 during attempts to induce reactivation of herpes simplex virus in eyes of rabbits with healed herpetic corneal lesions, several instances of spontaneous virus release were observed.1 In these studies eyes were not examined for evidence of accompanying herpetic disease. Spontaneous shedding of virus from the rabbit eye was subsequently also noted to stimulate reactivations of herpetic keratitis in this host. It was observed at that time that spontaneous shedding of virus (that is, without preceding stimulation) occurred either with or without accompanying evidence of corneal disease.2'3 The present report provides information as to the long-term natural history of herpes simplex ocular infection in the rabbit. It is based on periodic examinations of the rabbit eye for presence of virus and lesions over a 3 year period after primary infection. Results of attempts to induce reactivation of virus during the last year of this period isease caused by herpes simplex virus in man is frequently recurrent in nature. Although the rabbit is not a natural host for this virus, it has been employed for studies with herpes simplex ever since Griiter demonstrated that this virus could replicate in rabbit tissue. From the Cornea Service, Wills Eye Hospital and Research Institute of Temple University Medical Center, Philadelphia, Pa., and the Evans Memorial Department of Clinical Research, University Hospital, and the Departments of Microbiology and Medicine, Boston University School of Medicine, Boston University Medical Center. Supported by Public Health Service Grants NB 06355 from the National Institute of Neurological Diseases and Blindness and AI-04305 from the National Institute of Allergy and Infectious Diseases. Presented in part at the meeting of the Association for Research in Ophthalmology, Tampa, Fla., April 28, 1968. Manuscript submitted Aug. 13, 1968; manuscript accepted Sept. 4, 1968. 346 Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017 Volume 8 Number 3 are also described. These data indicate that once herpetic infection of the rabbit eye occurs, the virus persists as a chronic infection for up to 3 years (the length of time the animals were studied). Materials and methods The initial herpetic corneal infection was produced by the Rodanus strain of herpes simplex virus. A 0.1 ml. suspension of virus containing approximately 105-5 TCD50 per 0.1 ml. for human amnion tissue culture was placed in the inferior cul-de-sac of albino male and female rabbits and the lids were gently massaged against the cornea for 30 seconds. Typical dendritic figures were noted in 48 to 96 hours, with many eyes proceeding to geographic corneal epithelial and stromal involvement with iridocyclitis. Viral cultures of each infected eye were obtained during the course of the infection to verify the cause of the keratitis. At least 4 weeks after the initial herpetic infection, cultures were obtained on the surviving animals, generally 5 or 6 days weekly. The mortality rate from the primary infection was approximately 25 per cent and was usually due to herpetic encephalitis. Cultures were taken from the eyes with a sterile cotton-tipped applicator which was rotated in the lower cul-de-sac, then across the cornea and into the upper cul-de-sac, without prior topical anesthesia. This technique did not denude the epithelium and the rabbits soon accustomed themselves to the daily routine. The conjunctival and corneal epithelium was examined with fluorescein for punctate, dendritic, or geographic staining and occasional animals were viewed with the biomicroscope. Swabs obtained from the eyes were either inoculated immediately into culture tubes or the specimens stored at -65° C , depending on the availability of human amnion or rabbit kidney tissue culture tubes. Virus isolations were performed according to standard procedures which have been described in a previous report.2 Identification of isolates was confirmed by neutralization tests in cultures of human amnion cells. Fifty rabbits constituted the study group, 38 infected bilaterally and 12 in one eye. All rabbits were individually caged and cross infection was not noted during these studies. Bilateral viral cultures were performed intermittently on the 12 rabbits infected in only one eye and viral isolation was never obtained from the uninfected eye. Rabbits were observed for periods ranging from 115 to 1,131 days after their initial infection, with variable interspersed periods of rest. These rest periods were employed to reduce the total number of daily cultures and observations to a level which could conveniently be handled. Due to the intermittent shedding of virus from Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017 Recurrence of herpes simplex virus 347 the rabbit eye, the following criteria were established to characterize any single episodes of viral reactivation. Positive cultures separated by 7 days or more of negative cultures were considered as representing distinct episodes of virus reactivation. When cultures for virus were positive in both eyes simultaneously (that is, separated by less than 7 days of negative cultures), this event was considered a single episode of virus reactivat.on for that rabbit. Although corneal lesions demonstrable by fluorescein staining were intermittently observed in most rabbits they were not considered as evidence of virus reactivation in the absence of positive cultures. Results In 50 rabbits, followed in some instances up to 3 years, there were 112 separate episodes of one or more days of spontaneous virus release. Spontaneous reappearance of virus occurred in all rabbits under study at least once, and one rabbit had seven separate episodes of virus release (Table I). Half of the spontaneous viral reappearances (57 of 112) were noted for only one day, and at the other extreme, during fourteen episodes of reactivation virus was Table I. Recurrence of herpes simplex virus in rabbit eyes (112 episodes of spontaneous reactivation in 50 rabbits) Episodes per rabbit 1 2 3 4 5 6 7 No. of rabbits 22 12 6 6 1 2 1 No. of episodes 22 24 18 24 5 12 7 Table II. Recurrence of herpes simplex virus in rabbit eyes (112 episodes of spontaneous reactivation in 50 rabbits) Duration of episodes (days) 1 2- 4 5- 7 8-11 >H No. of episodes 57 24 17 7 7 348 Laibson and Kibrick cultured for at least 8 days (Table II). Many of the reactivations occurring for just 1 or 2 days would have been missed had cultures not been obtained at least 5 or 6 days a week. As cultures were obtained only once a day, we still may have missed reactivation episodes occurring at other times on that day; therefore, the figures for virus isolation must be considered minimal. The number of rabbits observed for 3 years was limited but from Table III it is apparent that virus may be found in the eyes of rabbits for as long as 3 years after primary infection. Spontaneous reactivations were noted in 5 of 13 rabbits studied between 601 and 700 days after initial infection, in 3 of 8 rabbits between 701 and 800, and in 1 of 4 from 901 to 1,000 days. These "late" episodes of spontaneous reactivation varied in duration as did the earlier ones. Thus of 8 such episodes between Days 601 and 800, five persisted for one day or less and the remainder for 3, 8, and 10 days respectively. From 70 to 77 per cent of the rabbits developed spontaneous reactivations during the first 200 days after primary infection. By 400 days this had dropped to 35 per cent, and it remained at that level over the next year (Table III). After 2 years only a small number of rabbits was available for observation, and in this group spontaneous virus release and corneal disease were infrequently observed. Since previous experiments in our laboratories had shown that epinephrine in ointment form could induce reactivation of herpes simplex virus in the rabbit eye,4 this technique was employed to demonstrate persistence of virus in those rabbits which had survived more than 701 days after primary infection. Such treatment was associated with 7 additional episodes of virus release from these animals. The total of both induced and spontaneous reactivations during various intervals from 701 to 1,131 days is given in Table IV. Corneal staining with fluorescein was observed during 31 of the 112 episodes of Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017 In vestigative Ophthalmology June 1969 Table III. Recurrence of herpes simplex virus in rabbit eyes (spontaneous reactivations after primary herpetic infection) No. of No. of rab- Per cent with Days after rabbits bits with positive positive primary herpetic under nfection culture culture culture Day 40 31- 100 77 31 70 26 37 Day 101- 200 50 18 36 Day 201- 300 35 Day 301- 400 11 31 38 Day 401- 500 9 24 38 Day 501- 600 8 21 38 Day 601- 700 5 13 38 Day 701- 800 3 8 0 Day 801- 900 0 4 25 Day 901-1 000 1 4 0 Day 1.001-1 131 0 2 Table IV. Recurrence of herpes simplex virus in rabbit eyes (induced and spontaneous reactivations after primary herpetic infection) No. of rabbits with Days after primary No. of rabbits herpetic infection under culture reactivations Day 701- 800 Day 801- 900 Day 901-1,000 Day 1,001-1,131 spontaneous virus reactivation. This staining varied from minimal punctate dots with no conjunctival injection to marked geographic comeal ulcers accompanied by severe limbal injection and corneal vascularization. Dendritic figures were also observed in the transition of punctate changes to obvious ulceration. The appearance of punctate corneal staining in the absence of positive virus cultures was often noted, but it was unusual to find corneal ulceration without recovery of herpes simplex virus. Virus recovery was more common in the absence of visible ocular disease, and even on multiple consecutive days of virus isolation, demonstrable corneal disease was not always present. Twenty-three of the 50 rabbits developed at least one corneal ulcer during a spon- Volume 8 Number 3 taneous virus reactivation. The longest interval over which virus was recovered in any single reactivation period was 21 days, with a total of 9 positive cultures noted during this time, although 11 positive cultures were recorded during a shorter spontaneous reactivation episode. Episodes of spontaneous reactivation involving both eyes occurred 8 times. During 7 of these 8 episodes virus was isolated on 2 days or more from each eye. In these rabbits corneal ulcers were more common than in animals with unilateral involvement. Thus, 6 of the 8 developed such ulcers, and of these, 4 also developed corneal vascularization during the course of the reactivation. Discussion Although herpes simplex virus has been recovered intermittently from the eye in man and experimental animals, there have been no long-term animal studies to determine the possibility of spontaneous reappearance of this virus years after the initial herpetic infection. The rabbit, the laboratory animal of choice for ocular experimentation with herpes simplex virus, was long thought to recover from the initial ocular infection without developing recurrent herpetic keratitis. Recent evidence, however, indicates that herpetic keratitis in this animal is not a self-limited disease, and corneas which heal without scarring (by slit lamp examination) are subject to spontaneous reinfection with this agent.'10 Herpes simplex virus can persist for long periods at other sites in the rabbit. This agent was recovered from the saliva of 2 rabbits, 24 and 33 months after intraperitoneal inoculation.7 In addition, several investigators have reported reactivation of herpetic encephalitis well after the primary infection.8'9 The frequent and spontaneous reappearances of herpes simplex virus in the rabbit eye following primary infection indicate that this is the natural course of the experimental disease in this host. These recurrences of herpetic ocular infection in Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017 Recurrence of herpes simplex virus 349 the rabbit are similar to those naturally occurring with this agent in man. The physical characteristics of the corneal disease, the response to antimetabolite therapy, and viral persistence with or without keratitis also appear similar in man and rabbit. This study documents 112 episodes of spontaneous viral reactivation in 50 rabbits, over a period of almost 3 years. In addition to 4 spontaneous reactivations with onset between 701 and 1,131 days after primary infection, 7 episodes of reactivation were induced during the same period with topical epinephiine ointment as the incitant. The longer the interval after primary infection the less frequently were spontaneous reactivations observed. The fact that virus reactivation could be induced with epinephiine ointment following long periods during which virus was not demonstrable indicated that virus (or viral precursor) remained in these hosts. These findings suggest that once this agent is placed on the rabbit eye it produces a chronic inapparent infection which probably persists for life. During the 112 periods of virus recovery, coincidental pathological changes in the cornea were noted 31 times (28 per cent). Therefore, herpes simplex virus, isolated from the eyes of these rabbits, did not cause detectable disease 72 per cent of the time. The mechanism whereby this virus may persist in the eye in the absence of ocular disease is unknown. A similar phenomenon has also been demonstrated for herpes simplex virus in the upper respiratory tract10 and the human female genital tract.11 The relative role of host susceptibility and such local factors as trauma, anoxia, and fever in inducing reappearance of virus with or without disease still remain to be determined. The reisolation of virus from rabbit ocular tissue as long as 3 years after primary infection indicates persistence of virus in this host. Neither the state of the virus nor the site where it persists during quiescent periods, however, is presently known. Investigative Ophthalmology June 1969 350 Laibson and Kibrick REFERENCES 1. Anderson, W. A., Margruder, B., and Kilbourne, E. D.: Induced reactivation of herpes simplex virus in healed rabbit corneal lesions, Proc. Soc. Exper. Biol. & Med. 107: 628, 1961. 2. Laibson, P. R., and Kibrick, S.: Reactivation of herpetic keratitis by epinephrine in rabbit, Arch. Ophth. 75: 254, 1966. 3. Laibson, P. R., and Kibrick, S.: Reactivation of herpetic keratitis in rabbit. II. Repeated reactivations in the same host, Arch. Ophth. 77: 244, 1967. 4. Kibrick, S., and Laibson, P. R.: Chronic herpes simplex ocular infection in rabbits, in Antimicrobial agents and chemotherapy, 1967, American Society for Microbiology. 5. Nesburn, A. B., Elliott, J. H., and Leibowitz, H. M.: Spontaneous reactivation of experimental herpes simplex keratitis in rabbits, Arch. Ophth. 78: 523, 1967. 6. Kaufman, H. E., Brown, D. C , and Ellison, Downloaded From: http://iovs.arvojournals.org/ on 05/04/2017 7. 8. 9. 10. 11. E. M.: Recurrent herpes in the rabbit and man, Science 156: 1628, 1967. Ashe, W. K., and Rizzo, A. A.: Inapparent herpes simplex virus infection in inoculated rabbits, Proc. Soc. Exper. Biol. & Med. 124: 1150, 1967. Schmidt, J. R., and Rasmussen, A. F., Jr.: Activation of latent herpes simplex encephalitis by chemical means, J. Infect. Dis. 106: 154, 1960. Good, R. A., and Campbell, B.: The precipitation of latent herpes simplex encephalitis by anaphylactic shock, Proc. Soc. Exper. Biol. & Med. 68: 82, 1948. Lindgren, K. M., Douglas, R. C , Jr., and Couch, R. B.: Significance of herpes virus hominis in respiratory secretions of man, New England J. Med. 278: 517, 1968. Yen, S. S., Reagan, J. W., and Rosenthal, M. S.: Herpes simplex infection in female genital tract, Obst. & Gynec. 25: 479, 1965.