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 occasions in rabbits for up to 3 years after the primary eye infection. To detect these spontaneous reappearances of virus, s for herpes simplex virus were obtained 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 per cent of the viral reactivations occurred out coincidental corneal disease. Once the rabbit eye is infected 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. Disease 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 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 63 from the National Institute of Neurological Diseases and Blindness and AI-3 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 8, 968. Manuscript submitted Aug. 3, 968; manuscript accepted Sept., 968. In 96 during attempts to induce reactivation of herpes simplex virus in eyes of rabbits healed herpetic corneal lesions, several instances of spontaneous virus release were observed. 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, out preceding stimulation) occurred either or out accompanying evidence of corneal disease. ' 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 36 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933/ on //8
Volume 8 Number 3 Recurrence of herpes simplex virus 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. ml. suspension of virus containing approximately - TCD per. ml. for human amnion tissue was placed in the inferior cul-de-sac of albino male and female rabbits and the lids were gently massaged against the cornea for 3 seconds. Typical dendritic figures were noted in 8 to 96 hours, many eyes proceeding to geographic corneal epithelial and stromal involvement iridocyclitis. Viral s of each infected eye were obtained during the course of the infection to verify the cause of the keratitis. At least weeks after the initial herpetic infection, s were obtained on the surviving animals, generally or 6 days weekly. The mortality rate from the primary infection was approximately per cent and was usually due to herpetic encephalitis. Cultures were taken from the eyes 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, out 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 fluorescein for punctate, dendritic, or geographic staining and occasional animals were viewed the biomicroscope. Swabs obtained from the eyes were either inoculated immediately into tubes or the specimens stored at -6 C, depending on the availability of human amnion or rabbit kidney tissue tubes. Virus isolations were performed according to standard procedures which have been described in a previous report. Identification of isolates was confirmed by neutralization tests in s of human amnion cells. Fifty rabbits constituted the study group, infected bilaterally and in one eye. All rabbits were individually caged and cross infection was not noted during these studies. Bilateral viral s were performed intermittently on the rabbits infected in only one eye and viral isolation was never obtained from the uninfected eye. Rabbits were observed for periods ranging from to,3 days after their initial infection, variable interspersed periods of rest. These rest periods were employed to reduce the total number of daily s and observations to a level which could conveniently be handled. Due to the intermittent shedding of virus from the rabbit eye, the following criteria were established to characterize any single episodes of viral reactivation. Positive s separated by days or more of negative s were considered as representing distinct episodes of virus reactivation. When s for virus were positive in both eyes simultaneously (that is, separated by less than days of negative s), 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 s. Results In rabbits, followed in some instances up to 3 years, there were 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 ( of ) 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 ( episodes of spontaneous reactivation in rabbits) Episodes per rabbit 3 6 rabbits 66 episodes 8 Table II. Recurrence of herpes simplex virus in rabbit eyes ( episodes of spontaneous reactivation in rabbits) Duration of episodes (days) - - 8- >H episodes Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933/ on //8
38 Laibson and Kibrick In vestigative Ophthalmology June 969 d for at least 8 days (Table II). Many of the reactivations occurring for just or days would have been missed had s not been obtained at least or 6 days a week. As s 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 of 3 rabbits studied between 6 and days after initial infection, in 3 of 8 rabbits between and 8, and in of from 9 to, days. These "late" episodes of spontaneous reactivation varied in duration as did the earlier ones. Thus of 8 such episodes between s 6 and 8, five persisted for one day or less and the remainder for 3, 8, and days respectively. From to per cent of the rabbits developed spontaneous reactivations during the first days after primary infection. By days this had dropped to 3 per cent, and it remained at that level over the next year (Table III). After 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, this technique was employed to demonstrate persistence of virus in those rabbits which had survived more than days after primary infection. Such treatment was associated additional episodes of virus release from these animals. The total of both induced and spontaneous reactivations during various intervals from to,3 days is given in Table IV. Corneal staining fluorescein was observed during 3 of the episodes of Table III. Recurrence of herpes simplex virus in rabbit eyes (spontaneous reactivations after primary herpetic infection) s after primary herpetic nfection 3- - - 3- - - 6- - 8-3 6 8 9 9-.- 3 rabbits under 3 36 3 3 8 positive 3 6 8 9 8 3 Per cent positive 3 Table IV. Recurrence of herpes simplex virus in rabbit eyes (induced and spontaneous reactivations after primary herpetic infection) s after primary herpetic infection - 8 8-9 9-,,-,3 under reactivations spontaneous virus reactivation. This staining varied from minimal punctate dots 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 s was often noted, but it was unusual to find corneal ulceration out 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 rabbits developed at least one corneal ulcer during a spon- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933/ on //8
Volume 8 Number 3 Recurrence of herpes simplex virus 39 taneous virus reactivation. The longest interval over which virus was recovered in any single reactivation period was days, a total of 9 positive s noted during this time, although positive s were recorded during a shorter spontaneous reactivation episode. Episodes of spontaneous reactivation involving both eyes occurred 8 times. During of these 8 episodes virus was isolated on days or more from each eye. In these rabbits corneal ulcers were more common than in animals unilateral involvement. Thus, 6 of the 8 developed such ulcers, and of these, 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 herpes simplex virus, was long thought to recover from the initial ocular infection out developing recurrent herpetic keratitis. Recent evidence, however, indicates that herpetic keratitis in this animal is not a self-limited disease, and corneas which heal out scarring (by slit lamp examination) are subject to spontaneous reinfection this agent.' Herpes simplex virus can persist for long periods at other sites in the rabbit. This agent was recovered from the saliva of rabbits, and 33 months after intraperitoneal inoculation. 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 the rabbit are similar to those naturally occurring this agent in man. The physical characteristics of the corneal disease, the response to antimetabolite therapy, and viral persistence or out keratitis also appear similar in man and rabbit. This study documents episodes of spontaneous viral reactivation in rabbits, over a period of almost 3 years. In addition to spontaneous reactivations onset between and,3 days after primary infection, episodes of reactivation were induced during the same period 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 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 periods of virus recovery, coincidental pathological changes in the cornea were noted 3 times (8 per cent). Therefore, herpes simplex virus, isolated from the eyes of these rabbits, did not cause detectable disease 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 tract and the human female genital tract. The relative role of host susceptibility and such local factors as trauma, anoxia, and fever in inducing reappearance of virus or out 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933/ on //8
3 Laibson and Kibrick Investigative Ophthalmology June 969 REFERENCES. 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. : 68, 96.. Laibson, P. R., and Kibrick, S.: Reactivation of herpetic keratitis by epinephrine in rabbit, Arch. Ophth. :, 966. 3. Laibson, P. R., and Kibrick, S.: Reactivation of herpetic keratitis in rabbit. II. Repeated reactivations in the same host, Arch. Ophth. :, 96.. Kibrick, S., and Laibson, P. R.: Chronic herpes simplex ocular infection in rabbits, in Antimicrobial agents and chemotherapy, 96, American Society for Microbiology.. Nesburn, A. B., Elliott, J. H., and Leibowitz, H. M.: Spontaneous reactivation of experimental herpes simplex keratitis in rabbits, Arch. Ophth. 8: 3, 96. 6. Kaufman, H. E., Brown, D. C, and Ellison, E. M.: Recurrent herpes in the rabbit and man, Science 6: 68, 96.. Ashe, W. K., and Rizzo, A. A.: Inapparent herpes simplex virus infection in inoculated rabbits, Proc. Soc. Exper. Biol. & Med. :, 96. 8. Schmidt, J. R., and Rasmussen, A. F., Jr.: Activation of latent herpes simplex encephalitis by chemical means, J. Infect. Dis. 6:, 96. 9. Good, R. A., and Campbell, B.: The precipitation of latent herpes simplex encephalitis by anaphylactic shock, Proc. Soc. Exper. Biol. & Med. 68: 8, 98.. 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. 8:, 968.. Yen, S. S., Reagan, J. W., and Rosenthal, M. S.: Herpes simplex infection in female genital tract, Obst. & Gynec. : 9, 96. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933/ on //8