Adenovirus types 8 and 19 infection of rabbit corneal organ cultures. Harold S. Schwartz and Herbert M. Yamashiroya

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1 Adenovirus types 8 and 19 infection of rabbit corneal organ cultures Harold S. Schwartz and Herbert M. Yamashiroya Adenovirus types 8 and 19 are responsible for a large number of cases of acute follicular conjunctivitis, mainly in the form of epidemic keratoconjunctivitis (EKC). This study successfully demonstrates the establishment of adenovirus infection in rabbit corneal organ cultures. Normal rabbit corneas were maintained in culture without significant morphologic alterations for up to 7 weeks. Adenovirus types 8 and 19 replication in rabbit corneas was demonstrated by viral growth curves and subpassage studies, lntracellular localization of adenoviral antigens was confirmed with immunoflu ores cent techniques. The characteristic cytopathic effects of adenoviral infection were observed with the use of various histochemical stains. Thus an in vitro corneal organ culture model was established for the study of the pathogenesis of corneal lesions in adenovirus EKC. Key words: adenovirus, corneal cultures, organ cultures, keratoconjunctivitis, viral cytopathology, viral immunofluorescence Acute follicular conjunctivitis is commonly associated with several adenovirus types, as well as other viral and nonviral pathogens. Epidemic keratoconjunctivitis (EKC) is caused primarily by adenovirus types 8 and 19, and occasionally other adenovirus types. 1 Up to 5% of patients with EKC have been reported to manifest delayed corneal involvement. Among the corneal manifestations are subepithelial infiltrates and stromal opacities. It has been speculated that the nature and development of these corneal le- From the Illinois Eye and Ear Infirmary and the Virology Section, University of Illinois Hospital Laboratories, Chicago, 111. Supported in part by grants from Fight for Sight, Inc., New York, N. Y., and Illinois Society for the Prevention of Blindness, Chicago, 111. Presented at The Association for Research in Vision and Ophthalmology, Sarasota, Fla., May Submitted for publication Oct., Reprint requests: Harold S. Schwartz, M.D., Illinois Eye and Ear Infirmary, 1855 W. Taylor, Chicago, sions involve an immunologic phenomenon, since the infiltrates become evident only after some days to weeks following the onset of the acute follicular conjunctivitis. " 5 A similar mechanism involving an immune reaction in the corneal stroma has been proposed for the pathogenesis of herpetic keratitis. 6 Studies on the pathogenesis of adenoviral EKC have been hampered by the lack of an animal model of adenovirus ocular infection. Although the rabbit eye has long been the experimental model of herpetic keratitis, no animal model exists for adenoviral ocular disease. Experimental animals including the rabbit have been reported to be generally insusceptible to human adenovirus infection. 7 However, the susceptibility of isolated rabbit corneal organ cultures to adenovirus infection has not been determined. This study reports on the long-term cultivation of rabbit corneal tissue and the successful establishment of adenovirus types 8 and 19 infection in these cultures /79/9956+8$.8/ 1979 Assoc. for Res. in Vis. and Ophthal., Inc. Downloaded From: on 11/5/18

2 Volume 18 Number 9 Adenovirus infection of rabbit cornea 957 Methods and materials Preparation of corneal organ culture. Corneal cultures were prepared according to a modification of the technique of Summerlin et al. 8 New Zealand white rabbits were sacrificed by an intravenous lethal dose of sodium pentobarbital. Immediately after death, the rabbit eyes were enucleated and placed in sterile bottles with the cornea side up on cotton premoistened with sterile saline. Under aseptic conditions, the eyes were removed from the bottles, and the corneas with a mm rim of attached sclera were dissected with a straight corneoscleral scissors. The corneoscleral button was then placed epithelial side up onto a sterile prescored microscope slide contained in a mm Petri dish. The corneoscleral button was made to adhere to the slide by placing a drop of fibrinogen and a drop of thrombin beneath the button. The corneal cultures were cultivated in Eagle's minimum essential medium (EMEM) containing 1% fetal calf serum (FCS), 1% L-glutamine, 1 U/ml penicillin, 1 /Ag/ml streptomycin, and.5 /ng/ml amphotericin B. Maintenance medium consisted of EMEM plus % FCS and antibiotics. The cultures were incubated in a humidified CO incubator at 7 C, and the medium was changed at - to -day intervals. Sufficient medium was added to moisten the epithelial layer, yet allowing the epithelium to maintain an air-fluid interface. Cell cultures. Primary human embryonic kidney (HEK) monolayer cultures were purchased from Microbiological Associates, Inc., Bethesda, Md. Maintenance medium for these cells consisted of Eagle's basal medium (BME) containing % FCS and antibiotics. Rabbit iris monolayer cultures initiated by Dr. Marilyn Miller (Illinois Eye and Ear Infirmary) from iris tissue of a New Zealand white rabbit were obtained through the courtesy of Dr. Robert Pumper (Department of Microbiology, University of Illinois College of Medicine). The cells were epithelioid in appearance and had undergone more than 1 passages and had been frozen under liquid nitrogen for 16 years prior to use in our studies. The cultures were maintained in BME containing 5% FCS. All cultures were incubated at 7 C in a humidified CO incubator. Stock viruses. Prototype adenovirus types 8 and 19 used in this study were isolated from patients with EKC 9 and typed by the neutralization test in HEK cells. Both adenovirus types were serially propagated for up to six passages in rabbit iris cells prior to use in the organ culture experiments. Virus inoculation of corneal organ cultures. Four days after establishment of the corneal organ cultures, adenovirus types 8 and 19 were inoculated into duplicate cultures in the following manner: after the medium had been suctioned off,. ml containing 1 tissue culture infective dose 5% (TCID 5 ) of adenovirus was dropped directly on the corneal button of one set of cultures. The inoculum was allowed to adsorb to the corneal epithelium for min prior to the addition of fresh medium. In the second set of cultures,. ml of adenovirus inoculum was injected directly into the superficial cornea by means of a 7-gauge needle with a Zeiss dissecting microscope. The medium was removed prior to injection and replaced with fresh medium min after introduction of the inoculum. Control uninfected cultures were processed in similar fashion with sterile medium substituted for the viral inoculum. Titration of viruses. For up to 7 weeks after infection, the medium was removed on days, 7, 1, 1, 5, and 9 from the infected and control cornea cultures and frozen at 7 C until titrated. The TCID5 was determined in HEK or rabbit iris cells according to the method of Reed and Muench. 1 The end point of virus activity was determined by observation of characteristic adenoviral cytopathic effects (CPE). To further assess the susceptibility of corneal organ cultures, the infective viral fluids were passed serially six times at 1-day intervals in organ culture. Histochemical and itnmunofluorescent examination of corneal cultures. At, 7, 1, 1, 5, and 9 days after adenoviral infection, one half of each corneal culture was fixed in 5% buffered formalin for histochemical evaluation. Following fixation the tissue was routinely processed for staining with hematoxylin-eosin (H&E), Alcian blue, periodic acid-schiff (PAS), and van Gieson stains. The remaining half of reach cornea was immersed in a solution of isopentane-liquid nitrogen and rapidly frozen in a metal container. Cryostat sections were then prepared for immunofluorescence examination. The sections were placed on glass microscope slides, allowed to air-dry, and equilibrated with hemagglutination (HA) buffer (Difco, ph 7.) for min at room temperature prior to immunofluorescent staining. The immunofluorescence procedure with adenovirus types 8 and 19 has been previously described. 9 Results Adenovirus susceptibility of rabbit iris monolayer cultures. Rabbit iris cells infected Downloaded From: on 11/5/18

3 958 Schwartz and Yamashiroya Invest. Ophthalmol. Visual Sci. September 1979 Tltrotlon In HEI Tltratloi li HEI o li ectlob techilqoe adsorption techilqie o li octloa techilqio adsorption technique days post Inlectloi Fig. 1. Effect of inoculation by adsorption or injection on adenovirus type 8 replication in rabbit corneal cultures days post Infection Fig.. Effect of inoculation by adsorption or injection on adenovirus type 19 replication in rabbit corneal cultures. with adenovirus types 8 and 19 exhibited typical adenoviral cytopathogenic effects of rounding and "grapelike" cluster of cells. During the initial two or three passages, an average of 1 days elapsed between inoculation of the infective supernatant fluids and observation of viral CPE. However, in subsequent passages, advanced CPE was observed to 5 days after inoculation. After six serial passages in culture, the infectivity titers of adenovirus 8 and 19 were 1~ 6 and 1~ 1 /.1 ml, respectively, as determined in rabbit iris cells. Growth and maintenance of normal rabbit corneal organ cultures. Rabbit corneal organ cultures were placed on EMEM supplemented with either 1% FCS (growth) or % FCS (maintenance) for periods up to 9 days of incubation. The number of epithelial cell layers attained with growth medium, as observed in H&E stained sections, increased from to 6 layers by day 1 and decreased to layers on day 9. Cultures placed on maintenance medium attained a maximum of epithelial layers by day 1 and fell to layers on day 9. No difference was observed with growth or maintenance medium in the number of keratocytes, which increased from per high-power field (hpf) on day to per hpf by day 1 and fell to per hpf by day 5. The endothelium and Descemet's membrane were well preserved until day 9 of cultivation. The cultures were terminated on day 9 because of disruption of the endothelium, excessive stromal edema, and splitting of Bowman's membrane. Alcian blue stain for mucopolysaccharides revealed no significant alterations in the corneal cultures. Van Gieson stain for collagen did not show a significant increase in collagenous tissue throughout the 9-day period. PAS stain showed no increase in deposition of basement membrane material in Descemet's or Bowman's membranes. Adenovirus susceptibility of rabbit corneal organ cultures. The growth curves for adenovirus types 8 and 19 in rabbit corneal organ cultures infected with virus passaged in rabbit iris cells are shown in Figs. 1 and. With both adenovirus types, peak infectivity titers were obtained at 1 days after infection with a gradual decline in viral titer thereafter until termination of the cultures on day 9. The titers obtained in "injected' cultures were slightly higher than those obtained in "adsorbed" cultures. The peak titer for adenovirus type 8 injected into the corneal cultures was 1~ /.1 ml, whereas the peak titer for the same virus adsorbed onto the corneal cultures was 1~ /.1 ml. The peak titer for adenovirus type 19 by the injection technique was 1~ 5 /.1 ml, and the highest titer obtained by adsorption was 1~ /.1 ml. Infectivity titrations were done on adenovirus types 8 and 19 serially passaged in rabbit corneal organ cultures at 1-day intervals for six consecutive passages. An initial inoculum of 1 TCID 5 was employed for both Downloaded From: on 11/5/18

4 Volume 18 Number 9 Adenovirus infection of rabbit cornea 959 Table I. Effect of adenovirus infection on the corneal epithelial layer No. of epithelial layers* Days after infection Growth Noninfected Maintenance Growth Infected Maintenance * Average number of intact epithelial layers in 1 different microscopic fields. Table II. Cytologic alterations in infected epithelial cells Days after infection Morphologic alterations in infected cultures epithelial layer Slight cytoplasmic vaculation 7 Pronounced cytoplasmic vaculation 1 Intranuclear basophilic inclusions 1 Nuclear enlargement and loss of cytoplasmic volume 1 Degeneration and loss of epithelium 5 Complete denudation of epithelium; fragmentation of Bowman's 9 No further changes adenovirus types. The infectivity titers were 1- - /.1 ml and l(r - 5 /.1 ml for adenovirus types 8 and 19, respectively, at the third passage level. These increased to a final titer of 1~ - 8 /.1 ml for adenovirus type 8 and 1Q-.8/Q ^ ml for type 19 at the sixth passage level. Histopathologic changes in infected corneal cultures. The histologic observations with the H&E stain in the epithelial layer in infected and control corneal organ cultures are summarized in Tables I and II. The cytopathologic changes were similar in adenovirus types 8 and 19 infected corneal cul-,tures. The epithelium consisting of cell layers on day after infection was gradually denuded and completely lost at 5 days. In contrast, the epithelium in uninfected cultures gradually increased from layers on the fourth day to 6 layers on the thirty-fifth day of incubation. At days after infection, early Table III. Histopathologic changes in infected corneal stroma Days after infection *Endothelium intiict. Morphologic alterations in infected cultures stromal layer* No abnormalities Slight rarefaction of anterior half Fibrosis extending to posterior half Moderate rarefaction of anterior half Severe rarefaction of anterior half Slight fibrosis of posterior half No further changes viral cytopathogenic effects were noted, with occasional cells displaying cytoplasmic vacuolation which became more pronounced by 7 days after infection. At 1 days typical adenoviral intranuclear basophilic inclusions were seen. Nuclear enlargement and loss of cytoplasmic volume and cell degeneration were noted by day 1. At 1 days after infection, loss of epithelium was seen. Complete epithelial denudation and fragmentation of Bowman's membrane, as observed with the PAS stain, occurred at day 5. The histologic changes observed in the stromal layer of infected corneal cultures are recorded in Table III. Alcian blue staining revealed no abnormal changes in the character of acid mucopolysaccharides in infected compared to control preparations. The endothelium remained intact throughout the experimental observation period as observed with the PAS stain. As infection progressed, an increase in the collagenous tissue was detected by the van Gieson method. This in- Downloaded From: on 11/5/18

5 96 Schwartz and Yamashiroya invest, Ophthalmol. Visual Sen. September 1979 Fig.. Normal uninfected rabbit corneal organ culture at 1 days' incubation. Note the four epithelial layers and intact stroma containing spindle-shaped keratocytes. (H&E; x 16.) Table IV. Effect of adenovirus infection on number of corneal keratocytes Days after infection No. of keratocytes per hpf* Noninfected t Infected * High power field; average number of intact keratocytes in 1 different microscopic fields. ft 1 i 11 i crease was first seen in the anterior stromal layers during the first weeks after infection and progressively involved the entire stroma by week 7 after infection. In H&E preparations changes in the number of keratocytes observed in infected and uninfected corneal cultures are summarized in Table IV, A decrease in the number of keratocytes was observed in infected corneal cultures from per hpf at day 7 to none by day 1. In contrast, the numbers of keratocytes in control cultures were per hpf on days 1 and 1, and at day 9. Representative cytopathic changes are illustrated in Figs. to 5. lmmunofluorescent localization of adenovirus antigens. Both adenovirus types 8 and 19 were detected in the epithelial layer at days after infection. Approximately % of the epithelial cells displayed entire cell fluorescence at days after infection; at 1 days approximately 7% of the cells were infected. This pattern persisted until day 5, when the epithelium was lost. With the injection method, homogeneous fluorescence of Bowman's membrane was noted as early as days after infection. At 7 days after inoculation, focal fluorescence was observed in keratocytes and other extracellular areas of the anterior stroma. This focal fluorescence became diffuse in the outer half of the stroma by day 1 and involved the entire stroma on and after day 1. Cultures infected by the adsorption method showed delayed viral immunofluorescence of Bowman's membrane at 7 days. Focal fluorescence was detected in the outer stroma on day 1 and became diffuse in the outer half of the stroma by day 1. The entire stromal layer was involved by day 1. Thus adenovirus antigen was detected in the stromal layer following both viral injection and adsorption, although a slight delay in stromalfluorescencewas observed by the latter method. Nofluorescencewas observed in uninfected control corneal cultures and infected cultures stained with normal rabbit Downloaded From: on 11/5/18

6 Volume 18 Number 9 Adenovirus infection of rabbit cornea 961 Fig.. Corneal organ culture at 7 days following topical application (adsorption) of adenovirus type 19. Epithelial cells with vacuolated cytoplasm and hyperchromatic nucleii characteristic of viral infection. (H&E; X16.) Fig. 5. Uninfected corneal culture at 1 days' incubation. Approximately 5 or 6 epithelial layers are present. (H&E; x 16.) serum and fluorescent buffer controls maintained throughout the experimental period. Figs. 6 and 7 illustrate immunofluorescent localization of adenovirus antigens in infected rabbit corneal organ cultures. Discussion This study has demonstrated the long-term cultivation of normal intact rabbit corneas for periods up to 9 days of incubation. Summerlin et al. 8 first reported on the successful cultivation of normal human and animal corneas for periods up to 8 days, an arbitrary time of termination of the experiments. As in Summerlin's study, excellent viability and good growth of the epithelium of intact rabbit corneal organ cultures were observed. The endothelium was well preserved, with Downloaded From: on 11/5/18

7 96 Schwartz and Yamashiroya Invest. Ophthalmol. Visual Set. September 1979 Fig. 6. Rabbit corneal organ culture at 1 days following topical application (adsorption) of adenovirus type 8. The indirect immunofluorescence method reveals entire cell fluorescence in the epithelial layer along with anterior stromal focal fluorescence. Deep stromal fluorescence is not observed. (X1.) changes in the stroma consistent with a slight decrease in the number of keratocytes. The collagenous stroma was normal, with minimal edema and shrinkage. The growth curves obtained for corneal organ cultures infected with adenovirus types 8 and 19 clearly demonstrated the ability of both virus types to replicate in rabbit tissue. The infectivity titers of adenovirus injected into the cornea were slightly higher than those with the virus adsorbed onto the surface layer. This may represent a mechanical advantage for injected virus as a result of trauma or injury to the epithelial layer. The decline of adenovirus infectivity titers in infected rabbit corneal organ cultures after a peak at 1 to 1 days after infection was correlated histologically with progressive loss of the infected epithelial layer. By 1 days after infection, the epithelial layer was almost completely gone, and viral activity was confined to the deeper corneal layers. Virus growth in corneal organ cultures was associated with the appearance of typical adenoviral CPE. Further proof of adenovirus types 8 and 19 replication was shown by passage of infective cell fluids serially for six passages in corneal cultures. In the epithelial layer, the sequence of cytopathology from early cytoplasmic vacuolation to production of intranuclear basophilic inclusions was characteristic of adenovirusinfected HEK monolayer cell cultures. In the corneal stroma, the presence of adenoviral inclusions in the keratocytes demonstrated the susceptibility of these cells to the virus. Reactive changes were observed in the stroma consisting of fibrosis and rarefaction of the stromal collagen. The indirect immunofluorescence technique using homotypic and heterotypic adenovirus types 8 and 19 antisera confirmed the histologic observations made in sections stained with H&E. Fluorescence was initially observed in the epithelial and anterior stromal layers. Definite focal fluorescence was observed in the anterior stromal layers corresponding to the location of infected keratocytes seen by light microscopy. As infection progressed, and with loss of the epithelial layer, fluorescence was then observed deeper in the infected stroma and became more diffuse in nature. This pattern of fluorescence suggested that although the keratocytes had decreased in number, adenoviral antigens persisted in the extracellular spaces in large numbers. No focal subepithelial lesions were observed histologically, although the presence Downloaded From: on 11/5/18

8 Volume 18 Number 9 Adenovirus infection of rabbit cornea 96 Fig. 7. Corneal culture at 1 days after intracorneal injection of adenovirus type 19. Immunofluorescence reveals staining of Bowman's membrane and deep stromal fluorescence. The epithelial layers are no longer present. (xloo.) of adenoviral antigens was confirmed by immunofluorescence and the detection of typical adenoviral inclusions in infected keratocytes. This would indicate that other factors besides the presence of the virus per se are involved in the pathogenesis of epithelial lesions which are typical of EKC. Thus it appears that a host response lacking in the in vitro system may be required in the development of the corneal lesions. It has been postulated 11 that in human EKC, adenoviral particles attached to the stromal collagen participate in an immunologic reaction which produces the characteristic subepithelial infiltrates. This study has shown that adenovirus types 8 and 19 are capable of replicating in infected rabbit corneal organ cultures with the production of typical cytopathologic changes. Further studies will be directed toward determining the role of immunologic mechanisms in the pathogenesis of adenovirus subepithelial lesions. We thank Roberta Smith for technical assistance in virologic studies, Henrietta Bueno for histochemical preparations, and Marie Murphy and Kathy Jameson for typing of the manuscript. REFERENCES 1. Center for Disease Control: Keratoconjunctivitis due to adenovirus type 19, Morbid. Mortal. Rep. :185, Dawson, G.R., Hanna, L., Wood, T.R., et al.: Adenovirus type 8 keratoconjunctivitis in the United States. III. Epidemiological, clinical, and microbiologic features, Am. J. Ophthalmol. 69:7, Dawson, C.R., Hanna L., and Togni, B.: Adenovirus type 8 infections in the United States. IV. Observations on the pathogenesis in severe eye disease, Arch. Ophthalmol. 87:58, Jones, B.R.: Adenovirus infections of the eye in London, Trans. Ophthalmol. Soc. U. K. 8:61, Laibson, P.R., Dhiri, $., Oconer, J., etal.: Corneal infiltrates in epidemic keratoconjunctivitis, Arch Ophthalmol. 8:6, Dawson, S., Togni, B., and Moore, T.F.: Structural changes in chronic herpetic keratitis studied by light and electron microscopy, Arch. Ophthalmol. 79:7, Andrews, C: Viruses of Vertebrates, Baltimore, 196, The Williams & Wilkins Co., pp Summerlin, W.T., Miller, G.E., Harris, J.E., etal.: The organ cultured cornea: an in vitro study, IN- VEST. OPHTHALMOL. 1:176, Schwartz, H.S., Vastine, D.W., Yamashiroya, H,, et al.: Immunofluorescent detection of adenovirus antigens in epidemic keratoconjunctivitis, INVEST. OPHTHALMOL. 15:199, Reed, L.J., and Muench, H.: A simple method of estimating fifty percent end points, Am. J. Hyg. 7:9, Knopf, H.L., and Hierholzer, J.C.: Clinical and immunologic response in patients with viral keratoconjunctivitis, Am. J. Ophthalmol. 8:, Downloaded From: on 11/5/18