Vesicular stomatitis virus (VSV) infection in rabbit eye: Role of antibody and interferon

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1 Vesicular stomatitis virus (VSV) infection in rabbit eye: Role of antibody and interferon Ralph Pollikoff, Anthony DiPuppo, and Patricia Cannavale The effect of VSV infection in rabbit eye was investigated. The findings indicated that maximum virus growth in the rabbit cornea coincided with peak keratitis on postinfection day. Thereafter, keratitis was generally not observed, whereas infectious virus was detected in the cornea through day 6. Primary virus infection in the cornea of one eye produced serum-neutralizing antibody which did not protect the contralateral control cornea against an initial infection with homologous virus. Virus infection in the cornea did not spread to other segments of the eye and produced resistance in that cornea to reinfection with homologous but not heterologous virus. Resistance to reinfection of the cornea was correlated with presence of a virus-neutralizing substance. The data also suggested that resistance to or recovery from VSV infection in the cornea might be mediated by inducers of interferon, e.g., bacterial endotoxin (S. marcescens), statolon (Venicillium stoloniferum), and complexed polynucleotide RNA (polycytidylic: polyinosinic acids). Keywords: vesticular stomatitis virus, virus infections, cornea, keratitis, time factors, virus isolation, antibody formation, interferon, RNA, histopathology, rabbits. Ves esicular stomatitis virus (VSV) produces an explosive, unpredictable, and serious mucocutaneous infection in ruminants and is capable of causing acute systemic or local cutaneous disease in man. The disease in man is characterized by rapid onset, with recovery being uneventful and almost invariably without sequelae. Experimental disease with high mortality rates has been produced in laboratory From the Virus Laboratory at Wills Eye Research Institute, Wills Eye Hospital, Philadelphia, Pa. 90. Supported by United States Public Health Service Research Grant NB-A 00 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. 488 mice, guinea pigs, hamsters, rats, and ferrets, 4 ' 5 but data concerning experimental ocular disease have not been reported. Accordingly, investigations concerning VSV infection in the rabbit eye were undertaken, and the results presented in full in the following report. 0 Materials and methods s. Female albino New Zealand rabbits weighing to 4 kilograms were used for experiments concerning VSV keratitis. Weanling rabbits weighing approximately kilogram were employed for tissue cultures of kidney cells. Virus. The source and growth of the Indiana strain of VSV, vaccinia, and the HF strain of herpes simplex virus have been described previously. Determinations. Keratitis. This was evaluated according to the following scheme: = < staining defects;

2 Volume 8 Number 5 Vesicular stomatitis virus 489 = to 4 pinhead or small linear staining defects; = 5 to 0 pinhead or linear staining defects covering less than 5 per cent of the cornea; to = staining defects covering 5 to 5 per cent of the cornea, respectively. Corneal scarification was necessary in order to produce keratitis following ocular instillation of 5 ml. of 05-s t0 loos TCID M of virus on the cornea. Lesions, which followed the lines of scarification, were detected by ocular instillation of a drop of per cent aqueous methylene blue. All rabbits were devoid of any countable staining defects prior to infection of the cornea. Virus infectivity titrations and interferon (ITF) assays. These were carried out as described previously. Virus neutralization tests. These tests were performed with rabbit sera inactivated at 56 C. for 0 minutes and stored at -0 C. On the day of test, sera were thawed rapidly and the tests performed by mixing a volume of serum dilution with an equal volume of maintenance medium (Puck's medium fortified with per cent inactivated fetal calf serum) containing 0-5 to 0 TCID-.o of VSV. Following brief agitation, the mixtures were incubated at C. for 5 minutes and then at 4 C. for 45 minutes. A ml. volume of each mixture was overlaid on each of four 0 ml. capacity flasks (Falcon Plastics, Los Angeles, Calif.) containing monolayers of rabbit kidney (RK) cells. The test was completed as described in a previous report. A control titration using logarithmic dilutions of virus was carried out at the same time. The cultures were stained 48 to hours later and serum titers expressed as the final serum dilution producing at least 50 per cent plaque reduction. 8 Neutralization tests, ITF assays, and virus titrations were also carried out by the cytopathic effect (CPE) method utilizing tube cultures of RK cells. Interferon inducers. Bacterial endotoxin. The source and preparation of endotoxin from Serratia marcescens has been reported. Statolon. A preparation of statolon, lot B-9, was obtained through the kindness of W. J. Kleinschmidt of the Lilly Research Laboratories, Indianapolis, Ind. It was stored in a desiccator at 4 C. until the day of test. The lot contained 9.5 per cent nondialyzable solids (active statolon), 8 per cent glucose, and.4 per cent ammonium carbonate. For use, a stock solution of statolon consisted of 0 mg. per milliliter of drug dissolved in sterile saline, and a single ocular injection of ml. of stock preparation contained 8 Mg of active statolon. Complexed polynucleotide RNA. Single-stranded polycytidylic (PC) acid and polyinosinic (PI) acid was obtained from P-L Biochemicals, Inc., Milwaukee, Wis. Multistranded polynucleotide RNA was obtained by equimolar mixing of PC (molecular weight 4 x 0 5 ) and PI (molecular weight. x 0 5 ) in 06M phosphate-buffered saline containing 5 x 0" M MgCl s. > 0 Histopathologic studies. The method of preparing sections of rabbit eye for histologic study has been reported. Analysis of data. Significance of the data was determined according to standard t tests. Results Effect of VSV infection in rabbit eye. Table I summarizes the effect of instilling 5 ml. of 0 G 5 TCID 50 of virus on the scarified rabbit cornea. The data indicate that peak keratitis occurs on day after infection and then declines to a minimum or nondetectable level by day. In the following experiment 0 rabbits were divided into groups of 0 each and infected with virus via the scarified cornea or by injection with ml. of virus via the aqueous or vitreous. The results shown in Table II demonstrate the effect of route of ocular infection on virus growth, pathogenesis, and histopathology. Infectivity titers were determined according to the CPE method in tube cultures of RK cells as well as by plaque-forming units (PFU) in monolayer flask cultures of the same cells. Good correspondence in infectivity titers was obtained employing these two techniques of virus assay. Note that when the rabbit eye is infected via the scarified cornea, peak virus growth (0-5 TCID r)0 ) is detected on day after infection. Thereafter, the data indicate a log lower level of virus by day, a time when keratitis is minimal. Subsequent titrations for Table I. Effect of VSV infection in rabbit cornea 4 5 Lesion score post infection Dai Day Day OD* OS OD OS OD OS "Lesion scores determined as shown in text.

3 490 Polldkoff, DiPuppo, and Cannavale Investigative Ophthalmology October 969 Table II. Growth and pathogenesis of VSV in rabbit eye Vinn titer post infection" Part Route of infection of eye Day Day Day Histopathology, clay Cornea Cornea Edema and slight keratitis Aqueous < < < Normal Iris + CB < < < Normal Anterior chamber Cornea < Normal Aqueous < < < Num. WBC Iris + CB < Iridocyclitis Vitreous Cornea < < Normal Aqueous < < Num. WBC Iris + CB < Iridocyclitis "Virus titer determined by CPE method in tube cultures of RK cells and expressed as logio TCTDr.o per milliliter infectious virus content of pools of cornea harvested on day 4 through day 6, the last day of sampling, revealed levels of infectious virus of to log above that detected on day. Infection via the cornea does not appear to spread to other segments of the globe as indicated by failure to detect growth of virus in the anterior chamber, iris, and ciliary body. It can also be seen that following infection via the cornea, 0- TCID 50 of virus and histopathology is demonstrated on day in the cornea, whereas virus and histopathology are not detected in other segments of the eye at this time. When virus is injected via the anterior chamber, less virus growth is detected in the cornea on day after infection and in the iris and ciliary body on day and day, whereas the aqueous appears to be devoid of demonstrable virus. By day, infectious virus appears to be nondetectable in any segment of the globe. Despite the apparent failure to demonstrate infectious virus on day, iridocyclitis is present in the iris and ciliary body and numerous leukocytes are observed in the aqueous. The cornea appears normal. Finally, injection of virus intravitreously through the pars plana also produces less virus growth in the iris and ciliary body on day and day. The cornea appears to be devoid of detectable virus. Detection of virus in the aqueous on day is difficult to explain since virus is not demonstrated in the cornea. Although virus is apparently not detected on day, iridocyclitis is observed and numerous leukocytes are detected in the aqueous. The cornea appears normal. In order to determine if lesions in the cornea were the result of virus multiplication, 0 per cent suspensions of infected cornea were clarified by ordinary centrifugation and serially passaged times via the corneal route in groups of 5 rabbits. The corneas were observed for extent of keratitis on postinfection day prior to harvest for infectious virus content. The results indicated that each passage of infected suspension of cornea contained 0 r>0 TCID 50 of virus (CPE method in tube cultures of RK cells) and produced extensive keratitis. Control suspensions of cornea failed to produce this effect. Virusinfected and control cornea were also prepared for gross and histopathologic examination. The results are shown in Figs. and. It can be seen in Fig. that in VSV-infected rabbit cornea staining defects (keratitis) follow along the lines of prior scarification and the infected eye exhibits signs of a mild inflammatory response. Biomicroscopic examination confirms these findings. In Fig. it can be seen that in VSV-infected rabbit cornea the corneal epithelium consists of fewer cell layers and the basal epithelial cells are generally ballooned. Several areas are seen where there are craterlike excavations in the epithelium. The corneal endothelium appears normal. Role of antibody. Table III summarizes

4 Volume 8 Number.5 Vesicular stomatitis virus 49 Fig.. Effect of VSV infection in the scarified rabbit cornea at 4 hours after infection. Note that the staining defects follow the lines of scarification. Staining defects are not detected in the scarified cornea given virus diluent. Fig.. Effect of VSV in the rabbit cornea. A, Normal, noninfected rabbit cornea. (Hematoxylin and eosin; x00.) B, VSV-infected cornea observed 4 hours after infection. Note the lesion formation in the cornea! epithelial layer. (x00.) the effect of primary VSV infection in the OD cornea on re-exposure to the same virus. Observe that extensive keratitis is produced in the scarified OD cornea day following infection with 05 5 TCIDao VSV, whereas the scarified OS cornea treated with virus diluent does not produce this effect. Yet, after rechallenge of the OD eye, and primary challenge of the OS eye with VSV on day 5, keratitis is produced only in the OS cornea. Note that keratitis is produced in the OS cornea despite a detectable level of circulating antibody. Under these experimental conditions, the OD cornea did not resist infection on day 5 with heterologous herpes simplex or vaccinia virus. It can also be seen that following VSV challenge keratitis declines one day after peak lesion formation (day 6) in the OS cornea. As regards primary

5 49 PoUikoff, DiPuppo, and Cannavale Itwestigatioe Ophthalmology October 969 Table III. Effect of primary VSV infection in rabbit cornea Day OD* Lesion score joost infection OS Day 5f OD OS Day 6 OD OS Day OD OS "Lesion scores determined as shown in text. fod cornea received a second challenge with VSV. OS cornea received an initial challenge with VSV. Neutralizing antibody titer of pooled sera on day 5 = :8. infection via the anterior chamber of the OD eye, resistance was less complete following a second challenge via the OD cornea on day 5, i.e., compared to the extent of keratitis in the OS control corneas, keratitis in the OD corneas was not prevented but significantly reduced. Similar results were obtained when primary infection was initiated via the vitreous. It was also found that after primary infection of the OD cornea the cornea strongly resisted a second challenge with VSV on day 4. Virus neutralization tests were carried out in RK and L 99 mouse cells with supernatant fluids obtained from 0 per cent suspensions of washed cornea. The suspensions were centrifuged at 400 x g for 0 minutes and heated at 56 C. for 0 minutes. The data indicated the presence of significant (p < 5) but marginal levels of antibody or antibodylike substance in the cornea on day 4. Although the OD corneas strongly resisted reinfection on day, virus-neutralizing activity was not detected in the cornea. On day 8 following primary infection, the neutralizing titer of the cornea was :0, whereas the serum titer was :8. Role of interferon. Tables IV, V, VI, and VII summarize the antiviral efficacy of various substances inducing host formation of ITF. These investigations were carried out in a blind, coded manner. In Table IV, the data indicate the effect of injecting a single 0 /.<.g dose of endotoxin via the vitreous through the pars plana Table IV. Effect of a single intravitreous dose Treatment" -8 hr. + hr. Lesion score post infection Day It Dai) \ Dai/ OD OS OD OS OD OS 0 'OD injected intravitreously with 0 fig of endotoxin :ontained in ml. OS injected with placebo. Table V. Effect of a single intravitreous dose Treatment -8 hr. Lesion score OD* + 4 hours OS OD injected intravitreously with 8 fig of statolon contained in nil. saline. Treatment significant at p < 0. OS injected with drug diluent. Tenfold increase in drug level produced increased toxicity and reduced efficacy. Lesion scores determined as shown in text. Table VI. Effect of a single intravitreous dose Lesion score + 4 hours OD* OS 4.0

6 Volume 8 Number 5 Vesicular stomatitis virus 49 Table VII. Effect of ocular drops Lesion score +4 hours OD* OS "After + hours, OD treated over 4 hour period with drops of a solution containing,600 /xg of PC:PI per milliliter. OS treated with drug diluent. Treatment effect on OD eye significant at p < 005. Lesion scores determined as shown in text. 8 hours before or hours after virus infection in the cornea. The data indicate that, on day following infection with 0 r>>5 TCID 50, lesions in the cornea treated with endotoxin 8 hr. before or hr. after infection are strikingly less extensive (p < ) than in the control cornea. In the pretreatment group, this effect is abolished by day since keratitis is also declining in the controls. Similar results were obtained when endotoxins was injected hours before virus infection. However, in the group treated hours after infection, lesions in the majority of the corneas are significantly less extensive on day despite the declining keratitis in the control group. In this respect, keratitis in the drug-treated and control group is not observed by day. As regards growth of virus, the results indicated that in the group treated after infection there was a log lower difference in virus titer in the cornea of the endotoxin-treated group on day after infection. Thereafter, virus titers declined in the control group and no significant differences between the drug-treated and control groups were observed. Table V summarizes results of an experiment in which the OD eyes of a group of 6 rabbits were injected intravitreously 8 hours before infection with a single dose of 8 /.ig of statolon contained in ml. The OS control eyes received a similar volume of drug diluent. The data indicate a significant reduction in keratitis in the OD group on day (p < 0). Thereafter, keratitis declines in a similar fashion in the drug-treated and control group and is not observed by day. A tenfold increase in dosage increased drug toxicity and failed to augment efficacy. Finally, 8 /.ig of drug injected at hours after virus infection was ineffective. Table VI summarizes the effect of ocular injection with multistranded polynucleotide RNA (PC:PI) on production of keratitis. The results demonstrate that a single dose of 5 ^ig of complexed PC:PI contained in ml. injected intravitreously hours after infection does not prevent but significantly reduces keratitis 4 hours after infection (p < ). Similar results were obtained when drug was injected at 6 hours post infection. In addition, drug therapy via the vitreous failed to demonstrate a significant difference in growth of virus 4 hours after infection, but produced a log lower level in virus growth in the cornea by 48 hours compared to the infected control group. Under these experimental conditions, pooled cornea and pooled iris and ciliary process from eyes of a group of ten rabbits injected intravitreously with 5 /xg PC:PI or drug diluent were assayed for an ITF inhibitor. ' 0 The data indicate that a significant level ( units) of an ITF-like substance was detected in suspensions of the cornea, iris, and ciliary body 8 hours after drug treatment. Drug efficacy was also demonstrated by injecting drug via the cornea or intravenous route (p < ), but was ineffective via the aqueous. In this connection, the PC polymer per se was inactive, whereas the PI polymer appeared to demonstrate significant efficacy (p < ). Reduction in keratitis was also achieved when 5 /.ig of PC:PI was injected subconjunctivally and 6 hours post infection (p < 5). Drug treatment via the cornea produced a smoothedged geographic lesion, iritis, and cloudy aqueous. Drug treatment via other ocular

7 494 Pollikoff, DiPuppo, and Cannavale Investigative Ophthalmology October 969 routes caused a similar inflammatory response but without ulcer formation in the cornea. It is of interest to point out that staining defects were strikingly lighter regardless of route of drug administration even in those instances where no difference in extent of staining defects was observed between the treated and control groups. Finally, the effect of ocular instillation of drops of PC:PI on development of keratitis is demonstrated in Table VII. The data demonstrate that, starting hours after infection, instillation of a single drop of a solution containing,600 /.ig PC:PI per milliliter on the infected OD cornea every half hour during the working day and every hour overnight for a total of 4 hours of treatment does not prevent but significantly reduces keratitis (p < 005). Gross observation of the drug-treated eye revealed a moderate to severe iritis and conjunctivitis without lid closure. Treatment with drug diluent failed to provoke this response. Also, it is of interest that curtailing drug therapy to a single drop every hour for four hours starting hours post infection strikingly reduced keratitis (p < 005) and did not cause iritis and conjunctivitis. Discussion The experimental results herein presented indicate that VSV produces a selflimiting disease in the scarified rabbit eye which peaks on day after infection and then declines rapidly. However, despite the rapid decline and disappearance of keratitis by day, significant levels of infectious virus are detected in the cornea on day 6, the last day of sampling. The data also indicate that the optimal route of infection is via the cornea, and it is of interest to note that infection via this route produces only mild or minimal pathologic changes and does not spread to other segments of the eye. On the other hand, ocular injection of virus via the anterior chamber or vitreous produces iridocyclitis and results in a low-grade infection in the cornea, iris, and ciliary body, respectively. Three serial passages of undiluted infected suspensions of the cornea via the corneal route revealed that virus multiplication had occurred in the abraded cornea since the initial virus titer and that of each subsequent corneal passage was 0 50 TCID 5O per milliliter. Thus, keratitis did not result from the mere presence of virus in the scarified areas of the cornea. The nature of the VSV-produced keratitis was determined by gross and microscopic observations. The results shown in Fig. demonstrate that lesion formation appeared to follow along the lines of prior scarification and morphologically does not resemble the dendritic-type of lesion and inflammatory response produced by infection with herpes simplex or vaccinia virus. ' rj Microscopic examinations of stained sections of the eye reveal milder pathologic changes compared to the severe inflammatory and necrotizing effect observed in vaccinial or herpetic keratitis. Resistance of the rabbit cornea to a second ocular challenge with homologous virus is demonstrated as early as postinfection day. However, a heat-stable, virusneutralizing factor is not detected in the cornea at this time, whereas the presence of trace levels of such a factor is detected on day 4. Antibody or antibody-like substance is not detected in the contralateral noninfected control cornea but a significant low level of antibody is detected in the serum. By day 8, however, significantly greater levels of a virus-neutralizing substance are demonstrated in the previously infected cornea compared to that detected in the serum. Although clearcut evidence for the appearance of corneal antibody on day is lacking, it might have been present but masked by the presence of VSV antigen in the cornea. Early resistance to reinfection of the cornea with VSV might also have been mediated by the interfering effect resulting from the presence in the cornea of a specific component of the VSV particle. 4 Finally, the results clearly demonstrate that serum-neu-

8 Volume 8 Number 5 Vesicular stomatitis virus 495 tralizing antibody to VSV does not protect a normal noninfected eye from infection with homologous virus, nor does it concentrate in a traumatized but uninfected cornea. 5 Under these conditions, primary infection of the cornea results in resistance of that cornea to reinfection with the same virus. Thus, the evidence herein presented supports the concept of local antibody formation in rabbit eyes, and, in general, confirms the work of previous investigators. ' ir> - Another and perhaps more promising approach to the study of host resistance to and recovery from virus infection in the cornea is by ocular induction of ITF, a proven virus inhibitor. Along these lines, some encouraging results were obtained when it was shown that topically applied nontoxic ITF significantly reduced keratitis produced by infection of the cornea with vaccinia virus, whereas treatment with gamma globulin was ineffective. 8 Recently, it was shown that certain substances such as endotoxin and multistranded polynucleotide RNA induced formation of ITF and were capable of promoting recovery from virus-produced keratitis. ' 0 The present investigation confirms and extends these findings concerning induction of ITF or ITF-like substance, and demonstrates that treatment of a previously infected cornea with a microgram dose of PC:PI via ocular, subconjunctival, or parenteral injection strikingly reduces keratitis. Under these conditions, ocular injection of PC:PI produced a moderate to severe iritis, whereas drug toxicity was not apparent via the parenteral route. Therapy was also significantly effective (p < 005) when it consisted of a drop of a solution containing,600 nig PC:PI per milliliter instilled periodically on the infected cornea over a 4 hour or a 4 hour period. In this connection, it is of interest to note that therapy after infection every hour for four hours compared to therapy over 4 hours failed to cause obvious pathologic changes in the eye. Finally, it was observed that, even when the extent of keratitis in the drug-treated group was similar to the controls, staining of the lesions was always lighter in the group treated with the complexed PC:PI. Although the explanation for this is not apparent, the effect might possibly represent yet another parameter of drug efficacy. In sum, the experimental data herein presented characterizes the effect of growth and pathogenesis of an RNA-containing agent, VSV, in the rabbit eye. The experimental results suggest that infection with this virus may occur in man following ocular trauma. If this does occur, the evidence shown here with one strain of VSV suggests a mild disease of short incubation and of short duration. The data further indicate the importance of locally produced antibody in preventing subsequent infection with the same virus, and confirm the utility of therapy with ITF-inducers in virus infection in the cornea. The authors wish to acknowledge the excellent technical assistance of Mrs. Addie Wilson in the preparation of histopathological sections. REFERENCES. Brandly, C. A., Hanson, R. P., and Chow, T. L.: Vesicular stomatitis with particular reference to the 949 Wisconsin epizootic, Proc. Book Am. Vet. M. A. 6, 95.. Hanson, R. P., Rasmussen, A. F., Brandly, C. A., and Brown, J. W.: Human infection with the virus of vesicular stomatitis, J. Lab. & Clin. Med. 6: 54, Gibbs, H. E.: A report upon an outbreak of stomatitis contagiosa, Vet. J. : 4, Cox, H. R., and Olitsky, P. K.: Neurotropism of vesicular stomatitis virus, Proc. Soc. Exper. Biol. & Med. 0: 65, Kowalczyk, T.: The response of domestic and laboratory animals to initial and secondary exposure to vesicular stomatitis virus, MS thesis, University of Wisconsin, Madison, Wis., Pollikoff, R., Jankauskas, P., and DiPuppo, A.: Effect of vesicular stomatitis virus in rabbit eye, presented at the Eastern Sectional Meeting of the Association for Research in Ophthalmology, March 8, Pollikoff, R., Jankauskas, P., and DiPuppo, A.: Studies on the effect of bacterial endotoxin on primary herpetic keratitis, INVEST. OPHTH. : 9, Wagner, R. R.: Biological studies of inter-

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