Herpes Simplex

Virus in Ocular Disease Dieter


1 and 2

Neumann-Haefelin, MD; Rainer Sundmacher, MD; G\l=u"\nterWochnik; Brigette Bablok

\s=b\In a continuous series of 457 patients with presumed herpetic eye disease, virus isolation and typing revealed 154 patients with herpes simplex virus type 1 and three patients with type 2 infections. In 219 isolates that were examined for neutralization by specific antisera, for growth in human fibroblasts, and, in part, for temperature sensitivity, there were found substantial strain differences in addition to the type-specific characteristics. The clinical features of each of the three type 2 infections are described in detail. A suggestive correlation found between clinical courses and virus growth characteristics of the type 1 strains indicates that further virologic differentiation of these strains would be useful.

(Arch Ophthalmol 96:64-69, 1978) infections of the eye are considered to be by herpes simplex virus (HSV) type 1. Though the genital type 2 virus may be occasionally recovered from herpetic lesions at extragenital sites,' HSV isolates from eyes with herpetic

Herpetic generally caused

Accepted for publication April 27, 1977. From the Institut f\l=u"\rVirologie, Zentrum f\l=u"\r Hygiene der Universit\l=a"\t(Dr Neumann-Haefelin and Mr Wochnik), and the Universit\l=a"\ts-Augenklinik (Dr Sundmacher and Ms Bablok), Freiburg, West Germany. Reprint requests to Institut f\l=u"\rVirologie, Zentrum f\l=u"\rHygiene, Postfach 820, Hermann Herderstrasse 11, Freiburg, West Germany (Dr Neumann-Haefelin).

keratitis or conjunctivitis have rarely been shown to be type 2.'-' An excep¬ tion to this fact occurs in neonates1 · " by genito-ocular spread of HSV type 2 at the time of birth. Neonatal HSV type 2 ocular disease is frequently accompanied by CNS involvement. However, only two cases of HSV type 2 ocular infections in adults have been reported after genital herpetic dis¬ ease." These two cases did not show any extraordinary clinical courses. The purpose of this study was to determine the incidence of HSV types 1 and 2 ocular infections in a contin¬ uous series of patients and to evaluate the clinical characteristics of each. Furthermore, we tried to correlate virologie differences within the HSV type 1 group with clinical findings. MATERIAL AND METHODS

Subjects During a continuous 24-month period, patients at the Freiburg (West Germa¬ ny) Eye Clinic were seen for presumed herpetic ocular disease. Virus isolations were performed as previously described by Sundmacher et al.T Herpes simplex virus 457


obtained from tears, corneal conjunctival swabs taken from 157 patients. The number (219) of isolates exceeded that of the patients, since replicate isolates, HSV strains from recur¬ rent lesions, and some isolates from the lids of the same patients were included. were



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The patients received ophthalmologic follow-up,

intensive an since most of them took part in clinical studies for anti¬ viral therapy (R. Sundmacher, MD, et al,

unpublished data, January


July 1976).,s

With a few exceptions, the virus nonproducers exhibited scarring, stromal keratitis or keratouveitis that was suggestive of herpetic disease, and metaherpetic disor¬ ders." All HSV isolates were propagated and tested on cells of the same line of human diploid foreskin fibroblasts. The passage level of the cells ranged from eight to 11 after primary seeding. For further charac¬ terization, virus stocks with identical passage history (three passages) were prepared as previously described by Neumann-Haefelin et al."' Our reference strains (HSV type 1, strain Maclntyre's; HSV type 2, strain MS) and 12 HSV type 2 isolates of genital origin had a longer passage history. However, they were also subjected to three human diploid fibroblast passages prior to stock preparation. Antisera against HSV type 1, strain Maclntyre and HSV type 2, strain MS, were prepared by repeated intramuscular inocu¬ lation of young adult rabbits with 10' mean tissue culture infective doses (TCID,,,) of HSV types 1 and 2 harvested from primary rabbit kidney cells. The virus (1 ml) was mixed with an equal amount of incomplete Freund's adjuvant before injection. Herpes simplex virus type 1 antiserum was subjected to immunoabsorption with HSV type 2 antigen, according to the method described by Jeansson." Herpes simplex

virus type 2 antiserum was only inacti¬ vated by heating at 56 C for 30 minutes before use. Microtitration and microneutralization tests were performed, according to the method described by Stalder et al,12 except that the tests were read microscopically for cytopathic effect, and the dose of virus to be neutralized was adjusted to approxi¬ mately 50 TCID.,,, in 0.05 ml for each microtiter well. Tests were discarded and repeated when the virus dose exceeded 89 TCID-,,, or fell short of 28 TCIDM per well. For variations within this range, the results of microneutralization were cor¬ rected by means of the control titration, according to the formula used by Pauls and Dowdle," and expressed as the mean neutralizing potency (pN) of the two distinct antisera against each virus strain. Temperature sensitivity of the HSV strains was evaluated in terms of the reproductive capacity at a temperature of 40 C (RCTJ compared with that at 36 C, according to the method described by Szanto," with the use of microtitration for assay of virus reproduced at the different


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temperatures. RESULTS

Virology The 219 ocular herpes simplex virus isolates were typed by microneutrali¬ zation tests with antisera of relativetype specificity for HSV types 1 and 2. When the pN values of HSV type 1 antiserum are plotted against the correspondent pN values of HSV type 2 antiserum, the points that belong to 216 strains form one cluster (Fig 1) that encompasses the points of the HSV type 1 reference strain. For all strains that correspond to the points of this cluster, the pN values of the HSV type 1-specific antiserum are greater than those of the HSV type 2specific antiserum. Thus, the strains are typed as HSV type 1. Another cluster of points is formed by the plotted pN values of twelve genital HSV type 2 strains, the mean obtained with the HSV type 2 refer¬ ence strain, and three of the ocular isolates that are typed, therefore, as HSV type 2. All of these HSV type 2 strains are characterized by the rela¬ tion of pN values, as well as by their low absolute pN values, with immunoabsorbed HSV type 1 antiserum

(Fig 1). Duplicate


triplicate HSV type






pN HSV Type 2 Antiserum

Fig 1.—Neutralizing potency (pN) of HSV type 1 antiserum immunoabsorbed with HSV type 2 (HSV type 1, antiserum) and of HSV type 2 antiserum, against 219 ocular HSV isolates and 14 controls. Open circle indicates ocular HSV isolates; +, HSV type 1, Maclntyre's strain (mean, 18 tests: SD for HSV type 1, antiserum 0.16 pN; HSV type 2 antiserum 0.12 pN); closed circle, genital HSV type 2 isolates; and crossed circle, HSV type 2, MS strains (mean, ten tests: SD for HSV type 1, antiserum 0.14 pN; HSV 0.12 pN). type 2 antiserum =




isolates were tested from 41 patients. Twenty-three pairs and triplets ren¬ dered pN values that differed by less

than the SD (2 SD) of HSV type 1, strain Maclntyre. In another 14 cases, the variation was within the range of 1.5 SD and in four cases, between 1.5 and 2 SD. By statistical analysis (t test), significance could not be demon¬ strated for the variation of any one of these cases.

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Most of the HSV types 1 and 2 strains displayed known mean typespecific growth characteristics in terms of virus yield under identical conditions (Fig 2, left) and in terms of

temperature sensitivity (Fig 2, right).

However, differences of virus yield between the individual ocular HSV type 1 strains exceeded the range of the HSV type 1 reference strain and all HSV type 2 strains tested. Temper-



Fig 2.—Growth characteristics of ocular HSV types 1 and 2 strains, genital HSV type 2 strains, and HSV types 1 and 2 reference strains. Left, Virus yield at standard conditions expressed as mean infectivity (TCID.,„/ml, rounded to nearest 0.25 log 10) of third virus passage on human diploid fibroblasts at submaximum cytopathic effect. Right, Temperature sensitivity expressed as difference of reproductive capacity of virus at 36 and 40 C ( log,,, titer at 40 C log,,, titer at 36 C, rounded to nearest 0.5 log,,,). Open column indicates ocular HSV type 1 isolates; closed column, ocular HSV type 2 isolates; shaded column, genital HSV type 2 isolates; column 1, HSV type 1, Maclntyre's strain ( ,„. mean of ten tests, SD 0.94); and column 2, HSV type 2, MS strain ( „, mean of ten tests, SD 0.79). =


ature sensitivity also was found to be not distinctly different in a randomly selected number of HSV types 1 and 2

strains (Fig 2, right), but some of the ocular HSV type 1 showed the degree of temperature sensitivity at 40 C, which is typical for HSV type 2.

Ophthalmology Of the 157 patients, 154 (98%) shed HSV type 1. No intratypic differences of pN values or temperature sensi¬ tivity were found which could be

correlated with the clinical course. However, six HSV type 1 strains yielded an extremely high quantity of virus in cell culture. Five of these were correlated with disabling or bilateral disease, whereas only two of nine isolates that yielded little virus in cell


culture showed such correlation. Three (2%) of 157 patients yielded HSV type 2. Their courses are de¬ scribed in the following case reports. REPORT OF CASES

21-year-old woman was first in September 1975, when dendritic keratitis complicating herpetic keratoiritis was diagnosed in her left eye (Fig 3). Herpes simplex type 2 was cultured from the dendrite. She gave a history of "mycotic vaginal disease" that had been treated seven weeks earlier by a gynecologist. This improved, but, one week later, both pharyngeal tonsils were removed because of acute tender swelling; however, the tonsils had never been previously diseased. At that time, vesicles were observed in the oral cavity, and the left eye became inflamed. Case l.-A


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One week later, keratoiritis was diagnosed and treated with 0.1% dexamethasone phosphate eye drops, three times daily for local effects. The eye did not improve, and, four weeks later, prednisolone, 20 mg/day, was added for systemic effects. After one more week, dendritic keratitis developed, and the patient was seen in referral. In the following months, she experienced many stromal recurrences, and, rarely, was the eye quiet. The patient moved away, at that time, and was not seen again until February 1977. She returned, and her cornea was totally scarred and vascularized (Fig 4). A perforat¬ ing keratoplasty was performed. The graft has, to date, remained clear under intensive postoperative steroid treatment and with trifluorothymidine eye drops (five drops of a 1% solution daily) as a prophylactic antiviral measure.

Case 2.-A 75-year-old man in April 1976. The history


was was

first diffi-

cult to assess, since the patient stated that he had never had a keratitis before. However, since childhood, both of his eyes had been far from normal. His left eye had never provided useful visual acuity, and he remembered that his right eye had been treated in childhood for a disease believed to be scrofula. During World War II, a cataract extraction was performed on the right eye, and, since then, he had not seen an ophthalmologist. He treated himself with homemade chamomile extracts when¬ ever his only functioning eye became blurred. His left eye was amblyopic from high myopia with extensive fundus degenera¬ tions. He also had a convergent squint, a dense cataract, and phlyctenular corneal scars. The right eye showed a severely ulcerated, interstitial keratitis from which HSV type 2 was isolated (Fig 5). In spite of local antiviral therapy with trifluorothymidine eye drops (five drops of a 1% solution daily), the corneal ulcer became deeper, and a donor cornea was sutured onto the diseased cornea to prevent its perforation. In January 1977, minor remnants of the donor cornea were surgically removed, and, one month later, a

Fig 3.—Dendritic HSV type infiltrates) and iritis (case 1).

2 ulcer

complicated by stromal disease (edema plus

perforating keratoplasty was performed. The graft has, as yet, remained clear under intensive postoperative steroid therapy with trifluorothymidine as an antiviral cover.

Case 3.—A 28-year-old man was seen in 1977. He suffered from atopic dermatitis from childhood. Eight days previously, his dermatologist had diag¬ nosed trigeminal zoster that involved all right divisions (Fig 6). On examination, an acute ipsilateral follicular conjunctivitis


present. The ipsilateral preauricular and submandibular lymph nodes were swollen and tender. Herpes simplex type 2 was isolated from skin and from the right conjunctiva. The facial lesions were pri¬ marily limited to the right side. However, on this side, the vesicles were also found in dermatomes Cl to C3 (C5), and even on the opposite side, some single vesicles were found on the shoulder. This zosteriform distribution of the lesions was thought compatible with "disseminated zoster," but, an unusual aspect was the sparing of the medial parts of the face and of nearly all of the nose. Clinically, HSV infection could not be ruled out; thus, steroids were not administered. No antivirale were given because the cornea was uninvolved. Two days later, a few additional vesicles appeared on the left side of the face (Fig 6), and virus cultures became positive for HSV. The course was uneventful, with healing of dermal rash and conjunctivitis within was

Fig 4.—Totally vascularized scarred cornea produced by many stromal within 18 months of beginning of HSV type 2 ocular disease (case 1). week without antiviral therapy. On further inquiry, the patient reported that he had never had any vesicular genital disease, but he had attended a carnival dancing party three days before the onset of the facial disease, and he remembered that he had scratched himself with his fingernail in front of his right ear. It was this region from which he observed the vesicles spread over face, neck, and shoul¬ ders. Paired sera were obtained on days 8 and one more

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36 of the disease. Neutralizing antibody titers against HSV type 1 were 1:640 and 1:960; against HSV type 2, the corre¬ sponding titers were 1:48 and 1:112. titers Complement-fixing antibody against HSV were 1:80 and against vari¬ cella-zoster virus, 1:10 in both sera. Thus, the HSV origin of the disease was confirmed from the serologie point of the virus too, whereas the question of primary or recurrent HSV type 2 infection could not be settled by those means.

The data presented here permit an estimate of the incidence of HSV types 1 and 2 ocular infections in a Middle European population that was surveyed for 24 months. Of all HSVpositive patients, 98% shed type 1, which confirmed previous reports of the overwhelming importance of this type for ocular disease.'-' However, three (2%) patients unsuspectedly yielded type 2, or genital HSV, which is known to account for most of the neonatal ocular herpetic disease.' In two of these cases, a genito-ocular spread was considered likely. In case 1, a genito-oral and genito-ocular spread seemed likely, while in case 3, the spread is presumed to have been genito-digi to-facial. In at least cases 1 and 2, it would seem as if the prediction of a more severe clinical course of HSV type 2 than of HSV type 1 ocular infections '1T was fulfilled.1 Both experienced multiple recurrences, both had to undergo keratoplasty in an attempt to restore visual acuity, and both cases belong to the least favorable prog¬ nostic keratoplasty group. However, one of two cases reported by Oh et al6 had only slight corneal involvement, and of our three cases, only case 3 exhibited no corneal involvement at all. Furthermore, disastrous courses were also seen in our patients with HSV type 1. However, the fact that three of five published HSV type 2 cases exhibited very severe courses suggests a less favorable outcome of established ocular HSV type 2 infec¬ tions. Case 3 is another example of the rare but important zosteriform HSV disease that has been reported in the literature.ia Our case appears unusual in that a facial eruption resulted from type 2 virus.4 The fact that the patient suffered from atopic dermatitis may have predisposed to the development of disseminated skin lesions after dermal infections.'" At admission the clinical picture was suspicious enough of eczema herpeticatum not to accept the diagnosis of disseminated herpes zoster; steroids were accordingly with¬ held while awaiting viral confirma¬ tion. Surprisingly, the extensive le¬ sions in this patient healed within less than three weeks without antiviral "

Fig 5.—Ulcerated HSV type

2 interstitial keratitis

(case 2).


which provides better discrimi¬ nation between HSV types 1 and 2 than that with unabsorbed sera (D. Neumann-Haefelin, MD, and col¬ leagues, unpublished data, November


Fig 6.—Zosteriform HSV type 2 skin rash plus ipsilateral HSV type 2 conjunctivitis in patient with atopic dermatitis (eczema herpeticatum) (case 3). COMMENT

The results of typing by microneu¬ tralization with the use of immunoabsorbed HSV types 1 and 2 antiserum were clear-cut. None of the viruses tested had to be designated as "inter¬ mediate," since the two clusters of HSV types 1 and 2 were found to be widely separated. This results from use of the immunoabsorption tech-

Temperature sensitivity, which has been accepted as a type-specific mark¬ er of laboratory strains,14 did not prove to be sufficient for differentia¬ tion between HSV types 1 and 2. The variability of temperature sensitivity for different ocular type 1 strains was considerable. It exceeded the wide range of antigenic variability (Fig 1) and corresponded to the variability of virus yield at standard conditions (Fig 2, left). All of these parameters indi¬ cate that there are ocular strains of HSV that belonged to the antigenic type 1, but they may display major molecular and biological differences. The kind of possible pathogenetic implications of these interstrain dif¬ ferences have not been elucidated thus far. We found that ocular HSV type 1 strains, which yield an abun¬ dance of virus in cell culture, are more often correlated with disabling or bilateral disease than HSV type 1 strains that have only poor reproduc¬ tive capability. This finding would seem to be a first step in this direc¬ tion. Additional careful, virologie findings will be necessary to confirm these observations.

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therapy. The cornea did not become involved in spite of an acute HSV type 2 follicular conjunctivitis. We have seen

this several times with HSV type

1 infections in otherwise healthy persons. Especially, in cases of pri¬ mary herpetic infections, the cornea seems to have a considerable degree of resistance to herpetic infections. It is important to recognize this, since


therapy of an unsuspected herpetic conjunctivitis may well trig¬ ger the development of severe herpet¬

ic corneal disease. Our cases do not shed any light on the efficacy of synthetic antivirals of HSV type 2 ocular infections.2"-' Case

3 did well without any special therapy, and the other two deteriorated from stromal disease that, as such, is less amenable to medical measures. Though small in number, the 2% of ocular infections caused by HSV type 2 promise interesting insights into the meaning of the type-specific proper¬ ties of HSV for the pathogenesis of

herpetic disease.

Our main concern, however, must be directed towards the 98% of HSV type 1 ocular infections. The investigated ocular HSV type 1 strains exhibited a considerable range of different prop¬ erties, and it may be of interest to investigate potential correlations be-

biochemical and biological markers of these strains and the indi¬ vidual clinical courses of the disease tween

they produce. This study was supported by grants from the Deutsche Forschungsgemeinschaft. Günter Wochnik provided major parts of his medical thesis for this study. J. Richard Barringer, MD, assisted in the preparation of this communication, and Anne Mattes, Ortrud Obrig, and Maria Schmidt, provided technical assistance. Karl E. Schneweis, MD, provided six genital isolates for this investigation. Statistical review was done by Jürgen W. Wagner (Dipl. Volkswirt), Institute for Medical Documentation and Statistics, University of


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AJ, Roizman B: Infection with viruses 1 and 2. N Engl J Med

289:781-789, 1973. 2. Kobayashi S, Shoji K, Ishizu M,


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et al:

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Acta Soc Ophthalmol Jpn 76:1454-1474, 1973. 3. Sawanobori S, Tanaka N, Goto E: Classification of herpes virus isolates from ocular lesions. Acta Soc Ophthalmol Jpn 77:521-530, 1973. 4. Wolontis S, Jeansson S: Correlation of herpes simplex virus types 1 and 2 with clinical feature of infection. J Infect Dis 135:28-33, 1977. 5. Hagler WS, Walters PV, Nahmias AJ: Ocular involvement in neonatal herpes simplex virus infection. Arch Ophthalmol 82:169-176, 1969. 6. Oh JO, Kimura SJ, Ostler HB: Acute ocular infection by type 2 herpes simplex virus in adults: Report of two cases. Arch Ophthalmol 93:1127\x=req-\ 1129, 1975. 7. Sundmacher R, Neumann-Haefelin D, Manthey KF, et al: Interferon in treatment of dentritic keratitis in humans: A preliminary report. J Infect Dis 133(suppl):160-164, 1976. 8. Sundmacher R, Neumann-Haefelin D, Cantell K: Successful treatment of dendritic keratitis with human leukocyte interferon: A controlled clinical study. Albrecht von Graefes Arch Klin Ophthalmol 201:39-45, 1976.

9. Sundmacher R, Neumann-Haefelin D: Keratitis metaherpetica\p=m-\klinischeund virologische Befunde. Klin Monatsbl Augenheilkd 169:728\x=req-\ 737, 1976. 10. Neumann-Haefelin D, Sundmacher R, Sauter B, et al: Effect of human leukocyte interferon on vaccinia- and herpes virus-infected cell cultures and monkey corneas. Infect Immun 12:148-155, 1975. 11. Jeansson S: Preparation of type specific herpes simplex antisera by an immunosorbent method. Acta Pathol Microbiol Scand B 83:48-54, 1975. 12. Stalder H, Oxman MN, Kenneth LH: Herpes simplex virus microneutralization: A simplification of the test. J Infect Dis 131:423\x=req-\ 430, 1975. 13. Pauls FP, Dowdle WR: A serologic study of herpesvirus hominis strains by microneutralization tests. J Immunol 98:941-947, 1967. 14. Szanto J: Reproduction in unadapted and adapted cells at 40 C of herpes simplex virus type 1 and type 2 strains. Acta Virol 19:287-292, 1975. 15. Oh JO, Moschini GB, Okumoto M, et al: Ocular pathogenicity of types 1 and 2 herpesvirus hominis in rabbits. Infect Immun 5:412-413, 1972. 16. Oh JO, Stevens TR: Comparison of types 1 and 2 Herpesvirus hominis infection of rabbit

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Herpes simplex virus types 1 and 2 in ocular disease.

Herpes Simplex Virus in Ocular Disease Dieter Types 1 and 2 Neumann-Haefelin, MD; Rainer Sundmacher, MD; G\l=u"...
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