Brit. J. Ophthal. (I 975) 59, 439

An epidemic of picornavirus and adenovirus conjunctivitis

U. C. CHATURVEDI, ASHA MATHUR, U. K. SINGH, A. K. KAPOOR,R. M. L. MEHROTRA, AND R. C. SAXENAFrom the Upgraded Department of Pathology and Bacteriology, K.G. Medical College, Lucknow, India

A pandemic of acute haemorrhagic conjunctivitisoccurred all over south-east Asia and Japan during1970 and 1971 (Kono, Sasagawa, Ishii, Sugiura,Ochi, Matsumiya, Uchida, Kameyama, Kaneko,and Sakurai, 1972). A similar epidemic occurred inI969 in Ghana (Chatterjee, Quarcoopome, andApenteng, I97oa, b) and Nigeria (Parrott, I971).In the Indian subcontinent the epidemic of con-junctivitis was first reported in Bombay during April1971, and later spread almost all over India. Casesof conjunctivitis started increasing at Lucknowduring the last week of May I97i, reaching a peakin June, and declining by July. Picorna-like viruseshave been isolated from cases in Singapore (Limand Yin-Murphy, 1971; Yin-Murphy, 1972) and inJapan (Kono and others, I972). The epidemic atLucknow was peculiar in that adenoviruses as wellas picornaviruses appear to have been present inthese cases.

Material and methods

The study was carried out by the ophthalmologist (RCS)on 253 typical cases of conjunctivitis which presented atGandhi Memorial and associated hospitals for treatment.Out of 253 patients 84-5 per cent were aged between I Iand 40 years. The youngest was 3 years old and the oldest65 years. The illness started unilaterally, and most of thepopulation at Lucknow was affected. There were multiplecases in a family and the infection could be transmittedeven by casual conversation. The incubation period was lessthan 24 hours in most cases. The chief complaints were a

gritty sensation in the eyes, watering, and photophobia.A few patients had upper respiratory symptoms. Ocularexamination revealed redness, oedema of the conjunctivaand lids, subconjunctival haemorrhage in a number ofcases, and enlarged preauricular lymph nodes. The cornea

remained unaffected. The detailed clinical findings havebeen reported by Saxena, Bhatia, and Chaturvedi (1972).

LABORATORY STUDIES

The laboratory investigations included total and differ-ential leucocyte counts and conjunctival swab cultureson blood agar and Robertson's cooked meat medium.

Address for reprints: Dr U. C. Chaturvedi, Upgraded Department ofPathology and Bacteriology, K.G. Medical College, Lucknow-226003, India

Bacteria were identified by their morphology, culturalcharacters, and biochemical reactions. Smears wereprepared from conjunctival scrapings of 6i patients andstained with Giemsa stain to study the pattern of cellularinfiltration and to determine whether inclusion bodiessuggestive of viral infection would be found.

VIROLOGICAL INVESTIGATIONS

Conjunctival swabs and scrapings were collected forvirological study within 48 hours of the onset of illness.Paired sera were collected from I5 patients for study ofantibody titres.

VIRUS ISOLATION

This was done on primary rhesus monkey kidney tissueculture. Each sample was inoculated into a group of fourtissue culture tubes. These were incubated stationary at370C and were examined daily for 15 days. Tubes showingcytopathic effects were subcultured.

ADENOVIRUS SEROLOGY

Haemagglutination testThe cytopathic agents were tested for haemagglutininusing albino rat and rhesus monkey erythrocytes. It wasfound that only rat erythrocytes were agglutinated by theisolates; therefore for further experiments only erythro-cytes from albino rats were used, taking the precautionsdescribed by Rosen (I960). Hyperimmune antisera wereraised against two of the isolates in rabbits by the tech-nique of Rosen (I960). After treating the hyperimmunesera with 25 per cent kaolin and absorption with raterythrocytes, a haemagglutination inhibition (HI) testwas performed on all the isolates. The HI tests wererepeated in the presence of heterologous antisera (Rosen,1960). With eight of the selected isolates chessboardtitrations were done using the hyperimmune sera describedabove. Paired sera collected during the acute phase and inconvalescence were tested for HI antibodies against oneof the isolates.

ELECTRON MICROSCOPY

One ofthe agents was studied under the electron microscopein the monkey kidney tissue culture fluid and after passagein HeLa cells at Birmingham.

IDENTIFICATION

This agent was finally identified at the Virus ReferenceLaboratory, London, by a neutralization test.

440 British Journal of Ophthalmology

PICORNAVIRUS SEROLOGY

In nine pairs ofsera, antibodies were studied by neutraliza-tion tests against the 15477/7I strain isolated by DrChang from an epidemic of conjunctivitis in Hong Kongin 197I and also against the EC2/7I strain isolated from acase of acute haemorrhagic conjunctivitis in Singaporein 1971, containing 30 to IOO TCD50 infective doses.Equal volumes of suspensions of these viruses and eachdilution of a doubling dilution series of sera were heldat 37°C for I hr before each of two HeLa tissue cultureswas inoculated with o22ml of mixture containing 30 toIOO TCD5o of virus. The inoculated cultures were rolledat 37°C and read on the third and fourth day.

Results

LABORATORY FINDINGS

Total and differential leucocyte counts were doneon I83 patients. Normal counts were found in 148patients (8o.8 per cent). Leucocytosis was observedin 34 patients (I8-7 per cent) and leucopenia inone. The differential leucocyte counts showed neutro-philia in three cases, lymphocytosis in 40, and eosino-philia in thirty.

BACTERIOLOGICAL FINDINGS

Staphylococcus aureus was isolated from seven patients(2-7 per cent).

EXAMINATION OF CONJUNCTIVAL SCRAPINGS

The findings from conjunctival scrapings are shownin Table I. Intranuclear masses suggestive of inclusionbodies due to adenovirus infection (Fig. I) were presentin conjunctival cells of 26 out of 6I patients (42-6 percent). In 49 cases both cytological examination andisolation of virus were attempted. In 24 cases adeno-virus isolation was positive, and nine of these hadintranuclear masses in conjunctival scrapings. Similarintranuclear inclusions were seen in nine of the 25isolation negative cases. Conversely, nine of the I8cases with intranuclear masses yielded adenovirus,and of the 3I cases without intranuclear masses,15 were positive for adenovirus. There was thus nocorrelation between the dense intranuclear massesseen and positive cultures for adenovirus. Infiltrationby mononuclear cells (Figs 2 and 3) was seen in 40patients (95-6 per cent).

FIG. I Smear of conjunctival scraping. Epithelial cell containing irregular intranuclear inclusion body (arrow). Giesma. x 500

Epidemic ofpicorna and adenovirus conjunctivitis 441

FIG. 2 Smear of conjunctival scraping. Collection of conjunctival epithelial cells with infiltration by mononuclear cells. Giesma. X 200

FI G . 3 Smear of conjunctival scrapingfrom another patient. Conjunctival epithelial cells and infiltrating mononuclear cells. Giesma. X 200

442 British Journal of Ophthalmology

Table I Microscopy of conjunctival smears

InfiltrationInclusions Percentage

Mononuclear Mixed None

Present 20 2 4 42.6Absent 20 3 12 57'4

Total 40 5 i6 100

VIROLOGICAL INVESTIGATIONS

Viruses causing a cytopathic effect (CPE) of the kindcaused by adenoviruses were isolated from con-junctival swabs from 74 patients. The CPE wasobserved after 7-I0 days' incubation of inoculatedcultures. All these isolates haemagglutinated rat,but not rhesus monkey, erythrocytes. Altogether 62 ofthe isolates (84.5 per cent) gave a positive haemag-glutination inhibition test with hyperimmune anti-serum raised in rabbits against two of the isolates.Table II shows the findings of the chessboard HItests done on eight isolates.

Table II Results of chessboard HI test

Specimen no. K no. HI

7 I 346 2779 128*/32t71379 2788 32/647I359 2790 64/12871320 2866 32/I 67I250 2876 64/327 I 445 2654 256/12871456 2658 256/ I 287I354 2940 64/128

* Numerator = highest antigen dilutiont Denominator = highest antibody dilution

Electron microscopy revealed particles of typicaladenovirus morphology in one of the isolates. Similarvirus particles were observed after passing this agentin HeLa cells. It was identified as adenovirus type2 by serum neutralization.

Paired sera collected from I5 patients were testedfor HI antibodies against one of the isolated adeno-like agents. A significant rise in antibody titre wasseen in seven patients. Paired sera from nine of thesepatients were tested for antibodies against the twostrains of picoma viruses and against adenovirus.Six pairs of sera showed more than a fourfold riseagainst EC2/71 strain, while in two pairs a small riseof less than 8 to a titre of 12 was seen. None of thesera had antibodies against the 15477/71 strain(Table III). Furthermore, adeno-like viruses were

Table III Picornavirus antibodies in paired sera

Date ofSpecimen no. onset

tn 1971

71179 -I I OJune-2

7II98 -I I4June-2

71290 -I 26 June-2

71315 -I 29 June-2

*7327 -I I July-2

71360 -I 4 July-2

71370 -I 4 July-2

71433 -I 8 July-2

71653 -I I 9 Aug.-2

Date ofcollectionof sera

I I June28 June17 June3 July

28 Junei 8 July

I Julyi8 July2 July

25 July5 July

25 July6 July

27 JulyI 0 July30 July20 Aug.6 Sept.

Picornavirus antibodies

15477/71 EC2/71

< i6 <8< i6 <8< I6 <8< i6 12< i6 <8< i6 32< i6 <8< i6 I2< i6 < i6<i6 I28NT <8NT 32<i6 <8<i6 24NT <8NT 96NT <8NT 128

* Rising levels of antibody to adenovirusNT = Not tested

isolated from conjunctival swabs of the two patients(71327 and 71370) in whose sera rising antibodiesagainst the EC2/7 I strain were found, as well asrising levels of antibody to adenovirus (< I0 to 8o and< I0 to 40 respectively).

Discussion

The findings of the present study demonstrate that62 antigenically identical cytopathic agents wereisolated from conjunctival swabs in monkey kidneytissue culture. One of them was found to be anadenovirus by electron microscopy and was finallyidentified as adenovirus type 2. Further evidenceof adenovirus infection was the rising antibody titresin seven pairs of sera. The findings thus point toadenovirus infection in a number of patients.The presence of dense intranuclear masses sugges-

tive of adenovirus inclusions did not correlate withthe isolation of adenovirus.From similar cases of epidemic conjunctivitis in

south-east Asia and Japan (Kono and others, 1972;Lim and Yin-Murphy, I971), two different strainsof picorna-like viruses have been isolated. We couldnot isolate any such agent from our specimens. There-fore, the paired sera collected during acute and con-valescent phases of illness were tested for the pre-sence of antibodies against picornaviruses. Risingtitres against EC2/7I strain (isolated at Singapore inI971) were observed in six out of nine pairs of sera

Epidemic ofpicorna and adenovirus conjunctivitis 443

tested. This indicates that the picorna-like virus wasalso implicated in the aetiology of the cases atLucknow. It may be mentioned that the EC2/7 Istrain is antigenically similar (Yin-Murphy and Lim,1972) to that isolated by Kono and others (1972) inJapan. The findings thus show that the epidemic atLucknow was similar to that in Singapore and Japan.Our failure to isolate the picornavirus may be becausewe used only primary monkey kidney tissue cultures,as was also found by Lim and Yin-Murphy (I97I).The interesting aspect of the epidemic at Lucknow

has been the presence of adenoviruses as well aspicornaviruses in many patients. In two of thepatients from whose conjunctival swabs adenoviruswas isolated, rising antibody titres against EC2/7Iwere also present. A similar double infection hasbeen observed by Dr M. S. Pereira (personal com-munication) in an epidemic of conjunctivitis inLondon. Another peculiar feature of this epidemicin India has been the observation of neurologicalcomplications. In three patients we observed paresisof the soft palate which developed after the con-junctivitis (Saxena and others, I972) had subsided.Similar findings of lumbar radiculomyelopathyhave been observed in Bombay by Wadia (personalcommunication). It is not certain whether these

complications were aetiologically related to theconjunctivitis or if they are merely coincidental.

Summary

A pandemic of acute haemorrhagic conjunctivitisoccurred in south-east Asia during I970 and I97I,and became epidemic in Lucknow in May I97I.From the conjunctival swabs adenovirus-like agentswere isolated in monkey kidney tissue culture; onewas typed as adenovirus 2. In paired sera risingantibody titres were found against an adeno-likeagent isolated and the picornavirus (EC2/7 I)isolated in Singapore. The epidemics in south-eastAsia were caused by a picomavirus while in Lucknowboth adenovirus and picornavirus appeared to beimplicated.

We thank Dr M. S. Pereira and Dr H. G. Higgins of theVirus Reference Laboratory, Colindale, London, fortyping the adenovirus, detection ofpicornavirus antibodies,and reading the manuscript of this paper; and Dr T. H.Flewett (Regional Virus Laboratory, Birmingham) whoalso read the paper and examined our isolates by electronmicroscopy. Part of the study was supported by a grantfrom the Indian Council for Medical Research.

ReferencesCHATTERJEE, S., QUARCOOPOME, C. O., and APENTENG, A. (I97oa) Ghana med. J., 9, 9

(I 97ob) Brit. J. Ophthal., 54, 628KONO, R., SASAGAWA, A., ISHII, K., SUGIURA, S., OCHI, M., MATSUMIYA, H., UCHIDA, Y., KAMEYAMA, K., KANEKO, M., and

SAKURAI, N. (I 972) Lancet, I, I I9ILIM, K. H., and YIN-MURPHY, M. (I97I) Singapore med. J., 12, 247PARROTT, W. F. (1971) Practitioner, 2o6, 253ROSEN, L. (I960) Amer. J. Hyg., 71, 120SAXENA, R. C., BHATIA, M., and CHATURVEDI, U. C. (1972) Orient. Arch. Ophthal., I0, 253YIN-MURPHY, M. (1972) S.-E. Asian J. trop. Med. publ. Hlth, 3, 303- and LIM, K. H. (I972) Lancet, 2, 857