295

and his assistants for careful observation of the technique ofcatheterisation described; and to Mrs. P. E. Strong and Mr. J.Maton for technical and clerical assistance.

REFERENCES

Gibson, E. (1958) Brit. med. J. i, 1326.McLeod, J. A., McLeod, J. W. (1959) J. Path. Bact. 77, 219.

— — (1961) Brit. J. exp. Path. 42, 171.McLeod, J. W. (1958) Lancet, i, 394.

— — Taylor, M. M. Unpublished.Pyrah, L. N., Goldie, W., Parsons, F. M., Raper, F. P. (1955) Lancet, ii, 314.Shackman, R., Messent, D. (1954) Brit. med. J. ii, 1009.Turk, D. C. (1959) Canad. med. Ass. J. 80, 194.

THE ISOLATION OF RESPIRATORY

SYNCYTIAL VIRUS FROM CHILDREN WITHACUTE RESPIRATORY DISEASE

A. HOLZELM.D. Prague, D.C.H.

READER IN CHILD HEALTH, UNIVERSITY OF MANCHESTER

L. PARKERM.A., B.M. Oxon.

CONSULTANT BACTERIOLOGIST,CRUMPSALL AND BOOTH HALL CHILDREN’S HOSPITALS, MANCHESTER, 9

W. H. PATTERSONM.D. Belf., D.C.H.

CONSULTANT PÆDIATRICIAN

L. L. R. WHITEM.D., B.Sc. Wales, D.C.H.CONSULTANT PATHOLOGIST

BOOTH HALL CHILDREN’S HOSPITAL

K. M. THOMPSONF.I.M.L.T.

J. O’H. TOBINM.A., B.M. Oxon., Dp. Bact. Manc.

OF THE PUBLIC HEALTH LABORATORY, MANCHESTER, 10

SINCE its first isolation by Morris et al. (1956), respira-tory syncytial (R.S.) virus has been shown by Americanand Australian workers (Chanock et al. 1961, Forbes et al.1961, Hamre and Procknow 1961, Reilly et al. 1961) tobe an important cause of severe respiratory disease inyoung infants, and milder infections in older subjects. Inthis country the virus has been isolated previously onlyby Peacock and Clarke (1961), who found it in twoinfants with bronchiolitis.This paper reports the isolation of R.s. virus from the

throats of thirty-six infants and young children admittedto hospital in the four months January to April, 1962,together with an account of the clinical symptoms andpathological findings in infection with this agent.

Materials and Methods

All children entering two of the medical wards at BoothHall Hospital had their throats swabbed in the receiving-room by the medical officer admitting the patient. Oneswab was used for the inoculation of a HeLa cell-culturetube and a monkey kidney cell-culture tube, and anotherswab was placed in Stuart’s transport medium for

bacteriological examination. The cell-culture tubes weremarked with the patient’s name and ward.The cell-culture tubes used for inoculation were kept in the

receiving-room, in stationary racks in an incubator held at33-34°C. Immediately after inoculation these tubes werePlaced in a roller-drum machine in the same incubator. Twiceweekly, inoculated cultures were transferred to the PublicHealth Laboratory, where incubation was continued undersimilar conditions. The cell-cultures were examined micro-scopically every 2 or 3 days, the medium being changed whenrequired. This was usually necessary 5 or 6 days after inocula-tion for the HeLa cells, and after a week for the monkey kidneycells.The monkey kidney cells were secondary cultures, prepared

at the Public Health Laboratory from primary cultures kindlysupplied by the Division of Immunological Products Controlof the National Institute of Medical Research (Mair and

Tobin 1960). The growth medium for these cultures was0-3% lactalbumin in Hanks’ solution (containing 0-08% sodiumbicarbonate, 5% calf serum, and 100 units per ml. of penicillinand streptomycin). Before the tubes were sent to the hospital,the medium was removed, the cells washed twice withHanks’ solution, and 1 ml. of " 199 " or lactalbumin Hanks’solution (without calf serum added). The HeLa cell-cultureswere also prepared at the Public Health Laboratory, from the" Bristol" line of cells (Peacock and Clarke 1961) kindlysupplied by the Common Cold Research Unit, Salisbury,Wiltshire. These cells were grown in the same medium as themonkey kidney cultures, except that 10% rabbit serum wasused instead of calf serum. Before dispatch to the hospital,the medium was changed, only 2% rabbit serum being incor-porated in the maintenance medium.The presence of respiratory syncytial virus was suggested

by the appearance of the characteristic cytopathic effect inthe HeLa cells, beginning 5-12 days after inoculation.This consisted of the formation of giant cells and syncytiawhich could be shown (by staining in situ with haema-toxylin and eosin) to contain cytoplasmic eosinophilicinclusion bodies. The virus was further identified byneutralisation with specific rabbit antiserum, and by lack ofhaemadsorption. The cytopathic effect produced in monkeykidney cells was not so characteristic as that in HeLa,and consisted mostly of rounding of cells with little or nogiant-cell or syncytial formation. Monkey kidney culturessuspected of containing R.s. virus were subcultured toHeLa cells for confirmation.The swabs in Stuart’s media were collected from the

receiving-room each morning and streaked on to platesof three culture media. This routine simplified the

organisation of the work for dealing with the swabs, andenabled the plates to be incubated for a standard time.The media and conditions of incubation were: (1)

layered 10% horse-blood agar, incubated aerobically, (2)chocolate agar, incubated in an atmosphere of approxi-mately 5% carbon dioxide in air, and (3) a layered 10%horse-blood agar containing 1/1,000,000 crystal violet inboth layers, incubated anaerobically. The plates wereincubated at 37°C, and inspected after 24 and 48 hours.The media used were considered to be suitable for theisolation of those bacteria commonly associated withinfections of the respiratory tract.The organisms were identified by standard practices

essentially similar to those described by Masters et al.

(1958).During the period covered by this report all the plates

were examined by one observer. The findings wererecorded without any knowledge of the clinical diagnosis,and before the results of the virological investigationswere known.

Results

R.s. virus was isolated from thirty-six cases admitted tohospital during January-April, 1962. The age-distributionand type of respiratory infection are given in table I. Theinfection affected mainly infants, 75 % of the cases beingunder 7 months; the oldest child in the group was 4 years.Twenty-three of the subjects were boys.

Coryza, cough, fever, and often an audible wheeze,

TABLE I-AGE-DISTRIBUTION AND CLINICAL SYNDROME OF 36 SUBJECTSYIELDING R.S. VIRUS

296

Fig. 1-X, aged 2 months. Consolidation in

right upper lobe.Fig. 2-Y, aged 7 months. Patchy consolida-

tion and atelectasis mainly in right lung.Increased right hilar markings.

were the initial clinical manifestations, the children beingadmitted to hospital after the development of respiratorydistress. In a few, vomiting had been reported by theparents, but this was not seen after admission. In a

quarter of the cases the illness had lasted less than 24hours; in half it lasted 3 days; and in the remaindersymptoms had been present for 4-7 days before admission.Fever of more than 100°F was present in twenty-fourcases, while in eleven infants the temperature never roseabove 99°F and in one case it was subnormal. The strikingfeature in all the cases was expiratory dyspnoea of varyingdegree, and in some cases the indrawing of the intercostalspaces indicated some inspiratory difficulty as well.

Cyanosis was relatively rare. In most cases localised areasof emphysema could be demonstrated on percussion, andfine rales and crepitations were audible over both lungs.In milder cases an expiratory wheeze was the predominantadventitious sound. In a few cases there were distinct

signs of consolidation: in three of these, X-ray examina-tion showed the whole lobe to be involved. In the

majority of patients, diffuse mottling-suggestive oflobular atelectasis-was found radiologically (figs. 1 and 2).

In conjunction with the clinical findings, the X-rayappearances formed a fairly characteristic pattern; butthese could not be regarded as diagnostic.

Forbes, Bennett, and Gray (1961) said that the white-cell count was characteristically low, with a predominance

Fig. 3-Z, aged 10 months. Hyperplastic bronchial epithelium.

of lymphocytes in the differentialcount. Leucocyte-counts were doneon twenty-five of our cases. Thecounts ranged from 4100 to 23,000 perc.mm., and in only nine was the totalnumber less than 10,000 per c.mm.In the differential blood-picture, lym.phocytes predominated in all exceptfive cases; but this finding is normalfor the age-group concerned. In fivecases the lymphocytes exceeded 75%of the total, and in two cases was morethan 80%. The erythrocyte-sedimen-tation rate (E.S.R.) was raised in nine.teen of the twenty-five cases, thehighest 1-hour reading being 57 mm.In five of the six infants with a normalE.S.R. the total white-cell counts wereunder 10,000 c.mm.

In all thirty-six cases symptomscleared in a relatively short time,twenty-seven of the babies being dis-

charged in less than a week. In five cases recovery took10 days; and in four a fortnight. The duration of illnessseemed shorter than that reported by Forbes et al. (1961),whose cases were in hospital from 11/2 to 3 weeks.

Otitis media developed in three infants. One of thebabies with R.s. virus infection had congenital monocytic

Fig. 4-Z, aged 10 months. Area of pulmonary consolidation.

leukxmia. This child recovered from its virus infection;but succumbed a month later to the blood disease.A 10-month-old boy, admitted to hospital with the

characteristic symptoms of obstructive bronchitis, died24 hours after admission, having been ill for 9 daysR.s. virus was isolated from his throat before death.At necropsy, the pharynx, larynx, and pleural sacs were normal;but there was mucosal congestion of the trachea and bronchi,with abundant, clear secretion in the lumen. Both lungs werevoluminous, with marked emphysema anteriorly: smallscattered areas of collapse and consolidation were present,chiefly posteriorly and near the hila. There was some cso-

phagitis with punctate gastric haemorrhage, and some alteredblood in the gastrointestinal contents. Other changes were:marked fatty change of the liver, and renal pallor. Mioo-scopically, what seemed to be the earliest lesions in the tracheo-bronchial tree consisted of ballooning of the goblet cells, anddesquamation of the columnar layer. In other areas thisprocess seemed to have progressed until only a few basal cdlsremained. There was appreciable mural infiltration bylymphocytes and plasma cells. The exudate within the lumiasincluded mucus, mononuclear phagocytes, and large eosiao-philic bodies (probably necrotic cell-debris). In what was

297

TABLE II-BACTERIAL FLORA OF R.S. VIRUS CASES AND OTHER

SUBJECTS UNDER 1 YEAR

considered an intermediate stage of the bronchial mucosallesion (fig. 3) the cells of the residual deeper layers were hyper-plastic and swollen, and occasional mitotic figures were seen.Some nuclei were swollen; and in them the chromatin was dis-placed peripherally, and the nucleoli were prominent. Nodefinite inclusion-bodies were seen. There was widespreademphysema of the lung parenchyma, with focal collapse andedema. In the small areas of consolidation (fig. 4) the exudateusually consisted of mononuclear phagocytes, and necroticcell-debris; but in places some fibrin and polymorph infiltra-tion were present. There was mononuclear proliferation in thealveolar walls.

The presence of proliferation, nuclear swelling,vesiculation, and cell necrosis, in the tracheobronchialmucosa-together with the nature of the focal consolida-tion-was regarded as consistent with a virus infection.The extensive emphysema suggested incomplete bronchialor bronchiolar obstruction as an important feature, butthis was not obvious on microscopy. The discrepancy maybe partly explicable by the nature of the exudate, andby damage to the bronchial mucosa (with consequentloss of ciliary activity).We limited our bacteriological studies to children

under a year old, admitted to hospital during the periodof isolation of R.s. virus. We believed that this wouldshow most clearly any difference between the findings inthe R.s. virus group and the control groups. The control

groups consisted of seventy children with respiratoryinfection but from whom R.s. virus was not isolated, andsixty-nine children without evidence of respiratorydisease. The results are given in table 11. " Other

hxmolytic streptococci " includes organisms of groups Gand 0, and unidentified strains. " Coliform bacilli "refers to gram-negative rods capable of growing on plainagar: they include the klebsiella group of organisms. Adetailed analysis is being made of these coliform bacillias part of a wider investigation into their occurrence inthe upper respiratory tract of children. In the presentseries about half the isolates were faecal strains ofEscherichia coli. Enteropathogenic coliform bacilli wereoccasionally found.The only obvious bacteriological differences between

the R.s. virus group and the groups of respiratory and

non-respiratory disease were in the incidence of a normalflora, of Hamophilus influenza, and of " other haemolyticstreptococci ". With H. influenza the differences betweenthe R.s. virus group and the other two groups were

significant-p < 0,05 in each case. Five strains ofH. influenza type b were isolated, two from each respira-tory group, and one from the non-respiratory group. Thedifferences in the number of cases with " other hxmolyticstreptococci " and with normal flora were not statisticallysignificant.

Discussion

R.s. virus was shown to be associated with a largeproportion of the respiratory infections occurring ininfants admitted to a children’s hospital during the first3 months of 1962. It caused respiratory disease of varyingseverity, from mild upper respiratory infections to onewhich was fatal.The clinical diagnosis in most of our patients with

lower respiratory infection was obstructive bronchitis, orbronchiolitis, or bronchopneumonia. Occasionally theclinical picture was indistinguishable from an acute

asthmatic bronchitis. The pattern remained fairlycharacteristic during the outbreak, so that in the laterstages the aaiology could be suspected merely on clinicalgrounds.The respiration-rate varied a great deal, but reflected

the extent of the pulmonary involvement rather than thedegree of pyrexia. The gastrointestinal tract did not appearto be affected, although vomiting and loose stools for aday or two had been noticed by parents: these manifesta-tions were not observed in hospital. The E.s.R. and thewhite-cell picture did not allow any definite conclusionsas to the presence or absence of secondary bacterialinfection.

Although R.s. virus was demonstrated in only one fatalcase, a re-examination of the history of two infants whodied on the way to hospital suggested that they too mighthave suffered from this virus infection.

R.S. virus was also isolated from two throat-swabsobtained from other sources. One isolate was obtainedfrom an infant with coryza, by direct inoculations of tissue-culture tubes immediately after swabbing in the doctor’ssurgery. The other was found in one of fifteen serologicallyproved cases (admitted to another hospital) from whichmaterial was extracted, frozen, and sent to us for virusisolation (Moss et al. 1963).The isolation of this agent was facilitated by the use of

Bristol " HeLa cells grown and maintained in rabbit-serum medium. These cells can detect R.s. virus at aconcentration of 1/100 of that needed to infect cell linesgrown and maintained in medium containing suckled-calf serum (Peacock 1962, Taylor-Robinson 1963).

All the R.s. virus isolations were made in " Bristol "

cells, but the corresponding monkey kidney cell-culturealso yielded virus in eleven cases. However, if thecharacteristic changes in HeLa cells occurred before any

TABLE III-NUMBER OF VIRUSES ISOLATED FROM THROAT SWABS TAKEN AT BOOTH HALL HOSPITAL, MANCHESTER, IN 1961-62

298

cytopathic change was recognisable in the monkeycultures, the latter were discarded after 10 days’ incuba- 1tion without further investigation. It is therefore

1

impossible to say whether monkey kidney cells are as goodas

" Bristol " HeLa cells, although there is little doubt thatthey are superior to some other HeLa lines.The apparent association of H. influenzce with R.S. virus

infections suggests that, if antibiotics are to be used in

suspected cases, chloramphenicol or tetracycline would bethe agents of choice.An increase in the incidence of H. infiuenzae in R.S. virus

infection is not peculiar to this virus, as experience withinfluenza has shown. It is likely that these bacteria aresecondary invaders of a mucosa already damaged by virus.The findings in our one fatal case show that such damagecan occur.

The isolation rate of hxmophili in the R.S. virus groupwas similar to that found by Masters et al. (1958) inchildren under 4 years, but the rate found in the other two

groups was considerably lower.The percentage of children from whom pneumococci

were isolated was surprisingly low. The superiority ofpernasal swabs for the isolation of pneumococci wasshown by Alexander and her colleagues (1941); but

although Masters et al. (1958) confirmed this, theirisolation-rate from throat swabs was also much higherthan ours.

R.S. virus has not yet been isolated in the Public Health

Laboratory from postmortem material; but it is probablethat the virus is not present in the respiratory tract atthe time of death, or else dies out by the time necropsysamples are taken. Isolations were obtained only duringthe first week of illness, and if the one fatal case is typical,it is possible that death due to this virus ensues only afterthis time.As can be seen from table 11, R.S. virus was the pre-

dominant one found during its period of isolation. Itsoccurrence was preceded by an outbreak of influenza-Bvirus infections, and followed by increased isolations ofadenoviruses, parainfluenza viruses (types 1 and 3), andenteroviruses (mainly Coxsackie B4). , An adenovirustype 21 was isolated in one instance-from cell-culture

passage material containing R.S. virus-by Dr. M. S.

Pereira, to whom most of our isolates were subsequentlysent. In our hands, " Bristol " HeLa cells failed to showa cytopathic change when inoculated with other isolatesof type 21 and with certain other types of adenovirus.

Although R.S. virus appeared to be the cause of at least30% of the acute respiratory infections in infants under1 year admitted to hospital during the months Januaryto March, 1962, further studies will be necessary to

define its exact importance in respiratory disease fromone year to another. An antibody survey showed thatinfection was widespread in the north-west of England(Moss et al. 1963), but whether it occurs yearly or inepidemics in this area, has yet to be determined.*

SummaryThe clinical features of respiratory syncytial virus

infection are described in thirty-six infants and youngchildren from whom this virus was isolated. Most of thecases were in infants with lower respiratory infections ofvarying severity. The method of virus isolation, thebacteria found in association with the virus infection, andthe necropsy findings in one fatal case, are described.* During the 7 weeks from December 6, 1962, R.S. virus has been isolated

from sixteen infants and young children with respiratory diseaseadmitted to Booth Hall Hospital.

Our thanks are due to the hospital resident staff for taking swabs,to Dr. M. S. Pereira for R.s. virus antiserum, and to a research granttowards this work from the endowment funds of the Booth Hall andMonsall Hospital Management Committee. The swab from a

patient in general practice was kindly supplied by Dr. D. Macdonald.

REFERENCES

Alexander, H. E., Craig, H. R., Shirley, R. G., Ellis, C. (1941) J. Pediat.18, 31.

Chanock, R. M., Hyun, W. K., Vargosko, A. J., Deleva, A., Johnson, K. M.,Cumming, C., Parrott, R. H. (1961) J. Amer. med. Ass. 176, 647.

Forbes, J. A., Bennett, N. McK., Gray, N. J. (1961) Med. J. Aust. ii, 933.Hamre, D., Procknow, J. J. (1961) Brit. med. J. ii, 1382.Mair, H. J., Tobin, J. O’H. (1960) Mon. Bull. Minist. Hlth, 19, 49.Masters, P. L., Brumfitt, W., Mendez, R, L., Likar, M. (1958) Brit. med.

J. i, 1200.Morris, J. A., Blount, R. E., Savage, R. E. (1956) Proc. Soc. exp. Biol., N.Y.

92, 544.Moss, P. D., Adams, M. O., Tobin, J. O’H. (1963) Lancet, i, 298.Peacock, D. B. (1962) Personal communication.

&mdash; Clarke, S. K. R. (1961) Lancet, ii, 466.Reilly, C. M., Stokes, J., McClelland, L., Cornfeld, D., Hamparian, V. V.,

Ketler, A., Hilleman, M. R. (1961) New Engl. J. Med. 264, 1176.Taylor-Robinson, D. (1963) Personal communication.

SEROLOGICAL STUDIES WITH

RESPIRATORY SYNCYTIAL VIRUS

P. D. MossM.B. Lpool, M.R.C.P., D.C.H.

CONSULTANT P&AElig;DIATRICIAN,BLACKBURN AND DISTRICT HOSPITAL GROUP

MARY O. ADAMSM.B. St. And.,Dp. Bact. Manc.

SENIOR BACTERIOLOGIST

J. O’H. TOBINM.A., B.M. Oxon.,Dp. Bact. Manc.

DIRECTOR

PUBLIC HEALTH LABORATORY, MANCHESTER, 10

RESPIRATORY syncytial (R.s.) virus infection produces,in man, specific antibody which can be demonstrated byeither neutralisation or complement-fixation. Such testsprovide a reliable method for diagnosing the infection(Parrott et al. 1961, Hilleman et al. 1962).We report here the results of serological tests (mainly

complement-fixation) with R.s. virus antigen on pairedsera from children suffering from respiratory disease

during last winter and spring; and on sera from otherpeople of all ages. ,

Materials and Methods

Infants and children, admitted to the Blackburn groupof hospitals with respiratory infection, were bled on

admission and again 7-14 days later. Their sera weretested against antigens prepared from the influenza andparainfluenza viruses, R.s. virus, adenovirus, and the

agents of psittacosis and Q fever. Sera sent up for routinevirus tests and other studies, and sera from women bledat antenatal clinics, were tested for R.s.-virus antibody inorder to determine the incidence of this antibody at

different ages.

Serological TechniquesComplement-fixation test.-Antigen was prepared in a similar

way to that described by Beem et al. (1960). HeLa cells weregrown in 4 oz. prescription bottles or Pyrex’ feeding-bottles,and maintained in 0-3% lactalbumin in Hanks’ solution(containing 2% rabbit serum and 0-08% sodium bicarbonate).These cultures were inoculated with the BH.1290 strain ofR.S. virus. When most of the cells were infected the cultureswere frozen and thawed three times. The culture fluid wasthen centrifuged at 3000 r.p.m. for 20 minutes, and the super-natant used as antigen. The optimal dilution was determinedby a chessboard titration, using a four-volume test (0’1 ml. pervol.), on ’ Perspex ’ plates.

Neutralisation tests.-0.6 ml. samples of serum, in four

dilutions starting at 1 in 10, were mixed with equal volumes ofvirus suspension containing approximately 30 T.c.iDse. Theywere held at room temperature C18-20&deg;C’) for 11/a hours, then