APPLIED MICROBIOLOGY, Nov., 1965Copyright © 1965 American Society for Microbiology

Vol. 13, No. 6Printed in U.S.A.

Sensitivity of Agar Overlay Method for theRecognition of EnterovirusesMARY 0. GABRIELSON AND G. D. HSIUNG1

Virus Diagnostic Laboratory, Department of Epidemiology and Public Health, Yale UniversitySchool of Medicine, New Haven, Connecticut

Received for publication 23 June 1965

ABSTRACTGABRIELSON, MARY 0. (Yale University, New Haven, Conn.), AND G. D. HSIUNG.

Sensitivity of agar overlay method for the recognition of enteroviruses. Appl. Micro-biol. 13:967-972. 1965.-Comparison of the agar overlay technique with the fluid culturetechnique for isolation and identification of enteroviruses showed the former to be use-ful: (i) for isolation of enterovirus when the number of virus particles was too smailto produce detectable cytopathic effect (CPE) in fluid cultures, (ii) for isolation ofechovirus 22 which did not produce detectable CPE in fluid cultures, (iii) as an aid torapid differentiation of enteroviruses, and (iv) for differentiation of viruses in mixedinfections. Nonpolio enterovirus isolation experience in the New Haven area over a4-year period is presented. It was concluded that the agar overlay technique is bothuseful and relatively simple for routine examination of clinical specimens in a diag-nostic laboratory.

Since the development by Dulbecco (1952) ofthe plaque technique for detection of animalviruses, this method has been used for assayinginfectivity of a variety of viral agents. The sub-ject was recently reviewed in detail by Cooper(1961). However, application of this techniquefor the detection and recognition of agents ofunknown nature has been limited.

During the past 4 years, the agar overlaymethod in bottle cultures (Hsiung, 1964) hasbeen used routinely on a variety of specimensreceived by our virus diagnostic laboratory,including throat swabs, rectal swabs, stoolspecimens, cerebrospinal fluid, and all autopsyspecimens. This report concerns our experienceswith the plaque technique for the isolation andidentification of enteroviruses from these clinicalspecimens, with a brief resume of enterovirusactivity in the New Haven area during a 4-yearperiod. A preliminary report of this work hasappeared (Gabrielson and Hsiung, Bacteriol.Proc., p. 132, 1964).

MATERIALS AND METHODSPreparation of bottle cultures. Rhesus monkey

kidney cultures were prepared by the usualmethod as described previously (Hsiung, 1964).The cells were suspended in growth medium of0.5% lactalbumin hydrolysate and 2% calf serum

1 Present address: Department of Medicine,New York University School of Medicine, NewYork, N.Y.

in Hanks' balanced salt solution (BSS). Flat pre-scription bottles (3 oz) were seeded each with 10to 12 ml of cell suspension containing 3 X 106 cellsper milliliter. After 4 to 5 days of incubation at37 C, the fluid was replaced with fresh medium.With an additonal 2 days of incubation, the mono-layer sheet was usually confluent, and the bottleswere ready for use.Agar overlay. Nutrient medium consisted of 60

ml of demineralized sterile water mixed with 18ml of Earle's solution (10 times concentrated;without phenol red or NaHCO3), 3.6 ml of calfserum, 5.4 ml of NaHCO3 (7.5%), 3.0 ml of neutralred (sterile, 1:1,000), and antibiotics. The mediumwas dispensed in 90-ml amounts into sterile stop-pered bottles prewarmed to 37 C before use.A 3.7-g amount of Noble Agar (Difco) was

mixed with 90 ml of distilled water, melted byboiling in a water bath, and sterilized by auto-claving at 15 psi for 15 min. The agar was thencooled to 43 C before use. Just before use, an equalamount of nutrient medium was added to theagar.

Preparation of clinical specimens. Throat andrectal swabs were immersed in 2-ml amounts ofHanks' BSS containing antibiotics, shaken for 30min, and then centrifuged at 3,000 rev/min for0.5 hr. The supernatant fluids were used for in--oculation. Stool specimens were prepared as 20%suspensions in Hanks' BSS. The suspensions wereshaken vigorously for 30 min and centrifuged at3,000 rev/min for 1 hr; the supernatant fluid wasthen removed, and penicillin was added to a con-centration of 500 units per ml before inoculation.Postmortem specimens were ground in a mortar

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GABRIELSON AND HSIUNG

with sterile alundrum. They were then made into10% suspensions in Hanks' BSS and centrifugedat 3,000 rev/min for 30 min; the supernatantfluids were collected and treated with penicillinto a concentration of 200 units per ml. Body fluids(pericardial fluid, cerebrospinal fluid, etc.) wereinoculated directly into tissue cultures withoutprocessing. At times when bottle cultures werenot available, all specimens were stored at -20 Cuntil inoculation.

Inoculation and overlay. When the bottle cul-tures exhibited a solid sheet of cells in monolayer,the fluid was removed and specimens were inocu-lated in 0.1- to 0.2-ml amounts per bottle. Theinoculated bottles were allowed to adsorb at 37 Cfor 1 hr; then, without removal of the inoculum,10 ml of the agar overlay medium were added toeach bottle to cover the cell sheet. Once the bot-tles were overlaid, it was necessary that they bekept in the dark to prevent photosensitization ofthe cultured cells in the presence of neutral red(Green and Opton, 1959). After solidification ofthe agar (about 10 min), the bottles were invertedand incubated at 37 C for up to 2 weeks with dailyobservation for the appearance of plaques.

RESULTS

Comparison of cytopathic effect (CPE) methodand plaque method for primary isolation. In anumber of instances, enteroviruses were isolatedby plaque technique when parallel fluid culturesremained negative (Table 1). Examples of isola-tions of the three types of polioviruses are shown

in the top line of Table 1. In all cases, the num-ber of plaque-forming units (PFU) was small,indicating a paucity of virus particles in thespecimens-perhaps too few to produce detect-able cytopathic effect (CPE) in tube cultures.Isolations of a number of nonpolio enteroviruses,including coxsackieviruses B-3, B4, B-5, andA-9, echovirus4, and echovirus-9, are alsoindicated in the table. Again, the number ofPFU was generally small.

Extremely low concentrations of virus weredetected by the plaque technique on threeautopsy cases. Tube cultures in all of these casesfailed to show CPE. J.E. was a noteworthy case.This 13-year-old girl entered the hospital with ahistory suggestive of myocarditis. The diagnosiswas supported by clinical findings, and the pa-tient expired on the 5th day of hospitalization.No virus was isolated from throat and rectalswabs taken at the time of admission. Studies onblood, cerebrospinal fluid, heart muscle, bowel,liver, brain, and kidney obtained at autopsy werealso negative. Pericardial fluid taken on the wardimmediately after the patient's death was inocu-lated into tube and bottle cultures of rhesus cells.No CPE was noted in the tube cultures. On the6th day after inoculation, six virus plaques ap-peared in one of the two bottle cultures inocu-lated. These were subsequently identified ascoxsackievirus B4. The patient's blood taken at

TABLE 1. Comparison of CPE and plaque method for isolation of enteroviruses

Method of testing

Case Virus type isolated Source of isolationCPE (no. posi- Plaque (avgtive/no. tested) PFU/0.1 ml)

M. P.* Poliovirus 1 Rectal swab 0/3t 2.0R. J. Poliovirus 1 Rectal swab 0/4 0.5D. G. Poliovirus 1 Rectal swab 0/6 4.5B. M. Poliovirus 2 Rectal swab 0/4 0.3D. D. Poliovirus 3 Rectal swab 0/8 1.5P. S. Poliovirus 3 Rectal swab 0/5 0.5F. B.$ Poliovirus 3 Stool (20%) 0/9 0.5

G. D. Coxsackievirus B3 Stool suspension, 20% 0/6 6.0B. K. Coxsackievirus B4 Stool suspension, 20% 0/3 2.5J. E. Coxsackievirus B4 Pericardial fluid 0/5 1.5M. L. Coxsackievirus B5 Stool suspension, 20% 0/5 2.0F. A. Coxsackievirus A9 Throat swab 0/5 1.0

P. P. Echovirus 4 Stool suspension, 20% 0/5 1.0A. R. Echovirus 9 Throat swab 0/5 0.25R. L. Echovirus 9 Throat swab 0/6 0.25N. W.t Echovirus 9 Spinal cord (20%) 0/5 0.5

* Paralytic poliomyelitis.t No. of tubes showing CPE/no. of culture tubes tested. Inoculum: 0.1 ml per tube or bottle.I Postmortem specimens.

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autopsy had a neutralizing antibody titer of1:160 against coxsackievirus B4. Postmortemexamination in this case showed myocardiallesions consistent with a coxsackievirus B infec-tion (Sanyal et al., 1965).

Isolation of echovirus-22. Two strains of echo-virus-22 which failed to produce CPE in tubecultures were isolated by plaque technique(Table 2). In each case, small, irregular hazyplaques appeared after about 10 days of incuba-tion. Repeated attempts at picking these plaquesand inoculating them into tube cultures failed toproduce CPE, although the virus could be re-covered from the culture fluid by reinoculationinto bottles for plaques. In both imstances, isola-tions would not have been made had not theplaque technique been used. The second case,C.M., was a 15-year-old girl who had been inexcellent health until 5 days prior to admission,at which time she developed generalized muscle

aches, shaking chills, and fever to 101 F; 1 dayprior to admission her fever rose to 105 F, andshe developed progressive difficulty in breathing.On admission she was described as practicallymoribund. She died within 5 hr. Her clinicaldiagnosis, staphylococcal pneumonia, probablysuperimposed on a viral infection, was supportedby bacteriological and viral studies in additionto the postmortem examination.Rapid diagnosis of enterotirus infection. Routine

use of the plaque technique resulted, in manyinstances, in more rapid presumptive identifica-tion of viral isolates. Preliminary diagnosis ofthe virus type was based on the plaque patternin conjunction with the host-cell susceptibility,as previously described (Hsiung, 1962). Table 3shows representative examples of time requiredfor presumptive and final identification of anumber of enterovirus types.

In the case of poliovirus (Table 3, first two

TABLE 2. Isolation of echovirus-22

Case l ClinlicalI diagnosis | Source of isolation (20% CPE (no. positive/ Plaque (agCase ~~~~~~~~~~~ ~~~~suspension) no. tested) PFU0.lml

D. I. Recurrent pneumonia Stool 0*/6 250C. M-t Staphylococcal pneumonia Lung 0/5 25

Colon content 0*/5 22

* Virus was recovered by the plaque method in subcultures.t Postmortem specimens.

TABLE 3. Time required for presumptive and final identification of enteroviruses

Days after specimenwas received

Patient Clinical diagnosis Plaque patter on rhesus Virus isolatedmonkey cellsPresumptive Final iden-diagnosis tification

P. J. Poliomyelitis Large, clear 6 10 Polio-iJ. L. C. DOA* Large, clear 5 9 Polio-3

S. J. FUOt Medium, hazy 5 85 Coxsackie BiL. M. FUO Medium, hazy 7 31 Coxsackie B4B. J. Poliomyelitis Medium, hazy 5 23 Coxsackie B5S. D. Pharyngitis Medium, clear 12 30 Coxsackie A9S. R. Pharyngitis, rash Medium, clear 8 30 Coxsackie A9J. S. FUO Medium, clear 5 21 Coxsackie A9

M. J. Aseptic meningitis Small, irregular, hazy 7 30 Echo4L. R. FUO Small, irregular, hazy 7 25 Echo4S. E. Pleurodynia Small, irregular, clear 5 28 Echo-8D. I. Recurrent pneumonia Small, irregular, hazy 10 270 Echo-22C. M. Staphylococcal Small, irregular 13 74 Echo-22

pneumoniaJ. B. Aseptic meningitis Two types-large, clear, 10 85 Polio-i and

and small, hazy echo4

* Dead on arrival.t Fever of undetermined origin.

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cases), the time required for final identificationwas only 4 to 5 days after the presumptive diag-nosis was made. However, final identification ofa strain of echovirus-22 was not made until 270days after its isolation. In the latter case, a pre-sumptive diagnosis of an echovirus infection wasmade on the 10th day, when small, irregular,hazy plaques appeared in cultures inoculatedwith the patient's stool specimen.Mixed infections and plaque variations. Mixed

infections can readily be recognized when two ormore types of plaques are produced in bottlecultures inoculated with a single specimen.Figure 1 shows an example of mixed infectionwith a poliovirus (large plaque) and an echovirus(small plaques). The two virus strains wereseparated by subculturing single virus plaquesinto fresh cultures. Final identification of thepurified viruses was made by serological tests.Dual infection with an enterovirus (coxsackie

B-1) and adenovirus type 7 was demonstrated influid from a tissue culture tube inoculated withcerebrospinal fluid of an infant recuperating fromHaemophilus influenzae meningitis. The presenceof the coxsackievirus was not suspected untilthe infected tissue culture fluid was plated intoagar overlay cultures in an attempt to produceadenovirus plaques. Two kinds of plaques subse-quently appeared-the minute plaques of adeno-virus type 7 and medium-sized plaques which.1E'-proved on serological identificatiosackie B-1. Apparently, the numbervirus B-1 particles in the original Ismall, and the cultures were overgadenovirus. (The original CSF speci

FIG. 1. Mixed infection. Bottle at lepoliovirus plaques. Bottle in centerinfection with poliovirus plaques (larvirus plaques (small, irregular). Bshows echovirus-8 plaques only.

FIG. 2. Plaque variation of coxsackievirus B-2in rhesus monkey kidney cell cultures 7 days afterinoculation. Bottle at left, stool sample obtainedfrom the sister of patient S.B. (6686). Bottle at right,rectal swab of S.B. (6686).

n to be cox- inoculated into agar overlay cultures because ofof coxsackie- insufficient quantity.)CSF was too Plaque variation led to erroneous suspicion ofrrown by the mixed infection in the case shown in Fig. 2. Bothimen was not large and small plaques were noted in bottles

inoculated with a rectal swab of the patient,S.B. (6686), but only the large-sized plaquesappeared in cultures inoculated with stool speci-mens from the patient's sister and father. Passagematerials from both large and small plaques wereneutralized by coxsackie B-2 antiserum, and se-rial passage of the small plaques into bottle cul-tures resulted in the production of large plaques

Enterovirus activity in the New Haven area,1961-1965. Figure 3 depicts isolation experiencesof nonpolio enteroviruses in the New Ha'ven

.-. area during the 4-year period, 1961 to 1965.Epidemics occurred in both the summers of1961 and 1962; 1963 proved to be a quiet yearwith respect to enteroviruses; in 1964 the numberof enterovirus isolations was somewhat greaterthan in 1963, but still failed to reach epiden4

eft shows large proportions.shows mixed In 1961, the predominant enteroviruses were

rge) and echo- coxsackievirus B4 (30 cases) and coxsackievirusottle at right B-5 (13 cases). In the summer of 1962, a few B

coxsackieviruses were isolated, but the epidemic

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20 -

lS -

10 -

19 61

11 rA me m I0~I

1962

1963

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O ECHO VIRUSTYPES 4, 6,8, 9, 11, 22

ol VIA V/IA 19 41 I

IE

s 4M I"^ IA F91 "|uf I..v InI0^v| t RCOXSACKIE 8 VIRUSCTYPESA,23, 4A5

[E] COXSACKIE A-9 VIRUS

FIG. 3. Nonpolio enterovirus isolations, 1961-1965, Virus Diagnostic Laboratory, Yale-NewHaven Medical Center.

virus was echovirus4. In both years, the clinicalsyndrome accounting for the largest percentageof cases was aseptic meningitis9-44% of totalcases in 1961 and 68% of total cases in 1962. Inboth years, nonspecific febrile illnesses accountedfor about 25% of the cases. Respiratory illnesses,particularly pneumonia, occurred in 25% ofthe cases in 1961, as opposed to only 5% in1962.Age distribution in the two epidemics was

striking. In 1961, 50% of all cases occurred ininfants under 2 years of age. In 1962, on theother hand, 75% of total isolations came frompatients aged 6 years or more.

In 1964, a total of 25 nonpolio enterovirusstrains were isolated. The most frequent typewas coxsackievirus A-9 (eight isolations), with

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several isolations each of coxsackieviruses B-1and B-2, and echovirus4. Clinical syndromesassociated with the majority of these infectionsincluded nonspecific febrile illness, upper respira-tory illness, and aseptic meningitis. There didnot appear to be any particular relationshipbetween the type of virus isolated and the clinicalsyndrome, nor was the age distribution remark-able.

DISCUSSIONThe data accumulated in our diagnostic labora-

tory during a 4-year period indicate the value ofroutine use of the agar overlay technique forclinical specimens. In numerous instances, includ-ing all of those reported in Tables 1 and 2, iso-lates would have been missed entirely had CPEin tube cultures been the only method used forisolation. The plaque technique provided a moresensitive method of detection when the numberof infectious particles in a specimen was toosmall to produce detectable CPE in fluid cul-tures.

Isolation of the two strains of echovirus-22 byplaque technique was noteworthy. Shaver,Barron, and Karzon (1961) noted that echo-virus-22 and -23 produced distinctive cyto-pathology in stained preparations; Wigand andSabin (1961) reported that echovirus-22 and -23showed higher titers when assayed by the plaquemethod than by the CPE method; however,failure of echovirus-22 to produce CPE has notbeen previously reported. Similar observationswere made with respect to certain arboviruses byHenderson and Taylor (1959) and Porterfield(1959), and for a strain of myxovirus by Hsiung(1959), but this phenomenon has not beenreported in enterovirus isolations.

Although it was not usually possible to make adiagnosis on the basis of plaque size and morphol-ogy alone, these observations, along with con-sideration of the host-cell spectrum of the virus,as demonstrated in different cell cultures, fre-quently resulted in a more rapid identification ofan agent. Thus, observation of plaque morphol-ogy can result in a presumptive diagnosis as soonas an agent is detected-usually within 5 to 10days. Final identification still depends uponserological tests and may take a considerableamount of time, depending upon the propertiesof the isolate.The current use of live poliovirus vaccine has

created additional problems in the dignosticlaboratory with the consequent increased possi-bility of mixed infections. When this occurred,the plaque technique proved useful in separatingand identifying the agents involved. In the caseof dual infection with coxsackievirus B-1 and

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an adenovirus, presence of the enterovirus wasinadvertently discovered and would have goneunrecognized had not the agar overlay techniquebeen used.

It should be noted, however, that variation inplaques produced by a single virus strain mayoccur, as in the case of coxsackievirus B-2 infec-tion; therefore, identification must always beconfirmed by serological means. In studies ofechovirus4 and -6, plaque variation was alsonoted by Barron and Karzon (1961) and byKarzon (1959).With the reduction in the number of cases of

poliomyelitis in this country in the past few yearsand the decline in the prevalence of wild polio-virus strains, there has been increased interestin other enteroviruses, i.e., the coxsackievirusand echovirus groups. These have been encoun-tered in a variety of clinical syndromes, includingaseptic meningitis, pleurodynia, myocarditis inthe newborn, and many others. As shown inFig. 3, the New Haven area experienced amarked variation in the prevalence of virustypes from year to year, not necessarily reflectedby the types of clinical illnesses. For example,aseptic meningitis was the predominant syn-drome seen in the sunmners of both 1961 and 1962,but virus isolations indicated that two differentgroups of enteroviruses, i.e., coxsackievirus B in1961 and echovirus4 in 1962, were involved.In other years, a variety of clinical syndromeswere encountered in association with a varietyof enterovirus types.

ACKNOWLEDGMENTS

We wish to express our appreciation to themembers of the resident staffs of the Departmentsof Pediatrics and Internal Medicine for their col-laboration in collecting specimens and for theirconstant interest. We also wish to acknowledgethe excellent technical assistance of Maria Zwengand Grace Tucker.

This investigation was supported by PublicHealth Service research grant AI-05577 from the

National Institute of Allergy and Infectious Dis-eases.

LITERATURE CITEDBARRON, A. L., AND D. T. KARZON. 1961. Charac-

teristics of ECHO4 (Shropshire) virus isolatedduring epidemic of aseptic meningitis. J. Im-munol. 87:608-615.

COOPER, P. D. 1961. The plaque assay of animalviruses. Advan. Virus Res. 8:319-378.

DULBECCO, R. 1952. Production of plaques in mono-layer tissue cultures by single particles of ananimal virus. Proc. Natl. Acad. Sci. U.S. 38:747-752.

GREEN, R. H., AND E. M. OPTON. 1959. Photosen-sitization of tissue culture cells and its effecton viral plaque formation. Proc. Soc. Exptl.Biol. Med. 102:519-521.

HENDERSON, J. R., AND R. M. TAYLOR. 1959. Ar-thropod-borne virus plaques in agar overlaidtube cultures. Proc. Soc. Exptl. Biol. Med. 101:257-259.

HsIUNG, G. D. 1959. The use of agar overlay cul-tures for detection of new virus isolates. Virol-ogy 9:717-719.

HsIUNG, G. D. 1962. Further studies on character-ization and grouping of ECHO viruses ... com-parative virology. Ann. N.Y. Acad. Sci. 101:412-422.

HSIUNG, G. D. 1964. Diagnostic virology, p. 28-35.Yale Univ. Press, New Haven, Conn.

KARZON, D. F. 1959. Phase variation in ECHO-6virus. Virology 9:564-576.

PORTERFIELD, J. S. 1959. Plaque production withyellow fever and related arthropod-borne vi-ruses. Nature 183:1069-1070.

SANYAL, S. K., M. MAHDAVY, M. 0. GABRIELSON,R. A. VIDONE, AND M. J. BROWNE. 1965. Fatalmyocarditis in an adolescent caused by Cox-sackie virus, Group B, type 4. Pediatrics 35:38-41.

SHAVER, D. N., A. L. BARRON, AND D. T. KARZON.1961. Distinctive cytopathology of ECHO vi-ruses types 22 and 23. Proc. Soc. Exptl. Biol.Med. 106:648-652.

WIGAND, R., AND A. B. SABIN. 1961. Propertiesof ECHO types 22, 23 and 24 viruses. Arch. Ges.Virusforsch. 11:224-247.

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