Journal of Clinical InvestigationVol. 44, No. 7, 1965

Response of Volunteers to Inoculation with Hemagglutinin-positive and Hemaggiutinin-negative Variants of

Coxsackie A21 Virus *

DAvmJ. LANG, THOMASR. CATE, ROBERTB. COUCH, VERNONKNIGHT,tAND KARL M. JOHNSON

(From the National Institute of Allergy and Infectious Diseases, Laboratory of ClinicalInvestigations, Bethesda, Md., and the Middle America Research Unit,

Balboa Heights, Canal Zone)

Coxsackie A21 virus has been associated withnaturally occurring human respiratory illness(1-4), and in this laboratory many of the re-sponses to inoculation with this agent have beenstudied in volunteers (5, 6).

In a previous report (7) the recognition andseparation of hemagglutinating and nonhemag-glutinating Coxsackie A21 particles were described.Evidence was presented that these were geneticvariants distinguishable by differences in theircapacity to grow in primary and continuous cellline tissue cultures. Passage in continuous celllines led to a predominance of hemagglutinin-negative (HA -) particles, whereas passage inprimary tissue culture slightly favored growth ofhemagglutinin-positive (HA+) particles.

To determine whether the HA+ and HA -variants of Coxsackie A21 virus caused differinghuman responses, volunteer inoculations withseparated pools of these variants were performed.The results of these inoculations will be described.

Methods

Preparation of inocula. The virus used for volunteerinoculations was derived from a throat swab specimenobtained from a Marine recruit with an acute upperrespiratory illness (no. 48560, Bouma) (7). Thisstrain of Coxsackie An was serologically indistinguish-able from the original Coe strain described by Lennette,Fox, Schmidt, and Culver (1). Virus was initially iso-lated in primary human embryonic kidney (HEK) tis-sue cultures. Separation of the HA+ and HA- par-ticles was accomplished by differential adsorption on hu-man type 0 erythrocytes at 40 C (7). The separated

* Submitted for publication December 7, 1964; ac-cepted March 4, 1965.

tAddress requests for reprints to Dr. Vernon Knight,Clinical Director, National Institute of Allergy and In-fectious Diseases, Bethesda, Md. 20014.

erythrocyte and supernatant fractions were further puri-fied by four terminal dilution passages in HEK tissuecultures. These separated pools which contained virusthat had been passaged nine times in HEK tissue cul-tures were centrifuged at 2,000 rpm for 20 minutes,filtered through gradocol membranes (830 mis), andstored at - 700 C until used.

An additional, further purified pool of each hemag-glutinin variant was prepared from individual plaqueharvests of the pools described above. Semicontinuousdiploid fibroblast tissue cultures derived from humanembryonic lung (WI-26) (8) were used for this proce-dure in a plaque assay technique similar to that describedelsewhere (9). Medium containing neutral red in a finaldish concentration of 1:50,000 was added after 60 hoursof incubation, and plaques were counted at 68 to 72 hours.The individual plaque harvests of each hemagglutininvariant were passaged an additional time in WI-26 tis-sue culture tubes to obtain sufficient volume for volunteerinoculations. These pools were centrifuged, submitted totreatment with trifluorotrichlorethane (Genetron) in aratio of 3: 1 (pool: Genetron) with a high speed Virtishomogenizer, separated from the trifluorotrichlorethaneby centrifugation, filtered, and stored at - 700.

Each of these four virus pools was safety tested (10)before volunteer inoculations and was reidentified asCoxsackie An in neutralization tests with hyperimmuneguinea pig serum prepared against the original Coestrain that had been passaged in human epidermoid car-cinoma (KB) tissue cultures. Virus titrations of thesepools on tissue culture revealed that each contained atleast 105 TCID0o' of virus per 0.2 ml, and hemaggluti-nation tests performed as described previously (11) indi-cated that the virus in each pool was of the expectedhemagglutinin type.

Serum inactivated inocula were prepared containingthe same concentrations of virus used for other inocula-tions, but in addition containing a 1:100 dilution ofspecific hyperimmune guinea pig serum. After 3 hoursincubation at room temperature, these mixtures wereadministered to volunteers and were subsequently shownto be free of infectivity for tissue culture.

1 TCID50 = that dose of virus which will infect 50%of tissue culture tubes.

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LANG, CATE, COUCH, KNIGHT, ANDJOHNSON

Procedure for inoculations. The HEKpools were ad-ministered nasopharyngeally by spraying 0.5 ml of virussuspension into each nostril with a hand nebulizer (De-Vilbiss no. 5) and instilling an additional 0.5 ml intoeach nostril by pipette. Each pool was diluted withveal infusion broth before inoculation. Simultaneoustitrations of the inocula indicated that the 2.0-ml doseper man contained 10-t°o2.7 TCID50 of HA+ virus or

1028 to 8.2 TCIDso of HA- virus.Inoculation by aerosol was performed with the plaque

derived pools as described elsewhere (6). The aerosolconsisted of particles ranging in size from 0.2 to 3.0 A indiameter. Estimates based on studies with other strainsof Coxsackie An1 suggest that about two-thirds of thevirus was carried by particles ranging from 1 to 2 u indiameter. Each volunteer inhaled 10 0.2 L of aerosol in9 to 12 consecutive deep inspirations- a total of 102.2TCID6o of virus. The serum inactivated virus inoculawere also administered in aerosol.

Volunteers. Participants in these studies were vol-unteers from federal correctional institutions. After an

indication of willingness to participate, subjects were

selected on the basis of absence of measurable serum

neutralizing antibody.Volunteers were isolated three per room 3 days be-

fore and 7 to 14 days following inoculation. Physicianswho were unaware of the specific virus administered or

the route of inoculation examined the volunteers daily.Temperatures were recorded four times a day after in-oculation. Oral values above 37.5° C were confirmed byrectal temperatures and were considered to be fever.Peripheral blood counts, liver function tests, chest X rays,

electrocardiograms, urinalyses, and nose and throat bac-terial cultures were obtained pre- and postinoculation.

Specimens for virus. Throat, nose, and anal swabspecimens were collected daily from volunteers inoculatedvia the nasopharyngeal route. In the aerosol experimentsonly throat swab specimens were collected. Swabs were

twirled in 2.5 ml of veal infusion broth containing 0.5%bovine albumin. The liquid was stored at -20° C untiltested; then 0.4 ml of the fluid was added to a cultureof HEK tissue. Cultures were placed on a revolvingdrum, incubated at 33 to 34° C, and observed for 9 to 14days for cytopathic effect. Cultures inoculated withanal swab specimens were washed with fresh mediumafter 12 hours to minimize contamination.

The identity of the first, the last, and an intermediateisolate from each volunteer was checked by neutralizationwith specific hyperimmune antiserum. All other isolateswere assumed to be Coxsackie A21 if they producedcharacteristic cytopathic effect.

Hemagglutination tests (11) were performed on allvirus isolates. To ensure that adequate virus was pres-ent for hemagglutination (106 TCIDso per 0.2 ml) alltests were performed on isolates from specimens in HEKtubes in which the cytopathic effect had progressed to100%, and all isolates with questionable hemagglutina-tion results were titered, and reisolation was performedif necessary.

Measurement of antibody response. Sera obtained be-fore and 3 to 4 weeks after inoculation were tested inboth neutralization and hemagglutination-inhibition testsas previously described (1). The Coxsackie A21 viruspool used for these tests contained both HA+ and HA-

TABLE I

Illness following inoculation of volunteers with HA+ and HA- Coxsackie A21*

Volunteers with clinical findings

UpperVolun- respir- Sys-

teers Volun- atory temic AverageInoculum and inocu- teers symp- symp- duration

dose (TCIDro) Route lated ill toms toms Fever of illness

daysHEK9, HA+,

102 3-lO2 7 Nasopharyngeal 11 9 8 5 5 4.4HEK9, HA-,

102 3-103 2t ' Nasopharyngeal 9 8 8 8 6 5.3

HEK9, WI2,HA+ 1022 Aerosol 6 5 5 3 5 5.3

HEK9, WI2,HA- 102.2 Aerosol 5 5 5 5 4 4.6

HEK9, WI2,HA+ 1022 Aerosol 3 0 0 0 0 0neutralized

HEK9, WI2,HA- 1022 Aerosol 3 0 0 0 0 0neutralized

t Three volunteers received 103.2 TCID50 of virus, the remainder receiving 102.3 to 102.9 TCID5o. No differences inresponse according to dose were detected.

* HA+ and HA- = hemagglutinin-positive and negative particles; HEK= human embryonic kidney tissuecultures; WI = human embryonic lung tissue cultures.

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HEMAGGLUTININVARIANTS OF COXSACKIEAn VIRUS IN VOLUNTEERS

TABLE II

Virus shedding by volunteers inoculated with HA+ and HA- Coxsackie A21

Volunteers with virus recovery (ies)

Volun- Pharyngeal and/or nasal swab specimensteers Anal swab Positive anal

Inoculum and inocu- Days Days Days specimens, swab specimens/route lated Total 1-7 8-14 15-21 total number tested*

HEK9, HA+,nasopharyngeal 11 11 11 10 7t 7 13/86 (15%)

HEK9, HA-,nasopharyngeal 9 9 9 9 6t 9 28/106 (26%)

HEK9, WI2,HA+, aerosol 6 4 4 4 4 ND: ND

HEK9, WI2,HA-, aerosol 5 4 4 4 4 ND ND

* Number tested refers only to specimens from volunteers with at least one anal isolate.t Three volunteers from group discharged on day 12.t Not done.

virus particles but was hemagglutinin positive. In ad-dition, each of the purified hemagglutinin variants wasused in neutralization tests with several sera.

Results

Illness. The number of volunteers who becameill and the characteristics of their illnesses areshown in Table I for each of the six inoculumgroups. As can be seen in the first four rows ofTable I, upper respiratory illness often with feverand systemic symptoms occurred in similar highproportions of volunteers receiving one or theother hemagglutinin types of Coxsackie A21,whether administered nasopharyngeally or byaerosol. There were no statistically significantdifferences in frequency of illness, upper respira-tory symptoms, systemic symptoms, or feveramong these groups. Represented in the bottomtwo rows of Table I are the volunteers who re-ceived serum inactivated virus pools. None ofthese latter men became ill.

Volunteers who became ill displayed a slightgranulocytosis and lymphopenia during the acutephase of illness. There were no significant changesin liver function tests, urinalyses, sedimentationrates, or bacterial cultures of the throat.

Two men given HA+ variant by aerosol didnot shed virus or show antibody rise. One ofthese became ill. It was found, however, thatsinusitis had been present before inoculation, andit is presumed that this illness was unrelated toCoxsackie A21 administration. One man in theHA - aerosol group also developed an unex-

plained illness without evidence of infection byvirus shedding or rise in antibody titer. It wasdiscovered that, although the latter volunteerpossessed no serum neutralizing antibody to Cox-sackie A21, his serum hemagglutination-inhibitingtiter was 1: 20 before inoculation. None of theother volunteers selected for absence of detectableneutralizing antibody possessed preinoculation he-magglutination-inhibiting antibody.

Virus shedding. In Table II are shown virusshedding patterns for the four inoculum groups ofvolunteers who received infectious virus. Therewas a similar high frequency of virus recoveryfrom the respiratory tract in each group regardlessof the hemagglutinin type of virus received, andpositive cultures were obtained with considerablefrequency throughout the 3 weeks of observations.

The amount of virus in several throat swabspecimens collected during the first 5 days afterinoculation was determined by plaque assay onWI-26 cells that are equally sensitive for detec-tion of the two hemagglutinin variants of Cox-sackie A21 (12). Observations were availablefor seven men given the HA+ variant and sixmen given the HA - variant. The average titerof Coxsackie A21 per 0.2 ml of specimen fluid was100911 (SE, 0.163) plaque forming units for theHA+ group, and 101.12 (SE, 0.312) units forthe HA - group. These values were not sig-nificantly different (Wilcoxon two-sample test,U = 16.5, p = 0.20; t test, t = < 1, p > 0.30).

However, virus recovery from anal swab speci-mens did differ according to the hemagglutinin

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LANG, CATE, COUCH, KNIGHT, ANDJOHNSON

TABLE III

Serologic response by volunteers inoculated with HA+ and HA- Coxsackie A21

Neutralizing antibody HI antibody

Volun- Volun-teers teers

Volun- Volun- with withteers teers rise Geometric rise Geometric

Inoculum and inocu- shedding in mean titer in mean titerroute lated virus titer* postinoc.t titer* postinoc.t

HEK9, HA+,nasopharyngeal 11 11 3 1:6 5 1:13

HEKR, HA-,nasopharyngeal 9 9 9 1:64 9 1:218

HEK9, WI2,HA+, aerosol 6 4 2 1:8 2 1:14

HEK9, WI2,HA-, aerosol 5 4 4 1:54 4 1:320

* Fourfold or greater, preinoculation versus 3- to 4-week sera.t Postinoculation titers only from volunteers who shed virus, all of whomwere free of both neutralization and HI

antibody preinoculation. Comparison of geometric mean antibody titiers (t test): HA+ versus HA-, nasopharyngealinoculation-neutralizing p < 0.01, HI p < 0.01; HA+ versus HA-, aerosol inoculation-neutralizing p = 0.17, HIp <0.01.

reaction of the inoculum (Table II). Among vol-unteers given the HA+ variant nasopharyngeallyvirus was recovered from anal swab specimens ofonly seven of the eleven infected volunteers, andfrom these seven volunteers only 15%o of the speci-mens were positive. In contrast, virus was re-

covered from anal swab specimens from all ninevolunteers who similarly received the HA -

variant, and 26%o of their specimens were positive.These differences were statistically significantwhen analyzed by the Wilcoxon two-sample test

(U= 13, p = 0.01).Antibody response. The frequency of fourfold

or greater rises in neutralizing and hemagglutina-tion-inhibiting antibody titers and the geometricmean values of convalescent titers are describedin Table III. The three men from whom no

virus was recovered exhibited no rise in antibodytiter with either type of antibody assay and were

not included in these calculations. Only three ofeleven and two of four men who were infectedfollowing inoculation with HA+ virus by thenasopharyngeal and aerosol routes, respectively,displayed both neutralizing and hemagglutination-inhibiting antibody responses, and an additionaltwo volunteers developed a rise in hemagglutina-tion-inhibiting antibody titer alone. In contrast,all men who were infected following inoculationwith the HA - virus by these routes exhibitedfourfold rises with both types of antibody assay.These differences are further emphasized by the

substantially greater geometric mean titers ofpostchallenge antibody from infected men who re-ceived HA- virus. In this calculation less thanmeasurable titers were considered as positive atone-half the value of the lowest measurable titer.The increases in postchallenge titers for the HA -groups over the HA+ groups were 7- to 10-foldfor neutralizing antibody tests and 15- to 23-foldin hemagglutination-inhibition tests, differencesthat were highly statistically significant, exceptfor neutralizing antibody titers in men inoculatedby aerosol where the numbers are small.

Although volunteers who received HA- virusnasopharyngeally also yielded more virus recov-eries in anal swab specimens than those who re-ceived HA+ variant, no correlation existedwithin either group between the frequency ofanal swab virus recoveries of individual volun-teers and final serum antibody titer. Similarly,there was no correlation of final antibody titerswith the number of virus recoveries from nasal orpharyngeal swab specimens, nor with titers ofvirus in pharyngeal swab specimens.

Duplicate neutralization titers were obtainedon sera from three HA+ volunteers and twoHA - volunteers employing the HA+ andHA - inocula as test viruses. The titers wereidentical in all cases.

Comparison of the relationship between hemag-glutination-inhibiting and neutralizing antibodytiters in all men given the two variants is shown

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HEMAGGLUTININVARIANTS OF COXSACKIEAn VIRUS IN VOLUNTEERS

in Figure 1. If all titers are considered withoutregard to the kind of variant given, there is a

close correlation between hemagglutination-inhibit-ing and neutralizing antibody titers (r = 0.697,p < 0.01). There is also a significant correlationbetween the two titers in men given HA+ virus(r = 0.646, p < 0.01). No significant relation-ship exists between the two titers, in men givenHA- virus, but it should be noted that maximaltiters were not determined in some tests and thatthe positive tests were all of relatively high titerso that only a narrow range of variation in titerswas available for the comparison.

Pattern of shedding of the HA + and HA -

virus particles. The hemagglutination reactionsof 294 virus isolates from volunteers inoculatedby the nasopharyngeal route were determined.All but 16 of these were of the same hemagglu-tinin type as the inoculum the volunteers received.From Table IV it can be seen that one isolate fromone volunteer changed from HA+ to HA -.

The remaining 15 isolates that differed in hemag-glutinin type from the inoculum were composedof one or more isolates from seven of nine volun-teers who received HA- virus.

For the first week following inoculation all iso-lates from each volunteer were of the same hemag-glutinin type as the inoculum. When shifts inhemagglutinin type occurred, they were often so

late that the final pattern of virus shedding in re-

gard to hemagglutinin type could not be deter-mined. For example, the single isolate conver-

sion from HA+ and HA- was from the lastspecimen obtained from that particular volunteer.Four volunteers with a single isolate conversionfrom HA -- to HA+ had at least one subsequentisolate that was of the original type, but conversion

LL

I-

I-

> 1:640

1:320

' 1:160

|o HA-|| HA*

0 0 00

0

0 0 0000

1:80k

1:40k

0

0

1:20 F 0

110F<1:10 @0

I--

<1:4 1:4 1:8 1:16 1:32 1:64 1:128 1:256NEUT. ANTIBODY TITER

FIG. 1. COMPARISON OF HEMAGGLUTINATION-INHIBI-TION (HI) AND NEUTRALIZING SERUMANTIBODY TITERSFOLLOWINGINOCULATION WITH HA+ AND HA- Cox-SACKIE An.

in hemagglutinin type of virus isolates from threevolunteers appeared to persist. Shifts in hemag-glutinin type were usually confined to isolatesfrom one specimen site (nose, throat, or anal)per man, and a majority were seen in isolatesfrom nasal specimens.

The hemagglutination reactions of 70 isolatesfrom volunteers inoculated by aerosol were deter-mined, and there were no conversions in hemag-glutinin type. However, this was not significantlyfewer than the conversions in isolates from volun-teers who received nasopharyngeal inoculation(p > 0.10).

There was no difference in antibody response

between men whose specimens remained un-

changed in hemagglutinin type and those whose

TABLE IV

Alteration in hemagglutination type of virus isolates from volunteers inoculatedwith HA+ and HA- Coxsackie A21

VolunteersVolun- Volun- with shift Isolates with shift in HA type/

teers teers in HA isolates from all volunteersInoculum and inocu- shedding type of

route lated virus isolates Days 1-7 After Day 7

HEK9, HA+,nasopharyngeal 11 11 1 0/68 1/77*

HEK,, HA-,nasopharyngeal 9 9 7 0/82 15/67*

*p < 0.01, Chi square test.

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LANG, CATE, COUCH, KNIGHT, ANDJOHNSON

isolates exhibited this late change in hemagglutinintype.

Discussion

This study has described the response of volun-teers to inoculation with purified pools of HA+and HA - variants of Coxsackie A21. Upperrespiratory illness, often with fever and systemicsymptoms, occurred in a large majority of vol-unteers whether inoculated by the nasopharyngealroute or by a small-particle aerosol. There wereno significant differences in the clinical responsesto the two variants, nor did they differ appreci-ably from responses to a wild strain of CoxsackieA21 tested earlier (5, 6).

In contrast, antibody responses after infectionwith the two hemagglutinin variants differed toa very considerable extent. Men who receivedHA - inocula had significantly higher postchal-lenge titers of both neutralizing and hemaggluti-nation-inhibiting antibody than those who re-ceived HA+ inocula.

The fact that anal swab specimens from volun-teers who received HA - virus yielded more virusrecoveries than anal swab specimens from volun-teers who received HA+ virus suggested thatincreased multiplication of HA - virus in theintestines might result in increased antigenic mass.However, among individuals receiving one or theother variant, there was no correlation betweenfrequency of virus recovery from anal swab speci-mens and final serum antibody titer. Virus re-coveries from nasal and pharyngeal swab speci-mens and the amount of virus in several of thelatter specimens were similar for the two hemag-glutinin variants, and individual antibody titersdid not correlate with any of these measurementsof virus shedding. Thus the contrasting sero-logical responses do not appear to be explainedby differences in multiplication of the two variantsand the resultant differences in antigenic mass thatthis would imply.

Simon and D6m6k (13) have reported separa-tion of two hemagglutinin variants of ECHOvirus 6 and have demonstrated an antigenic dif-ference between the two. Antiserum preparedin animals against an HA - strain of ECHOvirus 6 was of higher titer against the HA -variant than against the HA+ variant; antise-

rum prepared against the HA+ variant did notdistinguish between the two. In the presentstudy the neutralizing antibody in human serumfollowing an induced infection with one or theother hemagglutinin variant of Coxsackie A21virus was of similar titer when either variant wasused as the test virus. The sum of these studiessuggests that the HA+ and HA - particles ofCoxsackie A21 virus possess differing capacitiesto stimulate the production of antibody directedagainst antigens that are common to both.

The finding of nearly uniform shedding of thehemagglutinin type administered during severaldays after inoculation suggests that these inoculawere true genetic variants rather than HA+virus, the hemagglutinin of which had beenmasked by inhibitors in the suspending medium aswas suggested in studies by Schmidt, Fox, andLennette (14). These investigators found thattreatment of HA - populations with Genetrondeveloped or increased hemagglutinin reactionsin a number of strains of Coxsackie A21' Treat-ment of the present HA+ and HA - pools usedfor aerosol inoculation with Genetron did notalter these properties of the inocula, nor did itappear to alter the volunteer responses to theinocula.

There was a definite shift toward HA+ vari-ants in men given HA - virus beginning 8 daysafter inoculation. The reciprocal event was de-tected in only a single specimen. Although thereis a slight growth advantage of the HA+ particlein HEKtissue (7), which was used to test speci-mens for virus, this advantage alone could notaccount for the shift towards HA+ variants inthe absence of a true shift occurring in the volun-teers. The late development of this change inhemagglutinin type may merely represent the factthat HA+ variants were present in very smallnumbers in the HA - inoculum and requiredseveral days to increase to detectable concentra-tions. In a study in 1960 among military recruits(3), 227 of 238 (95%7o) isolates in HEK tissueculture were HA+. It is possible that this isindicative of a predominance of HA+ variantsin natural infection, and the present results mayreflect a return to the normally predominantvariant.

One question of possible practical interest is

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HEMAGGLUTININVARIANTS OF COXSACKIEAn VIRUS IN VOLUNTEERS

whether the HA - variant of Coxsackie A21would prove to be a potent vaccine. The naturaldisease is apparently not often serious, but use

for vaccine might be found in military populations,and increased knowledge of the epidemiology ofthe disease may disclose new areas for its ap-plication.

Summary

Volunteers without measurable antibody wereinoculated by the nasopharyngeal route or by in-halation of a small-particle aerosol with hemag-glutinin-positive (HA +) and hemagglutinin-negative (HA -) variants of Coxsackie A21 virus.Mild upper respiratory illness often with slightfever occurred in similar high proportions of bothgroups. Virus recoveries from nasal and pharyn-geal swab specimens were frequent and continuedabout equally in both groups throughout 3 weeksof observation, and pharyngeal swabs taken duringthe acute phase of illness from both groups yieldedabout the same quantities of virus. Anal swabspecimens from volunteers who received theHA - variant more frequently yielded virus thanthose from volunteers who received the HA+variant. There was a definite shift to HA+variants in men given HA - virus beginning on

the eighth day that rarely occurred reciprocally.Neutralizing and hemagglutination-inhibiting anti-body responses were many times higher in men

given the HA+ variant. Within each group

there was no correlation between antibody re-

sponse and frequency of virus isolates from thevarious specimens. The two strains gave identi-cal titers when used as antigen in neutralizingantibody tests of volunteer sera.

Acknowledgments

The authors gratefully acknowledge the statisticalhelp afforded by Dr. David Alhing and the technical as-

sistance of Mrs. Carol Uhlendorf and Mr. EdwardHarvey. Aerosol inoculations were performed by Mr.William Griffith and Mr. Edward B. Derrenbacher, U. S.Army Biological Laboratories, Ft. Detrick, Md. In ad-dition, we are grateful to Mr. James V. Bennett, Di-rector; Dr. Charles E. Smith, Chief Physician; and Dr.Harold M. Janney, former Chief Physician, FederalBureau of Prisons, for permission and assistance in ar-

rangements for volunteers.

References1. Lennette, E. H., V. L. Fox, N. J. Schmidt, and J. 0.

Culver. The Coe virus: an apparently new virusrecovered from patients with mild respiratorydisease. Amer. J. Hyg. 1958, 68, 272.

2. Pereira, M. S., and H. G. Pereira. Coe virus: prop-erties and prevalence in Great Britain. Lancet1959, 2, 539.

3. Johnson, K. M., H. H. Bloom, M. A. Mufson, andR. M. Chanock. Acute respiratory disease as-sociated with Coxsackie A-21 virus infection. I.Incidence in military personnel: observations in arecruit population. J. Amer. med. Ass. 1962, 179,112.

4. Bloom, H. H., K. M. Johnson, M. A. Mufson, andR. M. Chanock. Acute respiratory disease as-sociated with Coxsackie A-21 virus infection. II.Incidence in military personnel: observations ina nonrecruit population. J. Amer. med. Ass. 1962,179, 120.

5. Spickard, A., H. Evans, V. Knight, and K. Johnson.Acute respiratory disease in normal volunteers as-sociated with Coxsackie A-21 viral infection. III.Response to nasopharyngeal and enteric inocula-tion. J. clin. Invest. 1963, 42, 840.

6. Couch, R. B., T. R. Cate, P. J. Gerone, W. F. Fleet,D. J. Lang, W. R. Griffith, and V. Knight.Production of illness with a small-particle aerosolof Coxsackie An. J. clin. Invest. 1965, 44, 535.

7. Johnson, K. M., and D. J. Lang. Separation ofhemagglutinating and nonhemagglutinating variantsof Coxsackie A-21 virus. Proc. Soc. exp. Biol.(N. Y.) 1962, 110, 653.

8. Hayflick, L., and P. S. Moorhead. The serial culti-vation of human diploid cell strains. Exp. CellRes. 1961, 25, 585.

9. Kisch, A. L., P. A. Webb, and K. M. Johnson.Further properties of five newly recognized picor-naviruses (rhinoviruses). Amer. J. Hyg. 1964, 79,125.

10. Couch, R. B., R. M. Chanock, T. R. Cate, D. J.Lang, V. Knight, and R. Huebner. Immunizationwith types 4 and 7 adenovirus by selective infectionof the intestinal tract. Amer. Rev. resp. Dis. 1963,88 (suppl. 3), 394.

11. Johnson, K. M., H. H. Bloom, L. Rosen, M. A.Mufson, and R. M. Chanock. Hemagglutinationby Coe virus. Virology 1961, 13, 373.

12. Lang, D. J., and K. M. Johnson. In preparation.13. Simon, M., and I. Domdk. Studies on intratypic

variants of Echoviruses. I. Separation of haemag-glutinating and non-haemagglutinating virus par-ticles from Echovirus type 6. Acta microbiol.Acad. Sci. hung. 1963, 10, 293.

14. Schmidt, N. J., V. L. Fox, and E. H. Lennette.Studies on the hemagglutination of Coe (Cox-sackie A=) virus. J. Immunol. 1962, 89, 672.

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