JOURNAL OF BACTERIOLOGY, Sept., 1966 Vol. 92, No. 3Copyright © 1966 American Society for Microbiology Printted in U.S.A.

Growth of Myxovirus parainfluenza Type 3 inOrgan Cultures of Guinea Pig Tissue

J. E. CRAIGHEADDepartment of Pathology, Harvard Medical School, Bosto,,, Massachusetts

Received for publication 4 May 1966

ABSTRACTCRAIGHEAD, J. E. (Harvard Medical School, Boston, Mass.). Growth of Myxo-

virus parainfluenza type 3 in organ cultures of guinea pig tissue. J. Bacteriol. 92:751-761. 1966.-Organ cultures of adult guinea pig nasal mucosa, lung, and pleura wereinfected with Myxovirus parainfluenza type 3. Observations were made on the growthof virus at intervals after inoculation. An inoculum of 102-5 tissue culture infectiousdoses (TCID50) initiated infection in each of the tissues. Cultures of nasal mucosayielded up to 1060 TCID50 per 6 hr for periods of as long as 2 weeks. Virus productionwas not affected by the "immune" status of the animal used as a source of tissue. In-troduction of antiserum into the medium appeared to suppress virus release butfailed to "cure" the infection. Interferon was not detected in fluids bathing the nasalmucosa. Cultured fragments of lung produced virus for 28 days after inoculation. Asmuch as 105 0 TCID50 per 6 hr was released by the tissue. Pleural mesothelial cells lin-ing the diaphragm yielded up to 1060 TCID50 per 6 hr over a 14-day period. Histo-logical sections showed that the tissues retained differentiated morphological featuresduring maintenance in vitro. Cytological changes unequivocally associated withinfection were not recognized. The techniques described give reproducible, quantita-tive results. Organ cultures are feasible for the study of virus growth and cytopathol-ogy in differentiated tissues.

A wide variety of cultured animal cells supportthe replication of Myxovirus parainfluenza type3. Most investigations with this virus have beencarried out in monolayer cultures prepared fromtrypsin-dispersed cells. Although this workprovides valuable information, it fails to definethe growth characteristics of the virus in differ-entiated tissues. This report records studies onorgan cultures of guinea pig nasal mucosa, lung,and pleura infected with parainfluenza virustype 3.

MATERIALS AND METHODSVirus. Parainfluenza virus type 3 was supplied by R.

Chanock of the National Institutes of Health. It hadbeen grown in monkey heart and Hep-2 cell culturesand was passaged in HeLa cells five times in thislaboratory. Titrations of the virus were carried outroutinely in tube cultures of HeLa cells, with the useof both cytopathic effect and hemadsorption todetermine infectivity end points (TCID5o).

Tissue preparation. Young adult guinea pigs sup-plied by a local breeder were anesthetized with Nem-butal and exsanguinated by cardiac puncture. Thenasal septum was removed with a saw and chisel, andthe mucosa was carefully excised from each side of theseptal cartilage by sharp dissection with a scalpel. The

diaphragm and lungs were then removed. The tissuefrom the nasal septum was trimmed to a convenientsize (approximately 7 by 10 mm of surface area). Itconsisted of the mucous membrane and a thin layerof the subjacent connective tissue and mucous glands.Lung tissue was minced into fragments (approximately1 mm3). The muscular diaphragm with its adherentpleural surface was cut into rectangular-shaped pieces(approximately 10 by 4 mm of surface area). Segmentsof rectus abdominis muscle of an equivalent size wereprepared and employed in all experiments with thepleura. This was done to exclude the possibility thatthe muscle of the diaphragm was supporting virusreplication. All tissues were washed thoroughly with asolution of5% heated (56 C for 30 min) chicken serumand Hanks balanced salt solution. After being washed,samples of most specimens were fixed for histologicalexamination in Bouin's solution.

Culture techniques. Individual tissues were trans-ferred to filter-paper strips and placed in 35-mm plasticpetri dishes on a bed of glass beads (8). A mediumcomposed of 5% heated chicken serum and Eagle'ssolution containing antibiotics was then added. Thequantity (2 to 2.5 ml) was adjusted so that the bathingmedium was flush with the sides of the specimen andmoistened but did not cover its surface. Except asnoted, cultures were incubated at 36 to 37 C in a hu-midified, 5% C02-95% air atmosphere.

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Infection of tissues. Inoculation was carried out ap-proximately 18 hr after specimen preparation. Virusdiluted in 2.5 ml of medium was added, and the cul-tures were incubated with the inoculum for 1 hr at 36to 37 C. Individual tissues were then removed, thor-oughly washed, and placed in freshly prepared petridishes. Release of virus from the cultures was deter-mined during the ensuing 6 hr and at intervals there-after. This was accomplished in the following manner.At the designated time, specimens were transferred toa clean dish, washed five times, and placed in a seconddish containing fresh medium. The cultures were re-turned to the incubator for a 6-hr period, after whichthe medium was withdrawn for virus titration.

Immunization of animals. Adult guinea pigs wereimmunized by footpad inoculation of virus mixed withFreund's incomplete adjuvant (106.0 TCID5o in 0.1 ml).Animals received weekly injections for 4 weeks andwere used for preparation of nasal mucosa cultures 3to 4 weeks after the last inoculation. The virus neu-tralization titer was determined on serum obtained atthe time of sacrifice.

Neutralization tests. Sera from all guinea pigs usedfor culture preparation were tested routinely for para-influenza virus type 3 neutralization antibodies at adilution of 1:4. Sera neutralizing the virus at thisdilution were titered by use of serial fourfold dilutions.HeLa cell cultures and 100 TCID5o of virus were em-ployed.

Interferon assays. Culture fluids were dialyzed for 48

Virus Yieldloq 10/ 6hrs.

6 1

5

4.

3.

2

0

hr at pH 2 in Sorensen's buffer and for an additional 48hr in Hanks solution (pH 7.4). Assays were carried outin guinea pig embryo cell cultures by use of the 50%plaque reduction technique and 30 to 50 plaque-form-ing particles of vesicular stomatitis virus (3). Samples(0.5 ml) of the preparation under test were incubatedwith the cell sheet for 24 hr before the challenge viruswas added.

Histological techniques. Tissues were fixed in Bouin'ssolution at intervals after inoculation and at the termi-nation of experiments. Alternate 5-p sections werestained with hematoxylin and eosin or by the periodicacid-Schiff technique.

RESULTSVirological studies of nasal mucosa. Parainflu-

enza virus type 3 grew in cultures of guinea pignasal mucosa after exposure of the tissue to awide range of virus dosages. An inoculum of102.5 TCID50 was sufficient to initiate infection.The amount of virus released at intervals afterinoculation appeared to depend upon the inputconcentration (Fig. 1). When relatively smalldosages were employed, a period of 24 to 48 hrelapsed before virus was recovered from the cul-ture medium. With much larger dosages, variablequantities of virus were released into the mediumduring the first 6 hr of incubation. It is likely

Inoculumtog ln/specimen

5.5

3 5

2*5

A

A

A 0*

A 0

A0

* )-Oa OPa a

06 24-30 46-54 ?2-7? 96-102

Ti me in HoursFIG. 1. Virus recovered from cultures of nasal mucosa at intervals after inoculation of 10O-', 1O1-, and 1O-

TCID50 ofparainfluenza virus type 3. Each point indicates the virus yieldfor a 6-hr test period.

752 J. B3ACTERIOL.

GROWTH OF MYXOVIRUS PARAINFLUENZA TYPE 3

that much of this virus represented portions ofthe inoculum which had not been washed free orhad adsorbed reversibly to the tissues. In repeatedexperiments, virus was not recovered from cul-tures inoculated with 102 TCID5o.The organ cultures supported parainfluenza

virus type 3 growth for extended periods of time.Substantial quantities of virus were elaboratedfor 342 hr in the longest experiment carried out.It is probable that virus replication would havepersisted for much longer periods. Histologicalpreparations of infected and noninfected tissueshowed that the mucosa was viable and intactafter maintenance for 2 weeks in vitro.

Serial passage of parainfluenza virus type 3 incultures of guinea pig nasal mucosa showed con-clusively that the tissue supported replication ofvirus. Fluids bathing infected tissues were har-vested routinely 72 hr after inoculation and wereintroduced into fresh cultures after dilution innew medium. Virus (105- TCID50) was recoveredafter eight serial passages. This represented a10-13 dilution of the original inoculum.The results of an experiment to compare virus

growth in tissues maintained in gas mixturesmade up of 5% CO2 and either 95% oxygen or95% air are shown in Fig. 2. Preparations ofnasal mucosa from the same animal were testedin parallel in one or the other of the two gasmixtures. Oxygen concentration did not appearto affect the morphological integrity of the mu-

cosa. Virus yields by tissues held in the differentgas mixtures were similar over a 222-hr period.All experiments recorded elsewhere in this paperwere carried out in a 5% C02-air environment.

Nasal mucosa obtained from animals possessinghigh titers of parainfluenza virus type 3 serumneutralization antibody (titer > 1:32) supportedgrowth of virus. Tissues from guinea pigs withnaturally acquired antibody and from immunizedanimals were challenged with 105- TCID5o ofvirus. In two different experiments, the viruselaborated by these cultures at intervals afterinoculation did not differ from the amounts pro-duced by similarly infected tissues obtainedfrom "nonimmune" animals. The virus inoculumin these studies was relatively large. It remainsto be determined whether cultures prepared fromguinea pigs with serum antibody are susceptibleto relatively small inputs of virus.

Infected cultures of nasal mucosa were not"cured" by introducing parainfluenza virus type3 antiserum into the medium. Two preparationsof mucosa from each of two animals were in-oculated with 10"' TCID50 of parainfluenza virustype 3. To confirm infection, the virus yield ofthe individual specimens was determined for theinterval between 72 and 78 hr. The tissues thenwere washed thoroughly. One culture of mucosafrom each animal was placed in a petri dish witha medium containing parainfluenza virus type 3antiserum (neutralization titer > 1:32) diluted

Virus Yieldlog 'OS 6hrs.

6 1

5

4

3

2

0

°~~0

A-A 5 % CO2 and 02O-O 5% CO2 and air

24-30 72-78 120-126

Time in Hours

168- 174 216-222

FIG. 2. Virus recoveredfrom cultures ofnasal mucosa maintained in gas mixtures made up of5% CO2 and either95% oxygen or air. Each point indicates the virus yieldfor a 6-hr test period.

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1 :10. The remaining specimens were handled ina similar fashion, except that guinea pig serumlacking demonstrable antibody was added to themedium. After incubation for 90 hr, the cultureswere washed and transferred to clean dishes. Themedium was subsequently harvested at intervalsto document virus production. As can be seen inFig. 3, release of virus by the mucosa was re-duced transiently after the tissue was removedfrom the antibody-containing medium. Althoughthe cultures were washed thoroughly, it is pos-sible that residual antibody adhered to the tissueand was available to bind virus released duringthe initial 6 hr. Nonetheless, virus replicationwas roughly equivalent to the controls at inter-vals thereafter. Results comparable to thoseshown in Fig. 3 were obtained in repeat experi-ments.

Studies were carried out to determine whethertissue from intranasally infected guinea pigswould support replication of virus in vitro afterremoval from the animal. Three Nembutal-anesthetized adult guinea pigs were inoculatedwith approximately 106 TCID50 of parainfluenzavirus type 3. After 3 days, the animals werekilled. The nasal mucosa from one side of theseptum was excised, homogenized, and titeredfor virus. The second, intact specimen of mucosawas cultured, and the medium was tested forvirus at intervals, in the manner described above.The results are shown in Table 1. Virus was pres-ent in the nasal mucosa removed from each

guinea pig 3 days after intranasal inoculation.Release of virus by the cultured tissues wasdemonstrated during the first 6 hr after excisionand persisted for the duration of the experiment(342 hr). The amounts of virus produced bytissues from the intranasally infected animalswere roughly comparable to the yields of mucosapreparations inoculated in vitro.

Interferon was not demonstrated in mediumharvested from individual cultures at intervalsafter inoculation. Fluids from several cultureswere concentrated and pooled before testing.These preparations also gave negative results.

Histological studies of nasal mucosa. Histo-logical sections of the nasal mucosa cultures wereprepared during the course of most studies androutinely at the termination of each experiment.This material provided an opportunity to eval-uate the integrity of the mucosa as well as todocument cytological alterations attributable toinfection. Figure 4 is representative of the pseu-dostratified nasal mucosa of the adult guineapig. The tissue retained a "normal" appearanceafter maintenance for roughly 48 hr in vitro.Pyknosis and lysis of basal and suprabasal cellswas evident by 96 hr (Fig. 5, 6). Focal necrosisof the stroma of the submucosal connectivetissue and glands usually appeared somewhatlater. By the 7th day, the superficial cell layers ofthe mucosa often were situated adjacent to thebasal layer (Fig. 7). Orientation of these cellsfrequently was lost. This appearance persisted for

Virus Yieldlogio/ 6hrs.

AA-

o

07

168-174 240-246 288-294 336-342

Time in HoursFIG. 3. Effect ofantiserum on virus yield by cultures ofnasal mucosa. Infected tissues were incubated with anti-

serum from 72 to 168 hr. Each point indicates the virus yield for a 6-hr test period.

A

0

6

5

4

3

2

E

.0 Nc-

.A

U S-

0.

A normal G.P serum

0 antiserum

72-78

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GROWTH OF MYXOVIRUS PARAINFLUENZA TYPE 3

the duration of most experiments, althoughoccasionally small foci of necrosis became evi-dent. Figures 8 and 9 show the morphologicalfeatures of two cultures of nasal mucosa whichwere yielding large amounts of virus when fixed222 and 342 hr, respectively, after inoculation.Comparison of histological sections from simul-taneously cultured, infected and noninfectedtissues failed to reveal alterations unequivocallyassociated with virus growth. In these studies nosystematic attempt was made to document thepresence or absence of cilia and mucus.

Virological studies of the lung. Parainfluenzavirus type 3 grew in fragments of guinea piglung for extended periods of time. Figure 10summarizes results from the longest experimentthus far conducted. In this study, three of fourlung fragments supported virus growth for 28days after inoculation of 105 TCID50. Virus wasrecovered from the fourth culture for 21 days.Tissue receiving an inoculum of 1025 TCID5o alsoproduced virus for this period, although intervalsof up to 120 hr elapsed before virus could berecovered from the medium. Two of the threespecimens receiving the smaller dosage were in-fected.

Histological studies of the lung. The fragmentsof lung employed in these studies were approxi-mately 1 mm3. Histological sections showed thatthe morphological integrity of the parenchymawas retained during maintenance in vitro for 48to 96 hr. Necrosis gradually appeared in thecenters of the fragments thereafter. However,

TABLE 1. Virus yield of nasal mucosa homogentatesand cultures at intervals after intranasalinoculation of 106 TCID30 of parainfluenza

virus type 3

Animal no.Prepn ~ Time afterPrepn inoculation

1 2 3

hr

Homogenate 72 4.5a 3.5 3.0

Culture 72-78 4.0 1.0 4.096-102 2.0 2.5 3.0240-246 5.0 5.5 3.0336-342 4.5 4.5 4.5

a Log1o TCID50 per nasal mucosa homogenate.b Log10 TCID50 per 6 hr.

FIG. 4. Uninfected nasal mucosa. The tissue was excised from the septum and washed in the routine fashionbefore fixation. Cilia and mucus are not present. Presumably, they were removed during preparation. Hematoxylinand eosin stains. Original magnification, X 50.

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FIG. 5. Uninfected nasal mucosa after 96 hr ofincubation. Note the alterations in the basal and suprabasal layersof cells. Hematoxylin and eosin stains. Original magnification, X 50.

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~~~~~~ow"A"m _'

FIG. 6. Tissue shown in Fig. 5 stained by the periodic acid-Schiffmethod. Note the layer ofmucus on the surfaceand the actively secreting submucosal glands. Goblet cells are not present. Original magnification, X 50.

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GROWTH OF MYXOVIRUS PARAINFLUENZA TYPE 3

FIG. 7. Uninfected nasal mucosa after incubation for 168 hr. There is a decrease in thickness of the epitheliumand a loss o, the pseudostratified arrangement ofcells. Cilia are present but mucus is no longer demonstrable. Hema-toxylin and eosin stains. Original magnification, X 50.

..,6 _..::

FIG. 8. Infectednasal mucosa after 222 hr ofinicubation. This tissue yielded 1O5-5 TCID5o ofvirus during the 6-hrperiod which precededfixation. Cilia are intact. The "inclusion"-like structures are believed to represent pyknoticnuclei ofdead or dying cells. Hematoxylin and eosin stains. Original magnification, X 125.

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FIG. 9. Infected nasal mucosa after incubation for 342 hr. This tissue yielded 106.0 TCID50 of virus during the 6-hrperiod which precededfixation. The organization of the epithelium is lost. Necrotic debris is evident on the surfaceand in the submucosa. Hematoxylin and eosin stains. Original magnification, X 50.Virus Yield

log0 / 6 hrs.

5.

4

3

2

/A

O

0

noculumlog 10/specimen

A-A 5.5

0- 0 2-5

4-0-048-54 144-150 240-246 336-342 432-438 528-534 624-630 672-678

Time in Hours

FIG. 10. Virus recovered from cultures oflung at intervals after inoculation of 10-5 and 10J-5 TCID50 ofpara-influenza virus type 3. Each point indicates the virus yield for a 6-hr test period.

o

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GROWTH OF MYXOVIRUS PARAINFLUENZA TYPE 3

the alveolar pattern of the lung usually was evi-dent for 360 hr or more after inoculation (Fig.11). Cells resembling those of the mesotheliumof the pleura occasionally lined surfaces of thetissue. Only a few clusters of viable parenchymaremained at the termination of the experimentshown in Fig. 10.

Rarely, multinucleate cells were found in in-fected lung fragments. These structures resembled-the syncytia formed by parainfluenza virus type3 in monolayer cell cultures. The morphologicalfeatures of these multinucleate cells were difficultto evaluate because of the overall distortion anddisorganization of the tissue. Their significanceis not known. Similar structures were not ob-served in uninfected lung tissue.

Virological studies of pleural mesothelium.Parainfluenza virus type 3 grew in mesothelialcells lining the guinea pig pleural cavity forperiods of as long as 342 hr (Fig. 12). Culturesof the rectus abdominis muscle were included inall experiments to exclude the possibility thatthe muscle of the diaphragm was supportingvirus replication. Both tissues released smallquantities of virus during the first 6 hr after in-oculation. Subsequently, virus was produced bydiaphragm cultures, but not by those of therectus abdominis muscle.

Histological studies of pleural mesothelium.Mesothelial cells are large and difficult to dem-onstrate adequately in routine histological prep-arations. Tissue sections, 10 , in thickness, wereused in an attempt to evaluate alterations attrib-utable to infection. Although this work was notpursued exhaustively, no consistent changes wereobserved. The mesothelial cells lining the dia-phragm were viable at the termination of ex-periments.

DiscussioNOrgan cultures provide a means for deter-

mining the growth properties of viruses in differ-entiated tissues. The technique permits the studyof virus replication and cytopathic changes in-dependent of modifying host factors. Only lim-ited use of organ culture has been made in thestudy of respiratory-tract virus infections. Bangand Niven (2), Hoorn (6), and Hoorn and Tyrrell(7) showed that cultures of mucosa from thenasal turbinates and trachea of human fetuses,monkeys, and ferrets supported replication ofadeno-, rhino-, and myxoviruses. Using frag-ments of human trachea, Tyrrell and Bynoe (9)isolated a "new" respiratory virus after recoveryattempts in conventional cell cultures had failed.

FIG. 11. Uninfected lung tissue after incubation for 360 hr. Alveoli are apparent. Hematoxylin and eosin stains.Original magnification, X 50.

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Virus Yieldlog lo/ 6 hrs.

/0 0

0

Inoculumlog 10 /specimen

A-A 5 50- 0 3*5

0-6 48- 54 120-126 192-198 240-246 288-294 336-342

Time in Hours

FIG. 12. Virus recoveredfrom cultures ofpleura at intervals after inoculation of J055 and 103"5 TCID50 ofpara-influenza virus type 3. Each point indicates the virus yield for a 6-hr test period.

Cultures of respiratory-tract tissue appear toretain a differentiated morphology over extendedperiods of time in vitro. I routinely have foundintact ciliated epithelium in the nasal mucosaafter 10 days of cultivation and well-definedalveoli in lung fragments after 15 days. Ciiaryactivity was observed in tracheal cultures byHoorn and Tyrrell for as long as 20 days (7).However, it might be asked whether tissuesmaintained in vitro for these long periods arecomprised only of differentiated cells. Hoorn andTyrrell (7) found mitotic activity and outgrowthsof "flattened or cuboidal cells" on the epithelialsurfaces of their cultures of tracheal mucosa.Similar observations have been made in thislaboratory. It may be that occasional organ cul-tures are comprised of mixtures of differentiatedand undifferentiated cells, particularly when theepithelial surface has been damaged duringpreparation of the tissue. Using Newcastle diseasevirus, Bang and Foard (1) showed that cells fromthe nasal mucosa of chicks, in organ culture, tookup substantially more virus than did identicalcells in the intact, intranasally infected animal.These workers suggested that disruption of themucous layer coating the epithelium of the cul-tures enhanced virus uptake. My histologicalobservations indicate that the mucous layer ofthe nasal mucosa often is lost during prepara-tion. Thus, in this regard, the organ cultures differfrom intact respiratory-tract tissues.

In this study, the virus yield of individualcultures was determined over an elapsed periodof 6 hr. This was done in an effort to documentthe ability of the tissues to support virus repli-cation at intervals after inoculation. It is believedthat thorough washing of specimens and theroutine changing of petri dishes removed muchof the residual virus which had been fabricatedduring earlier periods of cultivation.

Histological examination of infected tissuesfailed to reveal cytological alterations whichwere unequivocally associated with virus growth.Multinucleate cells suggestive of the syncytiainduced by parainfluenza virus type 3 in epi-thelial cells were observed in occasional culturesof lung. Although syncytia and necrosis regu-larly occur in monolayers infected with mosttissue culture-adapted strains of parainfluenzavirus type 3, substantial replication of virus canoccur in the absence of morphologically recog-nizable changes. Multinucleate cells and necrosishave been observed rarely in the respiratorytracts of mice and hamsters infected with thisvirus (4, 5).

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService research grant AI-5494 from the NationalInstitute of Allergy and Infectious Diseases.The assistance of B. J. Brennan and M. F. McLane

is gratefully acknowledged.

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LITERATURE CITED

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2. BANG, F. B., AND J. S. F. NIVEN. 1958. A study ofinfection in organized tissue cultures. Brit. J.Exptl. Pathol. 39:317-322.

3. BARON, S., AND C. E. BUCKLER. 1963. Circulatinginterferon in mice after intravenous infection ofvirus. Science 141:1061-1063.

4. BUTHALA, D. A., AND M. G. SORET. 1964. Parain-fluenza type 3 virus infection in hamsters: vi-rologic, serologic and pathologic studies. J. In-fect. Diseases 114:226-234.

5. CRAIGHEAD, J. E. 1966. Growth and interferon

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production by parainfluenza type 3 virus in infantand adult mice. Brit. J. Exptl. Pathol. 47:235-241.

6. HOORN, B. 1964. Respiratory viruses in modelexperiments. Acta Oto-Laryngol. Scand. Suppl.188:138-144.

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8. TAO, T. W. 1964. Phytohemogglutinin elicitation ofspecific anamnestic immune response in vitro.Science 146:247-248.

9. TYRRELL, D. A. J., AND M. L. BYNOE. 1965. Cul-tivation of a novel type of common-cold virus inorgan cultures. Brit. Med. J. 1:1467-1470.