INFECTION AND IMMUNITY, Dec. 1972, p. 958-964Copyright © 1972 American Society for Microbiology

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

Detection and Localization of Viruses in HumanFetal Intestinal Organ Cultures by

ImmunofluorescenceRAPHAEL DOLIN, NEIL R. BLACKLOW, RICHARD G. WYATT, AND MITZI M. SERENO

Laboratory of Infectious Diseases, National Institute of Allergy antd Infectious Diseases, National Institutes ofHealth, Bethesda, Maryland 20014, and Evans Memorial Department of Clinical Research, Boston University

School of Medicine, University Hospital, Boston, Massachusetts 02118

Received for publication 21 June 1972

Viral antigens from viruses belonging to four different viral groups were de-tected directly in human fetal intestinal organ cultures by the application of im-munofluorescent techniques. The time of appearance and the cellular localizationof fluorescent-stainable antigen varied with the type of virus under investigation.After infection with adenovirus or with adeno-associated virus, fluorescent-stain-able antigen was seen in the epithelial cells of the explants, though no light micro-scopic changes could be observed. In infection with herpes simplex virus andechovirus, fluorescence was noted in both the epithelium and the lamina propria,along with histological changes throughout the organ culture. These techniquesoffer promise for the investigation of possible viral agents implicated in gastro-intestinal disease.

The development of organ cultures suitable forin vitro cultivation of microbial agents offers anew and perhaps more sensitive medium for thegrowth of certain fastidious, occult viruses (7, 8).Human fetal intestinal organ cultures, recentlyestablished in several laboratories (2, 9, 11, 13),have been employed in the search for viral agentsinvolved in gastrointestinal disease. Recent evi-dence suggests that at least one such hithertoundetected agent, the Norwalk agent of acutegastroenteritis, replicates in organ culture (3).The growth of a variety of known viruses inhuman fetal intestinal organ culture has beendescribed (2, 11, 13); however, assay and identifi-cation of those viruses have required passage offluid harvests from the organ cultures into con-ventional tissue culture systems. These studiesdescribe methods for the rapid detection ofspecific viral agents directly in organ culture andfor the localization of those agents to cellular andsubcellular sites, employing immunofluorescenttechniques.

MATERIALS AND METHODSOrgan culture. Human fetal intestinal organ cul-

tures were prepared and maintained as describedpreviously (2). At daily intervals, an explant was re-moved, rapidly frozen in cryoform (IEC, Needham,Mass.), and 12-mi sections were cut on a IEC-CTFmicrotome cryostat. The sections were fixed for 10

min in cold acetone, and then tested by the indirectfluorescent-antibody (FA) method. Specimens forlight microscopy were prepared by fixing explants in2% glutaraldehyde in phosphate-buffered saline (pH7.4) for 24 hr. Paraffin-embedded sections were subse-quently stained with hematoxylin and eosin.

Viruses. Adenovirus 7 strain E46- was titered inhuman embryonic kidney cells (HEK), and 101median tissue culture infective doses (TC1D50) wereinoculated per organ culture dish. Echovirus 11 strainGregory was titered in W138 cells, and 103-5 TCID50were inoculated per dish. Herpes simplex virus (HSV)was a strain isolated in 1960 from a genital lesion; itwas titered in W138 cells, and 103 0 TCID50 wereinoculated per dish. Adenovirus-associated virus type2 (AAV2), prototype strain, was titered in HEK cells(6), and 104 0 TCID50 were inoculated per dish, alongwith 1040 TCID50 of adenovirus 7 helper. Each viruswas added to fluid medium and incubated for 48 hrwithout washinig, after which all media bathing theexplants were harvested and renewed.

Viral assays. To assay viral growth in the organcultures, fluids harvested at each time point werefrozen at -70 C. Subsequently all fluids were thawedand inoculated in WI38 or HEK tube cultures (in 10-fold dilutions, 0.2 ml in each of two tubes per dilution)which were observed for cytopathic effect. To assayfor AAV-2, fluid harvests were inoculated into HEKtube cultures coinfected with adenovirus type 7 helper,and tissue culture fluids were tested for the presence ofAAV-2 CF antigen as previously described (6). In-activation of virus in the absence of tissue was studiedby titration of fluids from dishes without organ cul-

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tures. All titers (TCID50) were calculated by the Reed-Muench method (10).

Immunofluorescence tests. Organ culture sectionswere tested by the indirect FA method using previ-ously described procedures (1). FA titers were con-sidered to be the highest dilution of the serum whichgave fluorescence in an indirect FA test with cellsinfected with the homologous virus. Organ culturesections were reacted with human anti-adenovirus 7serum, human anti-echovirus 11 serum, or humananti-herpesvirus serum, each containing 32 units offluorescent-stainable antibody against the homologousvirus as determined with infected cell culture cover slippreparations. Hyperimmune guinea pig anti-AAV2serum, containing 16 units of fluorescent-stainableantibody, was similarly employed. Fluorescein iso-thiocyanate-conjugated anti-human globulin andanti-guinea pig globulin (Antibodies, Inc., Davis,Calif.) were employed with the appropriate sera.

RESULTS

Virus growth. The growth patterns of variousviruses in organ culture are shown in Fig. 1.Adenovirus 7 and AAV2 grew to peak titer in6 to 8 days after inoculation, and maintained thistiter for up to 21 days. HSV reached peak titer bydays 4 to 6 and maintained this level until day 8,at which time the titer fell with the concomitantdegeneration of the organ culture. Echovirus 11reached peak titer at day 4, and maintained thistiter for 14 days.

Histopathology. The histology of uninfected

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human fetal intestinal organ cultures has beenpreviously described (2). When infected withknown viruses, organ cultures showed varyingdegrees of histopathology. Adenovirus and AAVgrew in organ cultures without appreciablealteration in morphology when viewed by lightmicroscopy (Fig. 2). HSV-infected organ culturesshowed disruption of cytoarchitecture, cell

degeneration, and lysis by day 4 (Fig. 3). Thesechanges began in the epithelial cells and spreadto the lamina propria and submucosa, with frankdestruction of the explants by day 8. Echovirus 11,though consistently growing to high titer in organculture, showed inconstant changes by lightmicroscopy. Generally, a cytoarchitectural dis-ruption was noted in the epithelium by days 8 to 9,with subsequent degeneration confined to themucosa. Occasionally, explants supported thegrowth of echovirus 11 without obvious histo-logical alteration. Intracellular inclusions were

not seen with the above viruses.Immunofluorescence. Examination of adeno-

virus-infected cultures by fluorescence microscopyrevealed that FA-stainable antigen was confinedentirely to the epithelial cells and initially ap-

peared in scattered foci by day 4 (Fig. 4). Theintensity and distribution of fluorescence grad-ually increased, reaching its maximum by day 10,and maintained this state until the study was

terminated on day 21. Fluorescence appeared to

B. Herpes Simplex Virus

Explant

C. Adeno-associated Virus Type 2 D. Echo Virus Type IIEpithelium destroyed

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DAYS AFTER INOCULATION

FIG. 1. Viral growth patternzs in human fetal intestinal organ cultures. Medium was harvested every 2 days and

assayed for the presence of infectious virus in tissue culture. Symbols: *, infected organ cultures; 0, inactivationof virus in dishes without organ culture.

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FIG. 2. Section ofhuman fetal intestinal organ culture, 10 days after infection with adenovirus 7, stained withhematoxylin and eosin. Villi including columnar epithelium are well preserved. X 460.

be primarily intranuclear, but cytoplasmic stain-ing was also present. AAV2 fluorescence followedthe pattern of adenovirus staining, but appearedsomewhat later in the time course of infection(day 6, Fig. 5). Fluorescence, however, was al-most entirely intranuclear within epithelial cells.HSV staining appeared earlier than with otherviruses. Nuclear and cytoplasmic fluorescencewas noted by day 2, with subsequent spread to thelamina propria. By day 5, the submucosa wassimilarly stained, and, by day 8, most of theentire explant fluoresced (Fig. 6). Echovirus 11fluorescence was noted first in the cytoplasm ofepithelial cells in discrete foci by day 3, with sub-sequent spread throughout the epithelium bydays 5 to 7 (Fig. 7). Occasional fluorescence wasdetected in the lamina propria.

In addition to the above experiments, the fol-lowing controls were performed:

(i) Uninfected organ cultures did not fluorescewhen reacted with anti-serum and fluorescein-labeled anti-globulin.

(ii) Infected organ cultures did not stain in

indirect FA tests with human sera that lackedneutralizing and FA-stainable antibodies againstthe homologous virus.

DISCUSSIONThese immunofluorescent studies showed that

specific viruses could be detected in intestinalorgan cultures in the absence of, or prior to thedevelopment of, any histological changes in theexplants. The appearance of maximum fluores-cence in the organ culture lagged behind theemergence of peak titers of infectious virus in thesurrounding fluid medium, suggesting the im-portance of assaying later time points with thistechnique. The fluorescence characteristicallyappeared in foci, which in many instances re-mained relatively separate until much later in thetime course of infection, further emphasizing thenecessity for the examination of multiple sectionsto accurately detect agents in this system.The growth patterns of the above viruses in

organ culture indicated that viruses which haveshorter replicative cycles in monolayer cell cul-

960 INFECT. IMMUNITY

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VIRAL DETECTION AND LOCALIZATION

FIG. 3. Sectionz ofhumant fetal intestinal organ culture at 4 days after infection with herpes simplex virus. Villiare absent and epithelium is denuded. X 460.

tures reached peak titer in the extracellular fluidearlier than those with longer replicative cycles.HSV and echovirus with replicative cycles of 5 to12 hr (12) and 5 to 10 hr (5), respectively, reachedpeak titers in 4 to 6 days, whereas adenovirusand AAV with replicative cycles of 12 to 24 hr(1) reached peak titers in 8 days. In experimentalinfection with orally administered adenovirus inman, significant recovery of adenovirus in thestool does not take place until 4 days after ad-ministration of the virus, and maximum recoveryof virus occurs at 7 to 10 days (4).Each of the viruses studied initiated infection

on the epithelial surface of the explants. Adeno-virus and AAV replicated solely in epithelial cells,whereas HSV and, to a lesser extent, echovirusreplicated in the lamina propria and submucosaas well. Most monolayer tissue culture prepara-tions, even those made from dispersed wholeorgans, appear to lose most of their epithelialcomponents (particularly after passage) andconvert largely to fibroblastic cell sheets. Thus,

organ cultures, in which epithelial cells remainintact, may provide a superior culture for thoseagents which replicate preferentially in the epi-thelium.The concept that organ cultures represent a

more sensitive culture medium for the cultivationof occult viruses was supported by recent evi-dence that certain respiratory viruses, i.e., typesof coronaviruses and rhinoviruses, replicate intracheal organ culture but not in standard mono-layer tissue culture (7, 8, 14). Presumably,tracheal organ cultures more closely approximatethe in vivo natural host target organ for theseagents. Similar reasoning is currently beingapplied to the search for occult agents implicatedin infectious gastrointestinal disease (3).

It is hoped that inoculation of human fetalintestinal organ culture with materials obtainedfrom naturally occurring outbreaks and experi-mentally induced disease, followed by studiesemploying FA techniques using antibodies con-tained in sera or secretions from the same cases,

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FIG. 4. Ultraviolet photomicrograph of frozen sections of human fetal intestinal organ caltuir? stained withhuman anti-adenovirus serum anid conjugate. X 260. A, Uninfected organ culture at day 10 after explanation; B,adenovirus 7-infected organ culture at day 4; C, adenovirus 7 at day 8; D, adellovirus 7 at day 10.

FIG. 5. Frozen section of' human fetal intestinal organ culture, 6 days after infection with adenovirus 7 andAA V-2, stained with guinlea pig anti-AA V-2 serum and conjugate. X 500.

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VIRAL DETECTION AND LOCALIZATION

FIG. 6. Frozen section of human fetal intestinal organ culture, 8 days after infection with herpes simplex virus,stained with huiman anti-herpes simplex serum and conjugate. X 400.

FIG. 7. Frozen section of human fetal intestinal organ culture stained with human anti-echovirus serum antdconjugate at 7 days after infection with echovirus 11. X 400.

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DOLIN ET AL.

or both, may aid in the detection of these occultagents.

ACKNOWLEDGMENTS

We thank Eleanor Stewart and Robert Chames for their assist-ance with these studies, and the obstetrics and pathology depart-ments of Church Home and Sinai Hospitals in Baltimore, Md.,for providing the specimens which were employed. Portions of thisinvestigation were supported by the U.S. Army Medical Researchand Development Command, Department of the Army (contractDADA 17-72-C-2071).

LITERATURE CITED

1. Blacklow, N. R., M. D. Hoggan, and W. P. Rowe. 1967. Im-munofluorescent studies of the potentiation of an adeno-virus-associated virus by adenovirus 7. J. Exp. Med.125:755-765.

2. Dolin, R., N. R. Blacklow, R. A. Malmgren, and R. M.Chanock. 1970. Establishment of human fetal intestinalorgan cultures for growth of viruses. J. Infect. Dis. 122:227-231.

3. Dolin, R., N. R. Blacklow, H. DuPont, R. F. Buscho, R. G.Wyatt, J. A. Kasel, and R. M. Chanock. 1972. Biologicalproperties of Norwalk agent of acute infectious non-bac-terial gastroenteritis. Proc. Soc. Exp. Biol. Med. 140:578-583.

4. Edmondson, W. P., R. R. Purcell, B. F. Gundelfinger, J. W. P.Love, W. Ludwig, and R. M. Chanock. 1966. Immuniza-tion by selective infection with type 4 adenovirus grown inhuman diploid tissue culture. J. Amer. Med. Ass. 105:453-459.

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5. Eggers, H. J., and I. Tamm. 1961. Spectrum and charactelis-tics of the virus inhibitory action of 2-a-hydroxybenzyl-benzimadazole. J. Exp. Med. 113:657-682.

6. Hoggan, M. D., N. R. Blacklow, and W. P. Rowe. 1966.Studies of small DNA viruses found in various adenoviruspreparations: physical, biological and immunological char-acteristics. Proc. Nat. Acad. Sci. U.S.A. 55:1467-1474.

7. Hoorn, B., and D. A. J. Tyrrell. 1966. A new virus cultivatedonly in organ cultures of human ciliated epithelium. Arch.Gesamte Virusforsch. 18:210-225.

8. McIntosh, K., J. H. Dees, W. B. Becker, A. Z. Kapikian,and R. M. Chanock. 1967. Recovery in tracheal organ

cultures of novel viruses from patients with respiratorydisease. Proc. Nat. Acad. Sci. U.S.A. 57:933-940.

9. Mitus, A., J. Folkman, J. F. Enders, and S. Driscoll. 1970.Cultures of segments of human fetal intestine: applica-tions to cytologic and virologic investigations. Proc. Soc.Exp. Biol. Med. 134:800-806.

10. Reed, L. J., and H. Muench. 1938. A simple method of esti-mating fifty percent endpoints. Amer. J. Hyg. 27:493-497.

11. Rubenstein, D., and D. A. J. Tyrrell. 1970. Growth of virusesin organ cultures of intestine. Brit. J. Exp. Pathol. 51:210-216.

12. Russell, W. C., E. Gold, H. M. Keir, H. Omura, D. H.Watson, and P. Wildy. 1964. The growth of herpes simplexvirus and its nucleic acid. Virology 22:103-110.

13. Stenhouse, A. C. 1970. Virus multiplication in organ culturesof human embryo small intestine. J. Gen. Virol. 8:235-239.

14. Tyrrell, D. A. J., and M. L. Bynoe. 1965. Cultivation of a

novel type of common-cold virus in organ cultures. Brit. J.Med. 1:1467-1470.

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