INFECTION AND IMMUNITY, Oct. 1973, p. 657-664Copyright 0 1973 American Society for Microbiology

Vol. 8, No. 4Printed in U.S.A.

Persistent Newcastle Disease Virus Infection inEmbryonic Chicken Tracheal Organ Cultures

J. F. CUMMISKEY, J. V. HALLUM, M. S. SKINNER, AND G. A. LESLIE

Department of Microbiology and Immunology, Tulane University School of Medicine,New Orleans, Louisiana 70112

Received for publication 11 June 1973

The persistent infection of embryonic chicken tracheal organ cultures withNewcastle disease virus (NDV) is described. Tracheal explants remainedmorphologically intact and were able to support the replication of NDV for 6months. Peak titers of released virus occurred at 1 week postinfection, whereasmaximal immunofluorescence was not observed until 30 days postinfection. Theinoculum titer was not critical, and viral persistence resulted with either of twostrains of NDV tested. Serum was not required in the medium for explantviability or to maintain the persistent infection, The presence of a contaminatingvirus morphologically resembling a leukovirus neither altered the course ofinfection nor affected the survivability of explants. Although interferon was notdetected in the culture medium, persistently infected explants were resistant toheterologous viral challenge, and a similar resistant state could be induced inuninfected explants with exogenous interferon or ultraviolet light-inactivatedNDV. No evidence was found to implicate antibody as a regulatory factor in theestablishment or maintenance of persistence. The results from electron micros-copy and immunofluorescence suggest the cells of the subepithelial connectivetissue as the site of NDV persistence.

Tracheal organ cultures support the growth ofa number of viruses and have been useful forviral studies in differentiated tissue (4, 8, 12, 17,24). Persistent paramyxovirus infections in tra-cheal explants have been described, and thesepersistent infections have been accompanied bythe local formation and secretion of eitherantibody (13, 14) or interferon (17, 20). Sinceviral persistence in certain cell cultures appearsto depend upon the presence of either interferonor antibody in the medium (23), the presence ofthese two substances in persistently infectedtracheal organ cultures raises the question ofwhether such substances are involved in theestablishment and maintenance of the persist-ent infections.

In this report an organ culture system ofembryonic chicken trachea persistently infectedwith Newcastle disease virus (NDV) is de-scribed. The course of infection was followed byviral assays, immunofluorescence, and electronmicroscopy. The effects of interferon pretreat-ment, titer of original inoculum, strain of NDV,and serum requirement on the establishment ofviral persistence were studied. The roles ofantibody and interferon as factors contributing

to the maintenance of persistence were ex-amined.

MATERIALS AND METHODSTracheal organ cultures. Fertile eggs from white

Leghorn chickens, DeKalb strain 161 (Pan AmericanHatchery, Hammond, La.), or specific pathogen-free,COFAL-negative eggs from Leghoms of the Mt. Hopestrain (Spafas, Inc., Norwich, Conn.) were incubatedfor 18 to 19 days at 37 C, and tracheal organ cultureswere prepared according to the procedure describedby Cherry and Taylor-Robinson (9). After excisionand sectioning, each explant was placed at the bottomof a screw-cap culture tube (16 by 125 mm) in 1 ml ofmedium and incubated in a stationary vertical posi-tion for 24 to 48 h at 37 C before infection.

Ciliary activity was observed with a Leitz bottom-view microscope, with 60x or 100x total magnifica-tion. Uninfected explants continued to show activelybeating cilia for at least a month in vitro, with anoccasional explant retaining activity for as long as 2months. In infected explants, complete ciliostasisroutinely occurred by the 4th or 5th day postinfection.Any given explant was considered viable if infec-

tious virus was present in the culture media, sinceelectron microscopy routinely confirmed the presenceof susceptible, viable cells within such explants.Among explants maintained in medium containing

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serum, 98% of the uninfected explants and 90% ofNDVp,-infected explants remained viable throughoutthe 6-month observation period. NDV,, is a smallplaque mutant of the Herts strain of NDV (NDVO).Among explants maintained in serum-free medium,the viability of uninfected explants was similar,whereas the viability of infected explants ranged from30 to 75%, depending upon the particular batchobserved. Some seasonal effect was suggested in thatthe lower percentage was among those explants putinto culture during the months of November andDecember of 2 successive years.

Infection of organ cultures. After preliminaryincubation, the culture fluid was removed from theexplants and replaced with 1 ml of medium contain-ing a dilution of virus. After a 1-h adsorption at 37 Con a rotary shaker (50 to 60 rpm), the virus suspensionwas removed and fresh medium was added. The tubeswere incubated at an incline of approximately 5° on aroller drum (0.20 rpm) at 37 C. The medium waschanged every 4 to 8 days, and the culture fluids fromlike groups (minimum of five explants per group) werepooled and stored at 4 C until assayed for infectivity.Medium. Eagle minimal essential medium (Grand

Island Biological Co., Grand Island, N.Y.) containing0.05 M N-2-hydroxyethylpiperazine-N'-2'-ethanesul-fonic acid buffer (Calbiochem, San Diego, Calif.),0.035% NaHCO3, penicillin (100 U/ml), and strep-tomycin (100 ,ug/ml) was used routinely for maintain-ing cultures and diluting inocula. The pH was ad-justed to 7.1 with 2 N NaOH and, when called for, 4%calf or fetal calf serum was added after sterilization.

Viruses. A stock pool of NDVO was prepared in theallantoic cavities of 10- to 11-day-old embryonatedeggs. The supernatant culture fluid from persistentlyinfected L-cell cultures served as the stock pool ofNDV,i (22). Pools of NDVO and NDV,i for infection ofexplants were prepared by infecting chicken embryomonolayer (CEM) cultures with a 102 dilution of thestock pool of NDVO or undiluted NDV,1 stock pool.Supernatant culture fluids were harvested when cyto-pathic effect was maximal (24 to 48 h for NDVO, 72 to96 h for NDV,i), clarified by centrifugation at 12,000x g for 20 min, and stored at 4 C. Pools of the Indianastrain of vesicular stomatitis virus (VSV) were pre-pared in L-cell monolayer cultures, and Sindbis viruspools were prepared in CEM cultures. Infectivitytiters were determined by plaque assay in CEMcultures. NDVO, VSV, and Sindbis virus plaques werecounted 48 and 72 h postinoculation; NDVpi plaqueswere counted after 72 and 96 h.

Interferon. Chicken interferon was prepared in9-day-old embryonated eggs infected with the WSstrain of influenza virus as previously described (25).Residual virus infectivity was inactivated by heatingthe harvested allantoic fluids at 56 C for 1 h. Superna-tant culture fluids from infected and control explantswere assayed for interferon at dilutions of 1:2 andhigher by either plaque reduction (25) or single-cycleyield inhibition in CEM cultures, using VSV as thechallenge virus. Before interferon testing, residualNDV,I in the supernatant culture fluids was inacti-vated by dialysis at 4 C against a glycine buffer, pH2.0, for 4 to 6 days, followed by dialysis against

INFECT. IMMUNITY

phosphate-buffered saline (PBS, pH 7.2; 0.01 Mphosphate, 0.15 M saline) for 24 to 48 h. Controlcultures were similarly treated. Single-cycle yieldinhibition was determined as follows. Twofold serialdilutions of the culture fluid under test were incu-bated with CEM cultures (3 ml/culture) for 18 h at37 C. After incubation, the cells were washed twicewith medium and challenged with VSV at an inputmultiplicity of infection of 1. Following a 1-h adsorp-tion, the monolayers were washed twice more, coveredwith 3 ml of fresh medium, and reincubated at 37 C.Fluids from the challenged cultures were harvestedafter 7 h and the VSV was assayed in CEM cultures.Controls consisted of CEM cultures incubated withmedium alone or culture fluids from uninfected ex-plants.Immunofluorescence antiserum. Fluorescein-

labeled horse anti-chicken globulin (Progressive Lab-oratories, Baltimore, Md.), at a 1:20 dilution in F.A.buffer (Difco Laboratories, Detroit, Mich.), pH 7.2,was used to detect immunoglobulin-containing cellsin frozen tracheal sections. At this dilution theantiserum could detect immunoglobulin-containingcells in frozen sections of trachea or spleen from a27-day-old chicken; the fluorescence could be blockedby pretreatment with goat anti-chicken globulin.Anti-NDV serum was prepared by wing web stab

immunization of young adult, specific pathogen-freechickens (Spafas, Inc., Norwich, Conn.) with theRoakin strain of live NDV vaccine (Salsbury Labora-tories, Charles City, Iowa). The globulin portion waslabeled (Sylvana Corp., Milltown, N.J.) with fluores-cein isothiocyanate and used at a 1:15 dilution in F.A.buffer for direct staining of NDV in frozen sections.The conjugate stained NDV-infected tissues; suchstaining could be blocked by pretreatment with unla-beled anti-NDV serum but did not stain uninfectedtissues or tissues infected with other viruses. Stainingoccurred in NDV-infected sections treated with post-immune serum and stained with conjugated horseanti-chicken globulin, but not in sections treated withthe pre-immune serum.

Immunofluorescence staining. Explants wereharvested at various times after infection, rinsed inPBS, submerged in Tissue Tek OCT Compound(Ames Co., Elkhart, Ind) in small aluminum foil cups,and quickly frozen in a dry ice-isopentane bath. Serial4-,um sections were cut on a cryostat maintained at-25 C, air-dried on albumin-glycerine-coated slides,fixed in acetone for 10 min, and stained for 20 min atroom temperature. They were rinsed twice for 10 mineach in F. A. buffer, mounted in buffered glycerine(pH 7.7), and examined on a Leitz fluorescencemicroscope with a BG 12 exciter filter and a blueabsorbing barrier filter, with an Osram HBO 200-Wmercury vapor bulb as the light source.

Electron microscopy. Whole tracheal explants,rinsed briefly in PBS, were fixed in cold 3% glutaral-dehyde with cacodylate buffer, postfixed with 1%osmium tetroxide in phosphate buffer, dehydrated,treated with propylene oxide, and embedded in Mara-glas epoxy resin (21). Thin sections were cut on aPorter-Blum MT 1 ultratnicrotome with a diamondknife, placed on uncoated copper mesh grids, stained

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with lead citrate alone or in combination with uranylacetate, and examined in a Philips EM 300 electronmicroscope.

Ultraviolet inactivation. NDVO, harvested frominfected allantoic fluids, was diluted 1: 10 in PBS andpassed through a membrane filter (0.2 um porediameter, Millipore Corp.). Samples (5.0 ml) wereplaced in 120-mm petri dishes and rotated for 3 min at10 cm from a Sylvania Germicidal G15T8 ultraviolet(UV) lamp, changing to a fresh petri dish after each1-min interval. The preparation was tested for infec-tivity in CEM cultures. Infectivity titers of NDVOfiltrates before inactivation were 107 and 108 plaque-forming units (PFU)/ml.

Poly(IC). Polyriboinosinic acid [poly(I)] and poly-ribocytidylic acid [poly(C)] (Miles Laboratories, Inc.,Elkhart, Ind.) were prepared by dissolving in PBS togive a concentration of 1 mg/ml of each. Equalamounts of the poly(I) and poly(C) solutions weremixed and annealed for 10 min at 37 C, cooled toroom temperature, and diluted to 50 gg/ml in PBScontaining 100 jug diethylaminoethyl-dextran/ml(Pharmacia, Uppsala, Sweden).

Radial immunodiffusion. Culture fluids (serumfree) were harvested from groups of infected anduninfected explants at intervals from 24 h to 23 daysafter excision and concentrated 100- to 200-fold byvacuum dialysis. Such concentrates were tested forthe presence of immunoglobulin (Ig)Y, IgM, and IgAby radial immunodiffusion on 1% agarose (MarineColloids, Inc., Rockland, Maine) plates with heavy-chain-specific goat anti-chicken IgY and IgM, orrabbit anti-IgA, as the developing antisera. Details ofthe antisera preparation and use in immunodiffusionhave been previously described (15). Precipitin ringswere measured to the nearest 0.1 mm with a Bauschand Lomb measuring magnifier and dark-field light-ing. Immunoglobulin concentrations in the culturemedium were determined by comparison of precipitinring diameters with those of a standard curve pre-pared from twofold serial dilutions of pooled wholechicken sera with previously determined immuno-globulin concentrations.

RESULTSEstablishment of persistent infection. After

infection with both NDVo and NDVpi, infec-tious virus was continuously recovered in theculture medium until the experiments wereterminated, usually 5 or 6 months later. Asshown in Fig. 1, which is typical of eight similarexperiments, NDVpi titers rose during the firstweek, then declined for 2 weeks until they some-what stabilized, usually within the range of 102to 104 PFU/ml. Serum was not required in themedium either to maintain the persistent infec-tion or to preserve explant viability, althoughviral titers were approximately 1 log lower whenserum was omitted (Fig. 1).

At 1 week postinfection, higher titers ofreleased virus occurred in those explants receiv-ing a higher initial dosage, but thereafter no

effect due to inoculum titer was seen (Fig. 1).Pretreatment of explants with 6 or 60 U ofchicken interferon for 18 h before infection hadno effect on the course of infection. Viral titersresulting from infection with NDVO were similarto those shown in Fig. 1 for NDVpi.A contaminating virus, morphologically re-

sembling a leukovirus, was present in certain ofthe explants derived from Pan American eggs.The leukovirus agent was about 100 nm indiameter and was readily distinguishable fromNDV. The agent was usually found in groupsand was located in and about all of the celltypes in the tracheas including the chon-drocytes. When certain of the experiments wererepeated in leukosis-free explants, no differ-ences in explant longevity or in the course ofpersistent NDV infection could be detected.Three to four weeks after the initiation of

infection, a change occurred in plaque morphol-ogy of the virus recovered from explants in-fected with NDVpi, but not with NDVO. Thenewly emerging plaques, at first present alongwith typical NDVpi plaques, were later in devel-oping in CEM cultures, had diffuse margins,and were difficult to see by reflected light.When this agent was isolated from a persist-ently infected explant 139 days postinfection,plaque-purified in CEM cultures, and used toinitiate a persistent infection in tracheal ex-plants, the pattern of released viral titers wasindistinguishable from that seen with NDVpi- orNDV.-initiated infections.No evidence was found to implicate antibody

as a necessary factor in the establishment ormaintenance of the persistent infection. Mater-

4% Serum

55SS X 103 PfUe- 5 X 0' PFU/s.pa0t

0-^-O 275 X105 PfU/e.piont75 X 103 PfU/c.plo.V

0-c 2 75 X 10' PfU/t.plov

6i

m.EZN

40Si_!

No Serum

DYOYS AFTER INFECTION

FIG. 1. Persistent infection of chicken embryo tra-cheal organ cultures infected with different amountsof NDVpO. The medium, either serum free or supple-mented with 4% calf serum, was renewed every 4 to 8days and assayed for infectivity in CEM cultures.

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nal antibody was not required to establishpersistence, and the course of infection was

similar in explants derived from immunized or

nonimmunized flocks. Immunoglobulin-con-taining cells were not detected by direct im-munofluorescence in frozen sections from eitherinfected or control explants harvested at inter-vals from the time of excision through 93 days inculture. IgM and IgA were not detected in theculture medium from either infected or controlexplants. However, immunoglobulins of the IgYclass were present in concentrations rangingfrom 0.08 to 0.16 jig/ml in the initially harvestedculture medium, either 24 or 48 h after excision,and thus only before infection. Once the me-

dium had been renewed IgY was no longerdetected.

Interferon activity was not detected in theculture medium of persistently infected ex-

plants at any time throughout the course ofinfection or in culture medium harvested fromuninfected explants 18 h after treatment witheither of two interferon inducers, poly(IC) (50ag/explant) or UV-inactivated NDV (1 ml/explant).

However, persistently infected explants were

resistant to challenge with either of two interfer-on-sensitive viruses, VSV or Sindbis virus. Theresistance to VSV developed gradually afterinfection with NDVpi and continued to in-crease for the 14 days of the experiment (Fig. 2).A transient resistance to VSV which could beinduced in uninfected tracheal explants by a

1-h treatment with UV-inactivated NDV (1ml/explant) was demonstrated when the ex-

plants were challenged immediately after treat-ment but not when the explants were returnedto fresh medium and challenged 3 or 7 days later(Table 1). A viral-resistant state could also be

:3

Jz 2-

I" .

NDVpi TITER

r ~~~~~~~~~~~E__-- RESISTANCE

TO VSV -I

12 3 4 5 6 7 8 9 10 12 13 14

DAY CHALLENGED AFTER INFECTION WITH NDVpi

FIG. 2. Development of resistance to VSV in per-

sistently infected chicken embryo tracheal organ

cultures. Tracheal explants were infected with NDVjL(5 x 10O PFU/explant) and challenged at intervalsafter infection with VSV (2 x 10 PFU/explant). TheVSV was harvested 24 h after inoculation and assayedfor infectivity in CEM cultures. Controls consisted ofthe VSV yield from uninfected tracheal explants.

TABLE 1. Induction of resistance to VS V withUV-inactivated NDVa

Days atter Logl,droptreatment from controls

0 1.051.09

3 0.09-0.07

7 -0.020.09

VSV challenge (1.4 x 105 PFU/explant) washarvested 20 h after inoculation.

TABLE 2. Replication of VSV in tracheal explantstreated with interferon

CelclueYield of' YieldCulture Cell culture VSV reductionassayed in (PFU/ml) ((')

Control CEM 1.8 x 107Pretreated with CEM 4.3 x 105 97.6

interf'eron'Control L cells 3.9 x 105Maintained in L cells 2.8 100

interferon'

Groups of unintected explants were incubated for18 h with chicken interferon (60 U/ml), washed inPBS, and challenged with VSV (2 x 104 PFU/explant). Af'ter a 1-h adsorption at 37 C, the challengevirus was removed and the cultures were washed inPBS and returned to fresh medium. The VSV washarvested 48 h after inoculation and assayed forinfectivity in CEM cultures. The VSV yield fromexplants treated with medium alone served as thecontrol.

'These cultures were pretreated with interferonand challenged with VSV as described above. How-ever, after challenge with VSV they were returned tofresh medium containing chicken interferon (60U/ml). The VSV was harvested 48 h after inoculationand assayed for infectivity in L cells to eliminate theeffect of chicken interferon.

induced in uninfected tracheal explants byexogenous interferon. An 18-h pretreatmentwith chicken interferon (60 U/ml) resulted in aVSV yield reduction of 98% (Table 2). Inexplants pretreated and maintained in the pres-ence of interferon after challenge, VSV replica-tion was inhibited 100%.Immunofluorescence. Direct immuno-

fluorescent staining of viral antigen in frozensections from tracheal explants was first de-tected at 48 h postinfection, when one to threesmall foci per tracheal ring were seen in theextracartilaginous connective tissue. A minoramount of fine granular staining was occasion-ally observed at the luminal surface as early as

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48 to 72 h after infection, but marked staining ofthe luminal surface was not seen until 20 dayspostinfection (Fig. 3). When immunofluores-cence observations were correlated with thefindings from light and electron microscopy, itwas our impression that staining of the mucosalepithelium was minimal, and the luminal sur-face staining seen later in the course of infectionmay have been located in the superficial con-nective tissue cells of the lamina propria ratherthan in the surface epithelium.With progression of time, staining of both the

lamina propria and extracartilaginous connec-tive tissue increased, and maximal fluorescencewas seen by day 30. Little change occurred inthe staining pattern thereafter. This late peakin immunofluorescence contrasts with the 7-daypeak in titers of released virus. Muscle andcartilage cells did not stain at any time, al-though they were autofluorescent.

FIG. 3. Immunofluorescent staining of NDV,, infrozen section from a tracheal explant 20 days afterinfection. x100.

Electron microscopy. Viral replication inexplants infected with NDVp, was initially de-tected by electron microscopy at 7 days postin-fection. Replication was seen in macrophagesand fibroblast-like cells located in the connec-tive tissue region just exterior to the trachealcartilage. Round and filamentous buddingforms, characterized by the presence of a triple-layered membrane and by the presence ofnucleocapsid beneath the membrane, werefound at the plasma membrane and in in-tracytoplasmic locations in infected cells (Fig. 4and 5). The dimensions of the rounded andfilamentous buds and of the extracellular vi-rions were variable. In general, the virion diam-eter was 120 to 170 nm for the rounded formsand 150 to 170 nm for the filamentous forms,whereas the diameter of viral nucleocapsid was11 to 15 nm. By 14 days postinfection, buddingNDV and mature virions could also be found inand adjacent to many macrophages and fibro-blast-like cells, which together with a rare lym-phocyte comprised the cell population of thelamina propria of the embryonic explants.Morphological evidence of NDV replicationwas confined to the fibroblast-like cells and themacrophages in both the lamina propria and ex-tracartilaginous connective tissue through the139 days that infected explants were examined.No evidence of viral replication was detected

in the epithelial cells, in the chondrocytes of thehyaline cartilage, nor in the scattered skeletalmuscle elements which were present at theextreme outer edge of the explants. It should benoted that the presence or absence of serum inthe medium of these explants had no effect onthe ultrastructural aspects of NDV replication.

r9''^''',''s;~~~~~~~i~'~~ ~~~* lo

FIG. 4. Connective tissue cell from explant at 7 days after infection with NDVi. Note budding virions inintracytoplasmic locations and at plasma membrane. x29,000. Insert, extracellular NDV. x39,000.

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Rather, the principal effect of the serum sup-plementation was one of prolongation of viabil-ity of the infected explants.During the course of examining the explants

infected with NDV, numerous fibroblast-likecells (occasionally infected) and several epithe-lial cells were noted to contain an intranuclearstructure. The structure was composed of com-pact parallel arrays of fibrils (Fig. 6) of varyinglengths which measured 8 to 10 nm in diameter.A similar structure, composed of fewer 7- to9-nm fibrils arranged in a rather loose array,was rarely encountered in control explantswhere they were found only in fibroblast-likecells.

In general, the course of infection in trachealexplants infected with NDVO paralleled that

seen with NDV,i. The following differences,however, were uniformly present. Viral replica-tion could be found in connective tissue cells inboth the extracartilaginous connective tissueand the lamina propria at 7 days postinfection.Similar to the findings with NDVpi, however,replication was detected only in fibroblast-likecells and macrophages of these regions duringthe 101 days that infected explants were ex-amined. Infection with NDVO was, in general,more devastating to the individual explant thanwas NDVpi. This was primarily evidenced by anincrease in cellular debris and disrupted cells inwhich nucleoprotein-like material was oftenapparent. Similarly, there was a concomitantdecrease in viable macrophages and fibroblast-like cells of the NDV.-infected explants as

FIG 5. Budding NDV at cytoplasmic membrane of infected cell. x 34,000.

..--~~~~~~~~..A<4-A.d4S -

FIG. 6. Nuclear fibrils in a cell from an explant infected with NDVpi. Evidence of viral replication was notdetected in this cell. x46,000. Insert, similar nuclear fibrils in a cell from an uninfected explant. x51,300.

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NDV IN CHICKEN TRACHEAL ORGAN CULTURES

compared with those infected for similar periodswith NDVpi.

DISCUSSIONChicken embryo tracheal organ cultures are

potentially valuable as an in vitro system for thestudy of persistent NDV infection, and perhapsother respiratory viruses as well. Viral persist-ence was the normal outcome of infection withNDV and required no special manipulations ofthe system. Unlike monolayer cultures, trachealexplants are structurally similar to the naturalin vivo host target organ, and remain intactwhile supporting the replication of NDV forlonger than 6 months. Blaskovic et al. havereported a spontaneous degeneration of simi-larly prepared embryonic chick tracheal organcultures after 15 to 20 days of in vitro mainte-nance and tentatively ascribed the degenerationto the presence of a passenger virus (5). Al-though certain of our cultures contained amorphologically similar agent, degeneration didnot occur in those explants during the 6-monthobservation period.No evidence was found in this study to

implicate antibody as a regulatory factor in theestablishment or maintenance of the persistentinfection. There was no requirement for anti-body in the medium or for maternally derivedantibody. The IgY which was present afterexcision was most likely maternally derivedimmunoglobulin. This conclusion is supportedby the absence of immunoglobulin-containingcells in the explants and the presence of only arare lymphocyte. These latter findings mayreflect an immunological incompetence of theembryonic chick trachea.Although released interferon was not detected

in the culture medium, the resistance of persist-ently infected explants to heterologous viralchallenge suggests that an interferon-like mech-anism may be operating. This viral resistancewas apparent by the third day postinfection; yetcell damage was not apparent by electron mi-croscopy until 7 days postinfection, suggestingthat a loss of susceptible cells was insufficient toaccount entirely for the magnitude of resistanceseen within the first week. This resistance mayhave been due to small amounts of interferon,perhaps intracellular, present but not detecta-ble by the assay system. In certain cell culturesa viral resistant state, seemingly interferon-mediated, can occur before, or in the absence of,detectable amounts of released interferon (2,11). A similar phenomenon of resistance with-out detectable interferon may be relevant to thepresent experiments. The tracheal explantswere clearly able to respond to exogenous inter-

feron, and the transience of the UV-NDV-induced resistance is compatible with an inter-feron-mediated resistance.The minimal epithelial staining seen with

immunofluorescence and the absence of in-fected epithelial cells by electron microscopywere surprising since other investigators havedescribed NDV in the epithelium (3, 12, 14).The early and uniform ciliostasis observed inNDVpi-infected explants implies an involve-ment of the virus with the epithelium. Bang andFoard noted a 100-fold decrease in the numberof NDV-infected cells from the epithelium ofchick tracheal explants within the first 5 h afterinfection and attributed this to a sloughing ofinfected cells from the mucosa (1). Reed andBoyd suggested that a rapid detachment ofrhinovirus-infected cells occurred in calf tra-cheal epithelium (18). Possibly the presentexperiments represent a similar situation andindividual epithelial cells became detachedsoon after infection. Such a loss of infected cellswould explain the apparent lack of epithelialparticipation in the persistent infection.The nuclear fibrils observed by electron mi-

croscopy in infected explants bear some mor-phological resemblance to the "smooth fila-ments" described in the nuclei of cells infectedwith SSPE agents (16). These fibrils, althoughoccasionally seen early in the infection, becamecommon 14 to 21 days postinfection. Although itis tempting to speculate upon the significance ofthe intranuclear fibrils in the cells of infectedexplants, considerable caution is warranted byat least two factors. First, although of a slightlydifferent array, an almost identical intranuclearstructure was present in certain of the cells incontrol explants. Second, remarkably similarintranuclear structures have been reported in awide variety of "normal" cells in several species(6, 10, 19).

In the intact bird, infection with NDV is notalways self-limiting (7), and a carrier state mayresult when the virus establishes itself withinthe host. Our observations with immunofluores-cence and electron microscopy suggest thatserious consideration should be given to thesubepithelial connective tissue as the possiblesite of NDV persistence in the intact bird.Multiple mechanisms such as interferon, or adelay in maturation and release, may be operat-ing simultaneously or sequentially to preserve adelicate balance between viral replication andcell survival.

ACKNOWLEDGMENTSThis study was supported by grants from the National

Tuberculosis and Respiratory Disease Association, The JohnA. Hartford Foundation, Inc., and the National Institute of

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General Medical Sciences, Public Health Service microbiol-ogy research training grant GM-00079.

LITERATURE CITED

1. Bang, F. B., and M. Foard. 1964. Interaction of respira-tory epithelium of the chick and Newcastle diseasevirus. Amer. J. Hyg. 79:260-278.

2. Baron, S., K. T. Wong, C. E. Buckler, and F. Dianzani.1968. Comparative induction of the interferon systemby virus, statalon, and interferon, p. 129-145. In G.Rita (ed.), The interferons. An international review.Academic Press Inc., New York.

3. Beard, C. W., and B. C. Easterday. 1967. The influence ofthe route of administration of Newcastle disease viruson host response. III. Immunofluorescent and histo-pathological studies. J. Infect. Dis. 117:66-70.

4. Blaskovic, P., A. J. Rhodes, F. W. Doane, and N. A.Labzoffsky. 1972. Infection of chick embryo trachealorgan cultures with influenza A2 (Hong Kong) virus. I.Cytopathology, histopathology, immunofluorescence,hemadsorption, and titration of the released infectiousprogeny virus. Arch. Gesamte Virusforsch. 37:104-113.

5. Blaskovic, P., A. J. Rhodes, F. W. Doane, and N. A.Labzoffsky. 1972. Infection of chick embryo trachealorgan cultures with influenza A2 (Hong Kong) virus. II.Electron microscopy. Arch. Gesamte Virusforsch.38:250-266.

6. Boquist, L. 1969. Intranuclear rods in pancreatic islet Bcells. J. Cell Biol. 43:377-381.

7. Brandly, C. A., and R. P. Hanson. 1965. Newcastledisease, p. 633-671. In H. F. Biester and L. H.Schwarte (ed.), Diseases of poultry. Iowa State Univer-sity Press, Ames, Iowa.

8. Butler, M., and W. J. Ellaway. 1972. Comparativestudies on the infectivity of avian respiratory virusesfor eggs, cell cultures and tracheal explants. J. Comp.Pathol. 82:327-332.

9. Cherry, J. D., and D. Taylor-Robinson. 1970. Large-quan-tity production of chick embryo tracheal organ culturesand use in virus and mycoplasma studies. Appl.Microbiol. 19:658-662.

10. Clattenburg, R. E., R. P. Singh, and D. G. Montemurro.1972. Intranuclear filamentous inclusions in neurons ofrabbit hypothalamus. J. Ultrastruct. Res. 39:549-555.

11. Finkelstein, M. S., G. H. Bausek, and T. C. Merigan.1968. Interferon inducers in vitro: differences in sensi-tivity to inhibitors of RNA and protein synthesis.Science 161:465-468.

12. Finkelstein, M. S., M. McWilliams, and C. G. Huizenga.

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