INFEcriON AND IMMUNrrY, Apr. 1975, p. 770-782Copyright 0 1975 American Society for Microbiology

Vol. 11, No. 4Printed in U.S.A.

Bovine Parainfluenza Type 3 Virus Infection: Virus Replicationin Bovine Embryonic Cell Cultures and Virion Separation by

Rate-Zonal CentrifugationKIN-SON TSAI1 * AND R. G. THOMSON

Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

Received for publication 30 August 1974

Replicative sequences of a bovine strain of parainfluenza type 3 virus in bovineembryonic kidney and spleen cell cultures were investigated by light andfluorescence microscopy and by ultrathin section and negative-contrast electronmicroscopy. Observations from light and fluorescence microscopy showed thatintracytoplasmic inclusions were detected as small granules surrounding thenuclei of more than 90% of the cell population by day 2 postinoculation. With theincrease of postexposure times, these inclusions coalesced into larger bodieswhich occupied large portions of the cell. Ultrastructurally, the first sign of virusdevelopment was the appearance of aggregates of viral nucleocapsids in thevicinity of the nucleus. With the concomitant accumulation of viral nucleocap-sids in the cytoplasm, the virus maturation was expressed by budding processesthrough the cell membrane into round, oval, or elongated forms. Eosinophilicinclusions were demonstrable in many mitotic cells. Ultrastructurally, these cellswere observed to produce virus particles by a process identical to that of restingcells. Virions, prepared from infected culture fluid and negatively stained,appeared to be pleomorphic and their diameter ranged from 200 to 600 nm. Thevirions were separated, by rate-zonal centrifugation, into two subclasses in a

sucrose gradient (15 to 60%, wt/wt). The slowly sedimenting virions had a densityapproximately 1.20 gm/cm3 and an average size of 200 nm in diameter, whereasthe faster-sedimenting virions had a density of 1.24 gm/cm3 and averagediameter of 400 nm.

Parainfluenza type 3 virus (PI-3V) probablyis implicated more often than any other viralagents that have been associated with respira-tory disease in cattle. However, the role of PI-3Vin the pathogenesis of so-called "shipping feverdisease" is uncertain at present, and conflictingviews regarding its significance have been ad-vanced (2, 5, 10-12, 23, 24, 28).

In our continuing investigations on variousaspects of the pathogenesis of bovine respiratorydisease, it has become evident to us that aprime step in determining the significance ofthe action of the virus lies in obtaining ultra-structural data that concern the functionalimplication of replication of the virus and thealterations of cells initiated by virus infection.Until recently, only a few reports describingsubcellular changes in cells infected with PI-3Vhad appeared (1, 13, 17, 21). Consequently,more ultrastructural information is needed con-

'Present address: Connaught Laboratories Ltd., Willow-dale, Ontario, Canada.

cerning the etfects of PI-3V in its natural hostcells and tissues, the bovine species. The pres-ent study concerns the development of a bovinestrain of PI-3V and its mode of maturation. Inaddition, ultrastructural aspects of virus forma-tion in mitotic cells and characterization ofvirions separated by rate-zonal centrifugationare presented. To our knowledge this is the firstultrastructural observation of virus productionin mitotic cells that suggests that a persistentinfection can be established in PI-3V bovine cellculture systems.

MATERIALS AND METHODSVirus. A bovine strain of PI-3V was kindly pro-

vided by M. Savan, Department of Veterinary Micro-biology and Immunology, Ontario Veterinary College,University of Guelph, Guelph, Ontario, Canada. Thevirus was originally isolated from the nasal swab of a5-month-old calf (no. 4604-9) that was in a groupbeing studied for shipping fever and had been shippedfrom western Canada 2 weeks prior to isolation. Theisolate was subsequently identified as PI-3V by neu-

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tralization and hemagglutination-inhibition tests andimmunofluorescence and electron microscopy. Thevirus stock was prepared by two to four additionalpassages in bovine embryonic kidney (BEK) cells. Forsome experiments, virus was concentrated and par-tially purified by centrifugation. The medium con-taining released virus was removed from PI-3V-infected BEK cells, and the cellular debris wasremoved by centrifugation at 800 x g for 10 min. Thevirus was sedimented at 30,000 x g for 1 h. Theresulting pellets were suspended in a small volume ofphosphate-buffered saline (pH 7.2) and stored at -70C. The titers of stocks were in the range of 3.7 x 107 to2.3 x 10' plaque-forming units per ml.

Cell cultures. BEK and bovine embryonic spleens(BES) obtained from fetuses approximately 2 to 4months old were used for primary cell cultures. TheBEK cells in their second or third passages were usedfor most of the present work. Madin-Darby bovinekidneys, purchased from Grand Island Biological Co.,Grand Island, N.Y., were used occasionally for com-parative purposes. All cells were grown as stationarycultures in prescription bottles, Blake bottles, orLeighton tubes, depending on the experiments.Growth medium consisted of Eagle minimum essen-tial medium in Hanks balanced salt solution supple-mented with 5 to 10% fetal calf serum, streptomycinsulfate (100 ug/ml), penicillin G potassium (250IU/ml), and tyrosine (60 gg/ml). For maintainingmonolayer cell sheets, the fetal calf serum concentra-tion was reduced to 1%.

Virus infectivity titrations. The virus infectivitywas titrated either by cytopathic changes in culturetubes between 4 and 5 days postinoculation or byhemadsorbing activity (4) with calf erythrocytes.The virus infectivity was also titrated on BEK cells byplaque assay, and the virus titers were expressed asplaque-forming units per milliliter.Growth curves. Monolayers of BEK cells or BES

cells were infected with PI-3V at an infectivity titer of4 x 106 mean tissue culture infective doses per ml,and inocula were absorbed for 1 h at 37 C. Washed,infected cultures were incubated at 37 C and, atappropriate intervals, both culture fluids and cellswere harvested from single cultures. Cell-associatedand released virus fractions were obtained by amethod similar to that of Numazaki and Karzon (19).

Light microscopy and fluorescence microscopy.Cover slips were fixed in Bouin solution at appropri-ate intervals after infection and were stained withhematoxylin and eosin. For fluorescence microscopy,the direct method was used. The infected and controlcell cultures were fixed, at appropriate intervals, incold acetone, dried in air, and treated for 20 min atroom temperature with fluorescein isothiocyanate-conjugated antibody (goat generated), which waspurchased from Colorado Serum Laboratory, Denver,Colo. After being washed in three changes of phos-phate-buffered saline, the cover slips were mounted inbuffered glycerine and examined with a Zeiss fluores-cence microscope.

Thin-sectioning electron microscopy. The mostextensive series of observations by electron micros-

copy was made on BEK cells infected at an infectivitytiter of 4 x 106 mean tissue culture infective doses perml. Samples of infected cells were harvested at 24, 48,or 72 h after infection. Thin sections of normal andinfected monolayers for electron microscopy wereprepared as follows. The culture medium was re-moved from the monolayers and replaced by 20 ml ofphosphate-buffered saline. The cells were scrapedfrom the Blake bottles and centrifuged at 800 x g for20 min. The pellets were fixed in 2.5% glutaraldehydein 0.2 M phosphate buffer (pH 7.3) for 1 h. Afterfixation, 0.2 M sucrose-0.2 M phosphate buffer (pH7.3) was added to the pellets and left overnight at 4 C.The pellets were cut into approximately 1-mm3 blocksand postfixed in 1% osmium tetroxide in phosphatebuffer (pH 7.3) for 1 h. After dehydration in gradedacetone solutions, they were embedded in epoxy resinthat had been polymerized by incubation for approxi-mately 12 h each at 37 and 45 C and for 16 h at 60 C.Sections were cut with either glass or diamond knivesand stained with uranyl acetate and lead citrate. Thespecimens were examined with a Philips EM 200electron microscope.

Negative-staining electron microscopy. Fluidfrom infected cultures was examined for the presenceof released virus as follows. The fluid, after clarifica-tion, was centrifuged at 30,000 x g for 1 h at 4 C, andthe resulting pellets were suspended in a smallamount of phosphate-buffered saline. Some prepara-tions were dialyzed against distilled water for 1 h. Adroplet of the suspension was mixed with an equalvolume of 1% phosphotungstic acid, pH 6.8. Themixture was removed by a filter paper, and the gridwas left to air dry before examination in the electronmicroscope.

Rate-zonal centrifugation of virions. All rate-zonal centrifugations were performed in a SpincoSW41 rotor at 20 C. For analysis of virions, 2.5 mleach of 15, 30, 45, and 60% (wt/wt) sucrose in a buffercontaining 5 x 10- 3 M tris(hydroxymethyl)amino-methane-hydrochloride, 10- 3 M ethylenediamine-tetraacetic acid, and 10-2 NaCl (pH 7.4) (TEN buf-fer) was layered in a centrifuge tube and left over-night at room temperature. A 1-ml volume of virussuspension in TEN buffer was placed on the gradientand centrifuged at 200,000 x g for 40 min. The den-sity of virus fractions was calibrated by lambdapipettes (100 pl) standardized against water.

RESULTSGrowth of virus. Both BEK and BES were

productive for PI-3V replication (Fig. 1A andB). However, BEK cells made slightly morevirus than BES cells. The infectivity titer ofreleased virus was consistently higher than thatof cell-associated virus. The virus titer reacheda plateau between 2 and 3 days in both cellsystems.

Ultrastructure of cells before virus inocu-lation. The epithelial cells in our culture sys-tems were examined ultrastructurally. In brief,

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released virus.

the nuclei were oval or irregular in shape, withinfoldings of the nuclear envelope. The nucleuswas composed of rather homogeneously distrib-uted chromatin with a few aggregations alongthe nuclear envelope and in the nucleoplasm.One or two nucleoli were visible in a singlesection.

In the cytoplasm, the rough endoplasmicreticulum was distributed in the form of flat-tened cisternae. The Golgi complex was gener-ally prominent with flattened cisternae andassociated vesicles. A few membrane-boundbodies of high electron density, presumablylysosomes, were usually observed in the cyto-plasm. Mitochondria were small and numerous;they contained moderately electron-dense mat-rices and transversely oriented cristae.

Small vesicles, ribosomes, glycogen particles,and bundles of fine fibrils, 7 to 9 nm indiameter, were found randomly scattered in thecytoplasm.Light microscopy of cells after virus infec-

tion. The most significant feature seen in theearly infected cultures (1 to 2 days) stained withhematoxylin and eosin was the presence ofsmall eosinophilic inclusions in the cytoplasm(Fig. 2A). By fluorescence microscopy, theseintracytoplasmic inclusions were detected assmall granules surrounding the nuclei in morethan 90% of cell population by day 2 postinocu-lation (Fig. 3A). With the increase of postexpo-sure times, these inclusions coalesced intolarger bodies, which occupied large portions ofthe cell (Fig. 2B, 3B).Electron microscopy of cells after virus

infection: (i) Early cell alterations. The earli-est viral-induced alterations in BEK cells wereseen about 24 h after the initiation of infection.At this stage, the most obvious cytoplasmicalterations included a marked increase in thenumber of free ribosomes, an accumulation ofglycogen particles, and a dilated rough endo-plasmic reticulum with a low electron-densecontent. In some cells, multicentric stocks ofGolgi cisternae and scattered vesicular bodies ofboth low and high electron density were ob-served.At about the same time, aggregates of fila-

mentous structures were observed in the vicin-ity of the nucleus. These filaments, measuringabout 16 to 18 nm in diameter, were similar inappearance to the filamentous structures thathave been described in cells infected with vi-ruses of the parainfluenza-Newcastle disease-measles group and generally termed viral nu-cleocapsids (6, 8, 9, 16, 18). Extensive accumu-lations of nucleocapsids were occasionally seenin the cytoplasm of cells infected with PI-3V at24 h, but this was more frequent and marked by48 and 72 h. In some instances, masses of nu-cleocapsids were observed filling a large portionof the cytoplasm of infected cells (Fig. 4A).

(ii) Viral assembly and release. Althoughvirus buddings were observed in cells by 24 and48 h after infection, numerous budding proc-esses of virus particles were noted at the plasmamembrane ofBEK cells by 72 h (Fig. 4A). Thesebuds developed into round, oval, or elongatedforms, either free from the cell body or in theprocess of protruding from the cell membrane(Fig. 4B, C). The diameters of the round parti-cles and elongated forms ranged from 120 to 200nm, and both types had a unit membrane withan external coating that was considered to

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FIG. 2. Light micrograph of BEK cells infected with PI-3V. (A) Twenty-four hours after virus inoculation.Note granular inclusions (arrows) in the cytoplasm. (B) Seventy-two hours after virus inoculation the inclusionscoalesced into larger bodies (arrows). Hematoxylin and eosin. x1,400.

correspond to the spike projections (Fig. 4B, C).Beneath the viral envelope lay the nucleocap-sids which, in cross-section, appeared as ahollowed ring structure with the diameter rang-ing from 16 to 18 nm. In some instances, thenucleocapsid ribbons were seen parallel to theviral envelope.Two round forms of virus particles, light (L)

and dark (D), were usually distinguishable bythe arrangement and concentration of nu-cleocapsids within their envelopes and by theelectron density of the entire body. The Lparticles usually had a regular distribution ofnucleocapsid profiles immediately beneath themembrane envelope and an electron-translu-cent center. Possibly, some of the L particleswere the cross-sections of the elongated forms.

The D particles had tightly packed, randomlycoiled electron-dense nucleocapsids and werevaried in size but generally were larger than theL particles. Frequently, D particles were foundto be enclosed within vesicles of varying size inthe cytoplasm of cells that appeared to be in theadvanced stage of infection (Fig. 5).Virus infection in mitotic cells. Intracyto-

plasmic inclusion bodies were observed with alight microscope in many mitotic cells (Fig.6A-C). These inclusions were irregular, variedin size, and frequently appeared at the two endsof a dividing cell (Fig. 6A, B).

Forty-one mitotic cells were examined ultra-structurally, and the following characteristicfeatures were commonly observed. (i) Thesecells contained aggregates of viral nucleocapsids

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FIG. 3. Fluorescence micrographs of BEK cells infected with PI-3V. (A) Intracytoplasmic inclusions detectedas small granules surrounding the nuclei of more than 90% of cell population by day 2 postinoculation. (B)Larger inclusions seen at day 3 postinoculation. (A) x360; (B) x1,400.

that were often scattered in the cytoplasm butnot associated with chromatin materials. (ii)Some cells were actively producing virus parti-cles by a process identical to those of restingcells (Fig. 4A, 6D). Numerous virus buddingforms with various shapes and sizes projectedfrom a single mitotic cell in a given section (Fig.6D). (iii) When organelles, including the mito-chondria, rough endoplasmic reticulum, Golgicomplex, and general cytoplasmic matrix, werecompared with those of cells without virusinfection, it appeared that these mitotic cellswere not degenerating by fine structure criteria(Fig. 7).Virus structure and virions from rate-

zonal centrifugation preparations. Negative-contrast preparations of the resuspended pelletsfrom infected culture fluid contained pleo-morphic, round, oval, or irregularly shapedvirions, ranging from 200 to 600 nm in diameter.A cluster of helical nucleocapsids was seenwithin intact or partially ruptured particles,which may result from the physical damage ofcentrifugation. The negatively stained nu-cleocapsids were about 18 nm in diameter andhad the characteristic features described forother paramyxoviruses (27).The observations of small-sized virions from

previous viral preparations led us to questionwhether they might represent "incomplete" or

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FIG. 4. Electron micrographs of an infected cell. (A) Note the extensive accumulations of nucleoconsids fillinglarge portions of the cytoplasm (stars), Golgi complex (G), and the virus buddings at the cell surface (arrows).(B, C) Enlarged portions from A. (A) x11.450; (B, C) x33.1(00.

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"defective" virus particles. Our preliminaryresults by rate-zonal centrifugation indicatedthat at least two classes of virions were presentin the viral preparations. Virions with a densityof approximately 1.20 to 1.24 g/cm3 were iso-lated separately from sucrose gradients and

recentrifuged. Two such cycles of recentrifuga-tion were necessary to obtain reasonably homo-geneously sedimenting populations of virions.

Electron microscope examination by a nega-tive-staining method showed that the slowlysedimenting virions had an average diameter of

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FIG. 6. (A, B, C) Light micrographs of infected cells in mitosis, showing viral inclusions varied in size andshape and frequently at the two ends of a dividing cell. (D) Electron micrograph of an infected cell in mitosis.Note the virus buddings at the cell surface, an aggregate of viral nucleocapsids (empty arrows), and thechromatin substances of portions of isolated chromosomes (solid arrows). (A, B, C) x 1,100; (D) x24,600.

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200 nm, and virions sedimenting to the middle ofsucrose gradients had an average diameter of400 nm (Fig. 8). Virions from the bottomsediment were pleomorphic but generallyranged from 400 to 600 nm in diameter.

DISCUSSIONOne of the aims of the present study is to

establish a set of ultrastructural data thatconcern aspects of PI-3V infection in a permis-sive cell culture system of bovine origin. Thesedata will provide a basis for subsequent investi-gations on the pathogenesis of PI-3V infection inthe bovine respiratory tract and on the interac-tion between PI-3V and alveolar macrophages.The term "viral morphogenesis" has often

been used by morphological virologists to de-scribe a sequential development and matura-tion of an assigned virus. In this regard, manyexcellent papers have been published on para-myxoviruses. Our ultrastructural observationson the viral morphogenesis of PI-3V are compa-rable to those for other paramyxoviruses, whichinclude Newcastle disease virus (8), simianvirus 5 (6), parainfluenza type II virus (16), andmeasles virus (18). The intranuclear inclusionsobserved in our BEK cells infected with PI-3Vsupport the view that the intranuclear nu-cleocapsid formation suggests a terminal stageof virus infection (20). Since there is no directevidence that intranuclear tubules participatein the process of viral budding or morphogenesisof measles (18), it is difficult to accept theconclusion made by others (17), based on theintranuclear nucleocapsids, that "the mor-phogenesis of bovine parainfluenza 3 virus moreclosely resembles that of the serologically un-related measles virus than that of serologicallyrelated parainfluenza virus." In addition, longfilamentous virus forms were frequently ob-served in our PI-3V-infected cell cultures,whereas Nakai et al. (18) reported that "no longfilamentous budding particles were observed"in their cell culture system infected withmeasles virus.

It is apparent that factors from virions (infec-tivity titer, virulent or attenuated) as well asfrom cells (species, cell type, primary culture orestablished line) could influence the distribu-tion of nucleocapsids, the budding activity, andthe yield of the virus (7, 8, 14).

Results from the virus growth curve in addi-tion to the present ultrastructural findings indi-cate that, with diluted virus inoculum (104mean tissue culture infective doses per ml), aproductive infection usually occurred in thebovine embryonic cell cultures. Infectious virus

particles were demonstrated as released andcell-associated forms.

In the present ultrastructural study, the darkround particles are probably detached but re-mained as cell-associated virions. Althoughsimilar dark particles enclosed within mem-brane-bound vesicles were seldom described (6,8, 16), their frequent appearance in the presentcell culture system suggests that they probablyrepresent cell-associated forms of PI-3V. It isconceivable that, once virus particles are ac-tually detached from the cell surface, they areno longer topographically related to the cellbody. Thus, considerable numbers of releasedparticles may be no longer demonstrable in theultrathin sections under ordinary procedures.Only those membrane-enclosed virus particlesor particles trapped by the network of cytoplas-mic processes or microvilli are likely preservedin the ultrathin sections.

In ultrathin sections, large numbers of thefilamentous virus forms were observed in theinfected BEK cells. They are assumed to be atransitional form in the process of virus releasein the present study. This assumption is contra-dictory to the generally accepted ideas that theelongated viral elements are another form ofparamyxovirus and that they may be at-tenuated (3, 9) or more virulent (8) forms.Although it was not possible to determinewhether any of the light round forms that layseparate from the cells were actually continuouswith nearby cell membranes, some of them, atleast, are considered to be cross-sectioned formsof filamentous viral elements. Investigations byothers on the virus-cell interaction of a parain-fluenza virus, simian virus 5, indicated thatfilamentous forms were observed only in theultrathin sections and were usually not seen innegative-staining preparations (6). In studies onthe morphogenesis of Newcastle disease virus inchicken embryo, Donnelly and Yunis (8) alsofound that pellets from Newcastle disease virus-infected chorioallantoic fluid contained mostlythe dark, round, or ovoid virus forms. None ofthe virus particles was typically filamentous oras long as those found in the sections. Howe etal. (16) also stated that filamentous forms werenever encountered in their negatively stainedpreparation of type 2 parainfluenza virus. Thus,several possibilities with regard to virus bud-ding and maturation appear to be deduciblefrom previous findings by other (1, 8, 16) andour present observations. (i) The filamentousforms with regularly spaced nucleocapsids be-neath the viral envelopes observed in fixedultrathin sections are not entirely separated

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I.

SFIG. 8. Negatively stained PI-V3 prepared from rate-zonal centrifugation. The virions contain coiled

nucleocapsids and surface or spike projections and have average diameters of 400 nm. x69,300.

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from the cell body and represent transitionalforms of virus budding in action. (ii) Theseelongated forms transform into spherical typeswhen eventually pinched off into culture me-dium or later in the unfixed buffer solutionsused for negative staining. (iii) The previousregularly arranged nucleocapsids become ran-domly distributed within the virus envelope.(iv) The size of these spherical or irregularlyshaped particles in unfixed solutions dependsupon the length of the filamentous forms whenthey finally pinch off from the cell surface. (v) Ifthese viral elements pinch off when they are tooshort, with less than an adequate amount ofviral genome, incomplete or defective virusparticles may result.At the ultrastructural level, our data pro-

vided the first morphological evidence thatmitotic cells infected with PI-3V are activelyengaging in the production of virus particles. Insome infected mitotic cells, despite the presenceof aggregates of nucleocapsids in the cytoplasmand virus buddings at the cell surface, thesecells do not appear to be degenerating byordinary ultrastructural criteria. The presentobservations suggest that PI-3V can infect BEScells, replicate in cells, and transmit the virusgenome to daughter cells during cell divisionwithout the virus going through a surface trans-mission.

Persistent infections of cells in culture havebeen established with a variety of lytic viruses(25). These interactions are characterized bydetection of plaque-forming virus, accompaniedby cell destruction or a continual production ofvirus without massive destruction of cells.Mumps virus infection of human conjunctivacells resulted in infectious progeny and celldestruction. If, however, the growth conditionsfor these cells were such that cells were able todivide, a persistent infection was found (26). Inanimals, persistent or chronic viral infectionsresult in occasional bursts of detectable infec-tious virus as well as in continuous productionof virus (15, 22).Although two density classes of PI-3 virions

were separated by rate-zonal centrifugation inthe present study, the functional significance ofthe slowly sedimenting virions, which had adensity approximately 1.20 mg/cm3, remains tobe determined by further investigation.

ACKNOWLDGMENTSThis investigation was supported by the National Research

Council of Canada (grant A5749) and by the Ontario Ministryof Agriculture and Food.We thank Mary Halfpenny, Paul Coppin, and R. L.

Limbeer for their excellent technical assistance.

LITERATURE CITED

1. Ane, C. 1967. Etude au microscope electronique de lamorphogenese d'un myxovirus parainfluenza 3. J. Mi-crosc. (Paris) 6:31-40.

2. Baldwin, D. E., R. G. Marshall, and G. E. Wessman.1967. Experimental infection of calves with myxovirusparainfluenza 3 and Pasteurella hemolytica. Am. J.Vet. Res. 28:1773-1782.

3. Bang, F. B. 1953. The development of Newcastle diseasevirus in cells of the chorioallantoic membrane as stud-ied by thin sections. Bull. Johns Hopkins Hosp. 92:309-316.

4. Chanock, R. M. 1969. Parainfluenza viruses, p. 434-456.In E. H. Lennette and N. J. Schmidt (ed.), Diagnosticprocedures for viral and rickettsial diseases, 4th ed.American Public Health Association, New York.

5. Collier, J. R. 1968. Pasteurella in bovine respiratorydisease. J. Am. Vet. Med. Assoc. 152:824-828.

6. Compans, R. W., K. V. Holmes, S. Dales, and P. W.Choppin. 1966. An electron microscopic study of mod-erate and virulent virus-cell interactions of the Parain-fluenza virus SV-5. Virology 30:411-426.

7. Darlington, R. W., A. Portner, and D. W. Kingsbury.1970. Sendai virus replication: an ultrastructural com-parison of productive and abortive infection in aviancells. J. Gen. Virol. 9:169-177.

8. Donnelly, W. H., and E. J. Yunis. 1971. The morphogene-sis of virulent Newcastle disease virus in the chickembryo. An ultrastructural study. Am. J. Pathol.62:87-110.

9. Feller, U., R. M. Doughtery, and H. S. DiStefano. 1969.Morphogenesis of Newcastle disease virus in chorioal-lantoic membrane. J. Virol. 4:753-762.

10. Hamdy, A. H., A. L. Trapp, C. Gale, and N. B. King.1963. Experimental transmission of shipping fever incalves. Am. J. Vet. Res. 24:287-294.

11. Heddleston, K. L., R. C. Reisinger, and L. P. Watko.1962. Studies on the transmission and etiology ofbovine shipping fever. Am. J. Vet. Res. 23:548-553.

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