JOURNAL OF BACTERIOLOGYVol. 88, No. 3, p. 709-715 September, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

ELECTRON MICROSCOPY OF LATENT PSITTACOSISVIRUS IN McCOY CELLS

MASAHIRO KAJIMA, NEHAMA SHARON, AND MORRIS POLLARD

Lobund Laboratory, University of Notre Dame, Notre Dame, Indiana

Received for ptiblication 12 March 1964

ABSTRACT

KAJIMA, MASAHIRO (University of Notre I)ame,Notre Dame, Ind.), NEHAMA SHARON, AND MOR-RIS POLLARD. Electron microscopy of latentpsittacosis virus in McCoy cells. J. Bacteriol.88:709-715. 1964.-Replication of psittacosisvirus in McCoy cells was observed periodically byelectron microscopy, and was correlated with theappearance of inclusion bodies in infected cellsstained by acridine orange fluorochrome. Threewell-defined morphological stages characterizedthe replication cycle: (i) noninfectious initialbody, (ii) noninfectious intermediate body, and(iii) infectious mature or elementary body. Whenreplication of virus was interrupted by aminop-terin, the virus failed to develop beyond thenoninfective initial body stage; however, aftertreatment of such cultures with folinic acid, in-termediate and mature virus particles appearedin the cells. Latent psittacosis virus, as describedhere, represented a virus in which the replicationprocess was interrupted at a noninfective (eclipse)stage of the developmental cycle.

The morphology of psittacosis, lymphogranu-loma venereum, trachoma (PLT) viruses, in-cluding their developmental cycle, has beenstudied by many investigators with light andelectron microscopes. Electron microscopicstudies with thin sections of infected tissues werecarried out by Gaylord (1954); Tajima, Nomura,and Kubota (1957); Higashi, Tamura, andIwanaga (1962); Litwin et al. (1961); Arm-strong, Valentine, and Fields (1963); and Mitsui,Fujimoto, and Kajima (1964).

Higashi et al. (1962) observed the firststage of infection: virus particles were adsorbedon the surface of the host cell and then, by aninvagination process, invaded the cytoplasm.Tajima et al. (1957), Armstrong et al. (1963),and _Mitsui et al. (1964) described the virusmatrix appearing first in the cytoplasm of hostcell as a primary component from which viralparticles were produced, but did not describethe first stage of infection. The first part of this

report describes the developmental cycle ofpsittacosis virus (TT strain) in McCoy tissuecells, as examined by electron microscopy and ascompared with the results obtained by fluores-cence microscopy.McCloskey and Morgan (1961) demonstrated

a prolongation of the latent stage of psittacosisvirus in 'L"-cell tissue cultures by withholdingcertain vitamins and amino acids froin the nu-trient fluid. During this latent period, viralinclusion bodies and structures were not detectedby light or electron microscopy. Pollard andSharon (1963) showed that replication of psitta-cosis virus was inhibited at an early stage ifaminopterin (AP) was added to the culturemedium from the time of virus inoculation andthereafter. The AP inhibition could be reversedby addition of folinic acid to the culture medium,and mature, infectious virus p)articles appearedeven after 30 days of latency. The above resultswere determined by acridine orange fluorescencemicroscopy and by biological assay methods.The second part of this report describes the stageat which viral replication was interrupted byaminopterin, and the reversal of the effect byfolinic acid as observed by electron microscopy.

MATERIALS ANI) MIETHODS

Cell cultures and virus inoculation. The generalprocedures involving tissue cultures, virus inocu-lations, virus assay, and fluorescence microscopyhave been described (Pollard and Starr, 1962).McCoy cells were prepared as confluent mono-layer cell cultures in prescription bottles. Dupli-cate cell cultures were )repared on cover slipsin Leighton tubes, with which the infected cellswere examined by fluorescence microscopy. Thenutrient fluid consisted of mixture 199 supple-mented with 0.5% lactalbumin hydrolysate, 5%heat-inactivated calf serum, and 0.1 mg ofstreptomycin sulfate per liter. Each cell culturewas infected by 1.3 X 106 TT strain of psitta-cosis virus particles (Bone- et al., 1952), and

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KAJIMA, SHARON, AND POLLARD

was then incubated at 37 C during the course ofthe experiment.

Information acquired in previous experimentsindicated that addition of aminopterin to cellcultures at the onset of TT infection resulted ininterruption of virus replication (Pollard andStarr, 1962). Therefore, bottle cultures of McCoycells were inoculated with TT suspended innutrient fluid containing 25 ,ug of AP and 100,ug of thymidine per ml. The nutrient fluid wasreplaced every 4 days. Cultures of AP-interruptedpreparations were maintained for 7 days; then,one set of cultures was lysed by two cycles offreezing and thawing, and was assayed for viablevirus. In another set of interrupted cultures, thenutrient fluid was replaced with fresh nutrientfluid containing folinic acid (1 ,ug/ml). A thirdset of cultures served as control. All cultures wereexamined by electron and ultraviolet microscopy,and viable virus was assayed on fresh tissue cul-ture cells (Tanami et al., 1960).

Preparation for electron microscopy. TT-infectedcells were prepared for electron microscopy from10 min to 71 hr after onset of infection at inter-vals of 3 hr. AP-interrupted cultures were pre-pared for electron microscopy after 7 days ofincubation, to permit the virus to reach the lateststage of replication. The AP-treated cultureswhich received folinic acid were prepared forexamination 24 hr after the addition of the latterdrug. Uninfected cells processed from the samelot served as controls. Cells, detached from theglass wall by vigorous agitation with syringeand needle, were sedimented by centrifugation at1,500 rev/min for 5 to 10 min. The packedcells were fixed with 1% osmium tetroxide solu-tion with 0.045 g of sucrose per ml, buffered inVeronal acetate at pH 7.4, according to Caulfield(1957), and embedded in Epon, as described byLuft (1961). Ultrathin sections of embeddedtissues were prepared with a Porter-Blum micro-tome by use of a glass knife. The sections weretransferred to naked copper grids, stained for 1to 3 hr with a saturated solution of uranylacetate (Watson, 1958), and examined with anRCA EMU-3C electron microscope, operatingat 50 kv. The photographs were taken on Kodakprojector slide plates, and were subsequentlyenlarged as positive prints.

RESULTS

Normal cycle of replication. In the course of48 hr after onset of infection, TT virus was ad-

sorbed to the cell membrane. It penetrated into:the cytoplasm by an invagination process (Fig. 1),.and proceeded to replicate through a well-de--fined sequence of three morphological stages.(i) In the first stage of TT replication (12th hr),.the virus particle disintegrated in the cyto-plasm into fibrils (Fig. lb), and was assembledas a viral matrix (Fig. 2) around which a limit-ing membrane appears about 1,000 m, in diam-eter. The matrix constricted away from the outermembrane, thereby creating an inclusion cavity(Fig. 3). The large body was referred to as theinitial body, and corresponded to the ribonucleicacid (RNA)-staining matrix as observed by acri-dine orange fluorescence microscopy. (ii) Each ofthe large initial bodies (1,000 to 800 m,u in di-ameter) divided repeatedly into two smallerinitial bodies by constriction of the outer limit-ing membrane (Fig. 4a, b). The small initialbodies (300 to 500 m, in diameter) showed mar-ginal arrangement of granular material and a,mesh in the center made of fibrous material. Anelectron-dense spot was formed by coagulation ofthe mesh in the center of a fibrous electron-lucidarea (Fig. 4a, c). This small initial body with theelectron-dense spot in the center is referred to asan intermediate body, and corresponded to thetransitional viral inclusion which appeared yel-low-orange by acridine orange fluorescence mi-croscopy. Viral particles at stages i and ii werenot infectious. (iii) The electron-dense centerspot of the intermediate body combined with theperipheral granular material, and from this wasevolved mature virus particles or elementarybodies (250 to 300 m,), each consisting of adense nucleoid which was separated by a spacefrom a distinct virus membrane (Fig. 5a, b), andcorresponded to the green deoxyribonucleicacid (DNA)-staining virus material as observedby acridine orange fluorescence microscopy. Thevirus particles increased in number during thenext 48 hr and were infectious.

Effect of AP on replication of TT virus. Thefate of AP-treated TT virus in McCoy cellsfollowed two pathways, depending on the amountof virus in the inoculum. When 1.6 X 106 in-fectious virus particles were used as the inoculum,the developmental cycle of TT was interruptedcompletely. Only small cytoplasmic inclusionswhich contained few initial bodies (Fig. 7) wereobserved 7 days after inoculation. The structureof each initial body simulated the structuresobserved after 18 hr of propagation in an un-

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FIG. 1. Adsorption and invagination of psittacosis virus into McCoy tissue cell; X 20,000. Insert showsonset of virus disintegration; X 20,000.

FIG. 2. Reticulated matrix of psittacosis virus in cytoplasm of McCoy tissue cell. X 20,000.FIG. 3. Formation of initial body of psittacosis virus in cytoplasm of McCoy tissue cell. X 20,000.

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FIG. 4. Developmlent of smaller initial and intermtediate bodies of psittacosis virus. Note electron-densespot in center of the intermsiediate body. 4a: X 18,000; 4b, 4c: X 30,000.

FIG. 5. Psittacosis incluesion body with initial, intermediate, and mature stages of vir1us; X 17,000. In-sert is a higher magnification of the mature infectious viruis particle; X 90,000.

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FIG. 6. Initial body of psittacosis virus at 18 hr after inoculation into McCoy tissue cell. Virus controlfor Fig. 7. X 19,000.

FIG. 7. Psittacosis virus interrupted by aminopterin at the initial body stage of replication. X 19,000.FIG. 8. Aminopterin-inhibited psittacosis virus with the three stages of replication at 36 hr after addition

of folinic acid to the culture medium. X 19,000.713

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KAJIMA, SHARON, AND POLLARD

TABLE 1. Virus assay after one cycle of replication

Drug treatment No. of particles

Control* .................... 1.3 X 106Medium + APt.................. NegativeMedium + AP + FOAt.......... 7.4 X 105

* Assay result for virus particles per milliliter.t Aminopterin, 25 ,g/ml.I Folinic acid, 1 ug/ml.

interrupted virus replication (Fig. 6). But,when 1.3 X 107 infectious particles were presentin the inoculum, some of the viral inclusionscontained intermediate and elementary bodies.

Effect of folinic acid on AP-interrupted TTvirus. Addition of folinic acid to tissue culturescontaining AP-interrupted TT virus resultedin the appearance of mature, infectious virus inthe cultures within 24 hr. The virus inclusionbody enlarged and showed many intermediateand elementary bodies which were the same sizeand structure as in untreated infected cultures(Fig. 8). The reversal of the effect of AP on TTvirus by addition of folinic acid was confirmedby acridine orange fluorescence microscopy andby virus assay. DNA-staining particles appearedin the cytoplasm of stained cells, and infectivevirus was demonstrated in cell cultures whichhad been assayed in fresh cultures of McCoycells. A comparison of virus production, as

influenced by drug treatment, is indicated inTable 1.

DISCUSSION

The well-delineated morphological sequence

through which TT virus replicated in cells was

based on a synchronous type of propagation ofvirus in tissue culture cells, and on correlation ofthe results with the extensively studied acridineorange fluorochrome microscopy and biologicalassay procedures (Pollard and Starr, 1962).Three replication stages were described: initial,intermediate, and mature virus particles. Onlythe last stage was infectious. It is likely thatthe initial body is made up of DNA fibrils andRNA granules. Binary fission of the maturevirus particle was not observed. In AP-treatedcultures, TT virus growth cycle was interruptedat the noninfectious initial body stage, as ob-served by electron microscopy. This stage cor-

responded to the noninfectious RNA-staining

stage as observed in acridine orange-stainedpreparations and described by Pollard and Starr(1963). TT virus was thus visualized in theeclipse or noninfective stage. After treatment ofAP-inhibited cultures with folinic acid, TT virusresumed replication through the intermediateto the mature stage. Since the roles of AP andof folinic acid in DNA synthesis are known(Friedkin and Roberts, 1956), and since theAP-interrupted stage (the initial body) pre-ceded viral DNA formation, it may be assumedthat the intermediate and mature stages containnewly synthesized viral DNA, whereas the initialbody does not.

Interruption of TT replication by AP in re-lation to inoculum concentration may indicatethat the large amount of virus may contain (i)folinic acid in sufficient concentration to reverseprogressively the effect of AP on immatureparticles, (ii) AP-resistant virus particles inquantity sufficient to be detected by the electronmicroscope, or (iii) particles of different replica-tion speed. Since citrovorum factor (folinic acid)has been detected in purified preparations ofpsittacosis virus (Col6n, 1961), and in TT-in-fected cultures at the last stages of the repli-cation cycle (Pollard et al., 1961; Pollard andTanami, 1962), it is likely that the first alterna-tive is the logical explanation.

ACKNOWLEDGMENTS

Supported by funds from the St. JosephCounty Cancer Society and the National Insti-tutes of Health.

LITERATURE CITED

ARMSTRONG, J. A., R. C. VALENTINE, AND C.FIELDS. 1963. Structure and replication of thetrachoma agent in cell structures, as shownby electron microscopy. J. Gen. Microbiol.30:59-73.

BONEY, W. A., JR., L. C. GRUMBLES, J. P. DELA-PLANE, J. V. IRONS, AND T. D. SULLIVAN.1952. Another agent causing air sac involve-ment in turkeys. J. Am. Vet. Med. Assoc.121:269-270.

CAULFIELD, J. B. 1957. Effects of varying thevehicle for OS04 in tissue fixation. J. Biophys.Biochem. Cytol. 3:827-829.

COL6N, J. I. 1961. Enzymes for formation ofcitrovorum factor in members of the psitta-cosis group of microorganisms. J. Bacteriol.79:741-746.

FRIEDKIN, M., AND D. ROBERTS. 1956. Conversion

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of uracil deoxyriboside to thymidine of de-oxyribonucleic acid. J. Biol. Chem. 220:653.

GAYLORD, W. H. 1954. Intracellular forms ofmeningopneumonitis virus. J. Exptl. Med.100:575-580.

HIGASHI, N., A. TAMURA, AND M. IWANAGA. 1962.Developmental cycle and reproductive mecha-nism of the meningopneumonitis virus instrain L cells. Ann. N.Y. Acad. Sci. 98:100-121.

LITWIN, J., J. E. OFFICER, A. BROWN, AND J. W.MOULDER. 1961. A comparative study of thegrowth cycles of different members of psitta-cosis group in different host cells. J. Infect.Diseases 109:251-279.

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MCCLOSKEY, R. V., AND H. R. MORGAN. 1961.Latent viral infection of cells in tissue cul-ture. VIII. Morphological observation ofpsittacosis virus in L cells. Proc. Soc. Exptl.Biol. Med. 106:85-88.

MITSUI, Y., M. FUJIMOTO, AND M. KAJIMA. 1964.Development and morphology of trachomaagent in the yolk sac cell as revealed by elec-tron microscopy. Virology 23:30-45.

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POLLARD, M., AND Y. TANAMI. 1962. Cytochem-istry of trachoma virus replication in tissuecultures. Ann. N.Y. Acad. Sci. 98:50-61.

POLLARD, M., AND T. J. STARR. 1962. Study ofintracellular virus with acridine orangefluorochrome. Progr. Med. Virol. 4:54-69.

POLLARD, M., AND N. SHARON. 1963. Induction ofprolonged latency in psittacosis infectedcells by aminopterin. Proc. Soc. Exptl. Biol.Med. 112:51-54.

TAJIMA, M., Y. NOMURA, AND Y. KUBOTA. 1957.Structure and development of viruses of thepsittacosis-lymphogranuloma group observedin the electron microscope. J. Bacteriol. 74:605-620.

TANAMI, Y., M. POLLARD, T. J. STARR, AND R. W.MOORE. 1960. Quantitative interaction be-tween psittacosis virus and human cells.Texas Rept. Biol. Med. 18:515-522.

WATSON, M. L. 1958. Staining of tissue sectionsfor electron microscopy with heavy metals.J. Biophys. Biochem. Cytol. 4:475-478.

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