INFECTION AND IMMUNrry, July 1974, p. 251-256Copyright © 1974 American Society for Microbiology

Vol. 10, No. 1Printed in U.S.A.

Growth of Murine Cytomegalovirus in a Heterologous CellSystem and Its Enhancement by 5-Iodo-2'-Deoxyuridine

G. PLUMMER' AND C. R. GOODHEART

Department of Microbiology, Loyola Medical School, Maywood, Illinois 60153, and BioLabs, Inc., Northbrook,Illinois 60062

Received for publication 11 March 1974

Mouse cytomegalovirus replicated in rabbit kidney cultures, a cell system ofnonrodent origin. However, the sensitivity of these cultures, and the yields ofvirus therefrom, were lower than those of mouse cultures. Although a cytopathiceffect developed in rabbit kidney cultures inoculated with sufficient amounts ofthe virus, such cultures were unsatisfactory for plaque assay. This was also truewhen rabbit fibroblast cultures were used, even though the murine cytomegalovi-rus replicated much better in mouse fibroblasts than in mouse kidney cultures,the latter of which contained extensive areas of epithelial cells. Viral growth inrabbit kidney cells was considerably enhanced when those cells had beeninitiated and grown in the presence of 5-iodo-2'-deoxyuridine; not only were theviral titers increased, but also the clarity and distinctness of the inclusion bodies.

The taxonomic delineation of the cytomegalo-viruses from the other herpesviruses is of ques-tionable validity. There has been a tendency toseparate them as a distinct subgroup because oftheir slow replication, their attainment of lowtiters of infectivity in the fluid of infectedcultures, and their specificity for the cells oftheir host species. But there is indication thatherpesviruses with these properties form theend of a spectrum rather than a clearly distinctgroup, with viruses such as pseudorabies virusand the herpes simplex viruses at the other endof the spectrum. Also, it is by no means clearwhether the viruses at one end of such aspectrum are phylogenetically closer to oneanother than they are to the viruses at the otherend.

Specificity for the cells of the natural host iscertainly becoming questionable as a distin-guishing characteristic of the "cytomegalovi-ruses." Human cytomegalovirus can produce acytopathic effect and viral antigen in guinea pigcells or in bovine cells, although viable viralprogeny has not been produced (1, 10), andevidence is already available for the replicationof mouse cytomegalovirus in cells of nonmurineorigin (5, 7).We have studied mouse cytomegalovirus-

one of the "classical" cytomegaloviruses-incell cultures from rabbits. We have noted con-siderable enhancement of viral growth by priortreatment of the cells with 5-iodo-2'-deoxyuri-

I Deceased.

dine (IUdR), a procedure which St. Jeor andRapp (8, 9) found to increase the permissivenessof human cells to human cytomegalovirus. Wedo not mean to imply, however, that the presentdata indicate that IUdR enhancement is adistinguishing characteristic of "cytomegalovi-ruses"; subsequent work may show similar en-hancement of other herpesviruses.

MATERIALS AND METHODS

Viruses. The Smith strain of mouse cytomegalovi-rus was used in all the studies except where theFrench strain is specified. This latter virus (7) hadbeen passaged extensively in laboratory mice (Mus)and in tissue cultures from such mice, although priorto such passage the original agent was isolated fromthe French field mouse, Apodemus sylvaticus; thevirus had not been passaged in our laboratory, ourstock having been grown directly from the materialreceived from France. The actual origin of the Frenchvirus is somewhat open to question in view of itsextensive passage in laboratory mice, but it at leastrepresents a different isolate or strain of mousecytomegalovirus even if its origin be laboratory mice.Growth curves. Growth curves were done with the

Smith strain; the inoculum had been grown, andpassed 10 times, in rabbit kidney cultures. Primarykidney cultures were prepared in 60 by 15 mm Falconplastic petri dishes from kidneys of newborn rabbits ornewborn mice; Eagle medium with 10%c fetal calfserum was employed. These cultures consisted ofareas of epithelial cells interlaced with strands offibroblasts. Mouse embryos were used for the prepara-tion of cultures which appeared to be almost entirely

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PLUMMER AND GOODHEART

fibroblastic. The viral inoculum for the growth curveswas either 10; or 104 plaque-forming units (PFU)per petri dish. After 1 h of absorption at 37 C, the cellswere rinsed three times and then fed with 3 ml ofEagle medium containing 5% fetal bovine serum. Atappropriate intervals, petri dishes were removed,wrapped in Parafilm (American Can Co.), and frozenat - 70 C. Dishes were later thawed, the adhering cellsheet was scraped into the culture fluid, and themixture was then homogenized in a Ten Broeckgrinder. The homogenized cultures were then refrozenin tubes until titration. All plaque titrations weredone in mouse embryo cultures with a 199-methoceloverlay and read under the low power of the invertedmicroscope after 4 or 5 days.To test the effectiveness of IUdR, growth curves

were done in rabbit kidney cultures as describedabove, but in cells initiated and grown on mediumthat contained 100 ug of IUdR per ml; from the samepair of kidneys, normal cultures (no IUdR) wereprepared for the control growth curve. When thesecultures were confluent, at 6 or 7 days, they werewashed three times in Eagle medium, and parallelgrowth curves were commenced in the IUdR-treatedand nontreated cultures. No IUdR was, of course,present in the medium during the viral growth.

RESULTSGrowth in rabbit cells. Inoculation of mouse

cytomegalovirus in relatively small amounts(i.e., less than about 103 PFU, as measured inmouse fibroblast cultures) into tube or petri-dish cultures of rabbit kidney produced nocytopathic effect (CPE). If larger amounts wereinoculated, CPE developed after a period ofabout 7 days; this CPE could be transmittedfrom culture to culture provided the inoculumwas not diluted or was diluted only slightly. TheFrench isolate behaved the same as the Smithstrain in these respects.Comparative growth curves were therefore

done in rabbit kidney cultures, mouse kidneycultures, and mouse embryo cultures. Viruswhich had been passed 10 times in rabbitkidney cultures was used in each case as thevirus inoculum. As can be seen in Fig. 1, viralreplication in the rabbit cells is markedly lessthan in the mouse cultures.The mouse embryo cultures, which appeared

to consist entirely of fibroblasts, supported viralgrowth better than did the mouse kidney cul-tures, which consisted of areas of epithelial cellsextensively interlaced with strands of fibro-blasts. Figures 2A and B demonstrate the pref-erential development of CPE in the fibroblastsof a mouse kidney culture rather than in theepithelial cells. Figure 2C shows a rabbit kidneyculture at a similar number of days afterinfection; even though there are what seem to

be extensive areas of fibroblastic cells, no CPEis apparent.The mouse cytomegalovirus could not be

successfully plaque-assayed in rabbit kidneycultures, because not only did CPE developmuch more slowly than in mouse cells but the"plaques" varied widely in size and shape; itwas impossible to identify and count themaccurately, even under the low power of themicroscope. In contrast, the plaques whichdeveloped in mouse cultures could be readilydiscerned from one another and achieved adiameter of about 0.4 mm by 4 days post-inocu-lation.The attempts to titrate the virus in rabbit

cultures gave the marked impression that a10-fold dilution of the inoculum resulted inmore than a 10-fold decrease in the area of cellsheet involved in the CPE and in the number of"plaques." No precise dose-response curvecould be plotted because of the difficulty incounting the foci of CPE.

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FIG. 1. (A) Growth curves of mouse cytomegalovi-rus in primary cultures of mouse kidney and rabbitkidney. The viral inoculum was 1095 PFU per petri-dish culture. (B) Growth curves in mouse fibroblasts(i.e., the mouse embryo cultures) and rabbit kidney.The viral inoculum was 104- PFU per petri-dishculture. All of these growth curves represent com-bined virus from the culture fluid and the homoge-nized cells. The titers plotted are per 0.2 ml of thetotal 3 ml of homogenate.

252 INFECT. IMMUNITY

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FIG. 2. (A,B) Mouse kidney cultures stained with hematoxvlin and eosin 4 days after receiving a low inoculumof mouse cytomegalovirus. The CPE is largely confined to the fibroblasts. (C) Rabbit cultures similarly infectedand stained show no CPE after 4 days even though extensive regions offibroblasts are present in the culture.

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PLUMMER AND GOODHEART

In view of the apparent preference by mousecytomegalovirus for mouse fibroblasts and byhuman cytomegalovirus for human fibroblasts,rather than for epithelial cells, an attempt wasmade to assay the mouse virus in rabbit fibro-blast cultures prepared from lungs or skin ofvery young animals. The results were no differ-ent from those in rabbit kidney cultures. Onceagain, the areas of CPE were slow to develop,were irregular in shape, and seemed to be of alesser number than was obtained in mousecultures receiving a similar virus inoculum.Enhancement by IUdR. As can be seen in

Fig. 3, the mouse cytomegalovirus multipliedconsiderably better in rabbit kidney cultureswhich had been initiated and grown in thepresence of 100 gg of IUdR per ml prior to theinoculation of the virus. Similar results wereobtained in each of three such experiments.Even though the IUdR-treated cultures, as wellas the nontreated cultures, appeared to beconfluent at the time of virus inoculation, thecell counts in the treated cultures were between38 and 46% those of the untreated cultures. Ifone wished to compare the virus yields per givencell population, the IUdR virus titers wouldneed to be increased 0.4 log10, although theresults shown in Fig. 3 have not been so ad-justed.

Prior treatment of the rabbit kidney cellswith IUdR appeared also to enhance the clarityof the intranuclear and cytoplasmic inclusionbodies. In the untreated rabbit cultures, inclu-sions were difficult to discern, even though thecells were involved in CPE and many of thenuclei were elongated and curved in the charac-teristic fashion. However, in the IUdR-treatedcells; inclusions were easily seen when stainedwith hematoxylin and eosin and were remarka-bly similar to those caused by human cytomega-lovirus in human cells; Fig. 4B and C showmouse cytomegalovirus inclusions in IUdR-treated rabbit cells. For comparison, inclusionbodies formed in mouse fibroblasts are shownin Fig. 4A.

DISCUSSIONThe growth of the mouse cytomegalovirus in

rabbit cell cultures, even though somewhattardy, means that cell specificity cannot beused as a delineating characteristic of the "cyto-megaloviruses"; this is emphasized by the ap-parent specificity for cells of their natural hostsof the dog and cat herpesviruses, two viruseswhich replicate rapidly and which on that basiswould not be labeled cytomegaloviruses. Butwhen an attempt is made to correlate the

deoxyribonucleic acid (DNA) densities of theherpesviruses with the number of different spe-cies of tissue culture cells in which each herpes-virus will readily multiply, then a correlationbecomes tentatively evident (Fig. 5). A greaterproportion of viruses with DNA of a highdensity seems to replicate easily in a wide rangeof cell species than of viruses with a low DNAdensity. We wish to emphasize the very specula-tive nature of Fig. 5 and of the correlation whichit is meant to convey. Although there is littledoubt that viruses such as the herpes simplexviruses, pseudorabies virus, infectious bovinerhinotracheitis virus, and equine herpesvirustype 1 grow readily in a broad range of tissueculture species without prior adaptation, thereare a number of viruses for which we know theDNA density but for which we have poorknowledge of their behavior in tissue cultures;thus, the cottontail-rabbit herpesvirus is re-ported to be specific for the cells of rabbits (4),although we have evidence that it will grow inhamster cells but not in monkey cells. We areunable to define at this stage precisely whatshould be meant by "ready growth in a broadrange of tissue culture species," but we feel,nevertheless, that the possible correlation withDNA density should be pointed out. Figure 5also begs the question of whether the herpes-

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FIG. 3. Growth curves of mouse cytomegalovirus inrabbit kidney cultures pretreated with IUdR (A) anduntreated (0). The viral inoculum was 104. perpetri-dish culture. Each point represents combinedvirus from culture fluid and homogenized cells. Thetwo IUdR points and two control points shown at mostof the sampling times represent the titers of duplicateIUdR plates and duplicate control plates harvested atthose times. The IUdR-treated cultures contained2.5-fold less cells than did the control cultures. Tomake the results comparable per given cell populationthe IUdR points would need to be increased by 0.4log, but this has not been done in the above graph.

254 I NFECT. IMMUNrrY

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PLUMMER AND GOODHEART INFECT. IMMUNITY

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FIG. 5. The DNA densities of the various herpes-viruses are compared with the percentage of viruses ina given DNA density range which will readily grow in

a wide variety of tissue culture species. The viralinitials indicate the following viruses (in some in-stances, when confusion may arise as to the identity ofthe virus concerned, the provisional label recom-

mended by the Herpesvirus Study Group 13] is usedalone or is given in parentheses): CCV, channelcatfish virus (catfish herpesvirus 1); CRV, cottontail-rabbit herpesvirus; DoHV, dog herpesvirus; EBV,Epstein-Barr virus; Eql, equine herpesvirus 1; Eq2,equine herpesvirus 2; Eq3, coital exanthema virus

(equine herpesvirus 3); FeHV, feline rhinotracheitisvirus (feline herpesvirus 1); FV4, frog virus 4 (ranidherpesvirus 2); GPHV, an isolate ofguinea pig herpes-virus; HSVI herpes simplex virus type 1; HSV2,herpes simplex virus type 2; HuCMV, human cyto-megalovirus; HVT, herpesvirus of turkeys; IBR, infec-tious bovine rhinotracheitis virus; ILT, infectiouslaryngotracheitis virus of chickens; Lucke, ranid her-pesvirus 1; MDV, Marek's disease virus; MoCMV,mouse cytomegalovirus; PsR, pseudorabies virus;RaCMV, rat cytomegalovirus; RhCMV, an isolate ofcytomegalovirus from the rhesus monkey; SA6, theSouth African isolate of Cercopithecid herpesvirus 2;SpMV, a herpesviral isolate from the spider monkey;SqMVI, squirrel monkey herpesvirus (Cebid herpes-virus 1); SqM2, herpesvirus saimiri of the squirrelmonkey (Cebid herpesvirus 2); VZV, varicella-zostervirus.

viruses fall into four natural groups on the basisofDNA density; Nahmias (6) has also suggestedthe possible subdivision of the herpesviruses on

the basis of DNA density. But only when theDNA densities of many more herpesviruseshave been measured will it be unequivocally

clear if the four clusters remain discrete fromone another or if they merge together.The enhancement of viral replication in rab-

bit cells by pretreatment of the cells with IUdRis in harmony with the observations of St. Jeorand Rapp (8, 9) on the increased permissivenessof human cells to human cytomegalovirus, whenthose cells were pretreated with IUdR. Theysuggested that the IUdR prevented, or reduced,the formation of viral inhibitors by the cells; wehave no better hypothesis to offer.

ACKNOWLEDGMENTS

This investigation was supported by grants from the BrushFoundation and the National Science Foundation (GB-20250)and by contract no. N01-CP-22068 within the Virus CancerProgram of the National Cancer Institute.We thank Ann Ingerson for technical assistance.

LITERATURE CITED

1. Fioretti, A., T. Furukawa, D. Santoli, and S. A. Plotkin.1973. Nonproductive infection of guinea pig cells withhuman cytomegalovirus. J. Virol. 11:998-1003.

2. Goodheart, C. R., G. Plummer, and J. L. Waner. 1969.Density difference of DNA of human herpes simplexviruses, type 1 and 2. Virology 35:473-475.

3. Herpesvirus Study Group, International Committee forthe Nomenclature of Viruses (Chairman, B. Roizman).1973. Provisional labels for herpesviruses. J. Gen. Virol.20:417-419.

4. Hinze, H. G. 1971. New member of the herpesvirus group

isolated from wild cottontail rabbits. Infect. Immunity3:350-354.

5. Kim, K. S., and R. I. Carp. 1971. Growth of murinecytomegalovirus in various cell lines. J. Virol.7:720-725.

6. Nahmias, A. J. 1972. Herpesviruses from fish to man-asearch for pathobiologic unity, p. 153-182. In H. L.loachim (ed.), Pathobiology annual. Appleton-ten-turv-Crofts. New N'ork.

7. Raynaud, J., P. Atanasiu, C. Barreau, and M. Jahkola.1969. Adaptation d'un virus cytomegalique provenantdu mulot (Apodemus sylvaticus) sur differentes cel-lules heterologues, y compris les cellules humaines. C.R. Acad. Sci. Ser. D 269:104-106.

8. St. Jeor, S., and F. Rapp. 1973. Cytomegalovirus: conver-

sion of nonpermissive cells to a permissive state forviral replication. Science 181:1060-1061.

9. St. Jeor, S., and F. Rapp. 1973. Cytomegalovirus replica-tion in cells pretreated with 5-iodo-2'-deoxvuridine. .J.Virol. 11:986-990.

10. Waner, J. L., and T. H. Weller. 1974. Behavior of humancytomegaloviruses in cell cultures of bovine and ofsimian origin. Proc. Soc. Exp. Biol. Med. 145:379-384.

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