Vol. 36, No. 1INFECTION AND IMMUNITY, Apr. 1982, p. 310-3190019-9567/82/040310-10$02.00/0

Ultrastructural Study of Long-Term Canine Distemper VirusInfection in Tissue Culture Cells

HARASH K. NARANGRegional Public Health Laboratory, Newcastle General Hospital, Newcastle upon Tyne NE4, 6BE, England

Received 8 July 1981/Accepted 4 December 1981

The morphogenesis of canine distemper virus was studied in Vero cell culturesfor 43 days post-inoculation. Active replication of the virus was observed byelectron microscopy and assay from 12 h after inoculation on, and peak produc-tion was observed on days 5, 14, and 22. From day 28 on, constant but smalleramounts of infectious virus were detected. Two ultrastructural types of intracyto-plasmic nucleoprotein filaments were observed; although they first appeared atdifferent times, their subsequent chronological patterns of development were

similar. The cells apparently became free of virus by a mechanism of vacuolation.Intranuclear filaments were seen about day 11 and appeared to increase in numberthereafter, whereas the infectious titer declined. Possible mechanisms of persis-tence are discussed in the light of these findings.

Paramyxoviruses can cause subacute diseasesof the central nervous system, subacute scleros-ing panencephalitis (SSPE) in humans (3, 4), andcanine distemper in animals (10, 22). Measlesvirus, which belongs to this group, may producea slow or persistent infection, as shown byimmunopathological findings (6, 13), and hasbeen isolated from patients with SSPE who havehad childhood measles (5, 7, 11, 17, 18, 20). Themechanism of slow virus infection is not clear.Adams and Bell (1) suggested that after measles,virus infection RNA undergoes a reverse tran-scriptase-mediated change to a DNA form, andthat this change is brought about by coinfectionwith a leukovirus.Paramyxovirus nucleoprotein filaments occur

in large intracytoplasmic and intranuclear aggre-gates. The intracytoplasmic filaments have beendescribed as covered by fuzzy coats which havebeen resolved in a pentagon shape (15), whereasintranuclear filaments do not have fuzzy coats.In short-term (16) and long-term studies of SSPEand measles virus (19), intranuclear inclusionswere observed only in samples from patientswith chronic infections. This paper reports thefirst long-term study of chronic canine distempervirus (CDV) infection of Vero cells to reevaluatethe mechanism of virus persistence.

MATERIALS AND METHODSVirus. The Rockbam strain of CDV was obtained as

"Vaxitas D" vaccine (Tasman Vaccine Laboratory).This strain, which is grown in dog kidney cells, waspassaged in dog kidney cells on four further occasionsand in Vero cells on several occasions in our labora-tory.

Tissue culture technique. Vero cells (an African

green monkey continuous cell line) were obtainedfrom Flow Laboratories, Inc. Bottles (600 ml) wereseeded with 7 x 105 cells. Growth and maintenancemedia were minimal essential medium and Hanksbalanced salt solution containing 10 and 2% fetal calfserum, respectively, plus the following antibiotics:penicillin, 100 ,ug/ml; fungisone, 1 ,ug/ml; and kanamy-cin, 50 p±g/ml. Confluent monolayers were inoculatedwith 3 x 105 PFU of CDV. The virus was allowed toadsorb for 1 h at 37°C before maintenance medium wasadded; the resulting cultures were then incubated at37°C in 5% CO2 in air. CDV-infected cultures weresampled daily for up to 43 days after infection. Unin-oculated cultures were taken as comparable controlson the same days.

Viral assay. For the assessment of the amount offreeinfective virus produced each day, tissue culture fluidwas harvested daily, and the cells were washed threetimes with phosphate-buffered saline and replacedwith an equal volume of fresh maintenance medium.To determine the amount of all associated infectivevirus, I scraped the cells in the same amount ofmaintenance medium after washing them three timeswith phosphate-buffered saline.

Electron microscopy procedure. Cells were removedfrom the bottles after trypsinization, washed in Hankssolution, and pelleted at 250 x g for 15 min. A requiredpart of the pellet was fixed in 4% glutaraldehyde in 0.2M sodium cacodylate solution at pH 7.2 for 1 h,followed by 1% Dalton osmic acid (7) for 2 h. Afterdehydration, the tissue was embedded in Epon. Thinsections were cut with a diamond knife on an Ultra-microtome (LKB Instruments, Inc.). Sections werestained with uranyl acetate or lead citrate or a combi-nation of both (14) and examined in a Philips 300electron microscope.To avoid examining the same cell twice with the

electron microscope, I cut 10 blocks of each specimenand based the results on counting 100 cells and the freevirus particles between the extracellular spaces.

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LONG-TERM CDV INFECTIONVOL. 36, 1982

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FIG. 1. Growth ofCDV in Vero cell cultures whichwere inoculated with 2 x 105 PFU of virus. Thesupernatant fluids (0) and cell-associated virus (A)were assayed as described in the text.

RESULTSViral assay. The replication of small amounts

ofCDV in Vero cells, (Fig. 1 and 2) was detectedas early as 12 h postinfection. Significantamounts (Fig. 1 and 2) of virus were recoveredfrom the tissue culture media up to 28 days

Incubmion period in days

FIG. 2. Continuous production ofCDV nucleopro-tein filaments and virus particles, as determined byelectron microscopy over 43 days. The percentage ofcells containing different types of nucleoprotein fila-ments and virus particles on each day postinfection isshown. Symbols: 0, free and budding particles; A,

cell-associated virus type I; 0, cell-associated virustype II; U, nuclear filaments.

postinfection, and then low titers were observedon the remaining 15 days of the study (Fig. 1 and2). Virus was consistently associated with thetissue culture cells in titers ranging from 10-3 to10-6 (Fig. 1).

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FIG. 3. Electron micrograph of Vero cells at day 4 postinfection. Note that every cell contains at least one

CDV nucleoprotein inclusion body (arrows). x3,000; bar = 3,000 nm.

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--1., .- _.44tFIG. 4. High-magnification micrograph of a single infected Vero cell showing two intracytoplasmic inclusions

of CDV nucleoprotein filaments (arrows). x 11,000; bar = 1,000 nm.

Cytoplasmic changes. On examination of sec-tions after 12 h of incubation of the virus, about1 of 10 inoculated cells showed small intracyto-plasmic CDV nucleoprotein filaments. Some ofthese infected cells also showed budding parti-cles on the surface. After 25 h of incubation, thenumber of infected cells increased, and by day 4,almost every cell cross section contained at leastone intracytoplasmic inclusion of nucleoproteinfilaments (Fig. 3 and 4). Longitudinal and trans-verse sections of the CDV nucleoprotein fila-ments showed an electro-dense core coated withfuzzy material, as described previously for mea-sles virus (15). Cross sectioned, the central coreof the nucleoprotein measured about 16 nm.The number of budding particles seen also

increased with the incubation period and wasmaximal by day 5 (Fig. 2). The number ofparticles seen correlated with the viral assay(Fig. 1 and 2).From days 5 and 6 on, some of the infected

cells developed intracytoplasmic vacuoleswhich increased in size and were present in themajority of cells by day 8 postinfection (Fig. 5).

As the size and number of vacuoles in eachinfected cells increased, the virus filamentousinclusions were retained the cytoplasmic strands(Fig. 5), which eventually broke off from themain cell body: the cells thus became free of theinclusions (Fig. 6). At this stage, the cells be-came much smaller, about two-thirds the normalsize, and the number of cells containing the virusnucleoprotein inclusions decreased. The cellsthen increased in size, and, after division, con-fluent monolayers were produced. The secondcycle started when these inclusion bodies reap-peared in most of the cells, and peaks occurredon about days 14 and 22 (Fig. 2). From day 22on, the number of these inclusion bodies de-creased.On the day 7 postinfection, besides the typical

CDV nucleoprotein inclusion body describedabove (Fig. 3 through 5), another type of fila-mentous intracytoplasmic inclusion body ap-peared in some of the cells (Fig. 7A and B). Afew of the cells contained both types of inclu-sions. The number of cells containing the lattertype of inclusion reached a maximum by about

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LONG-TERM CDV INFECTION 313

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FIG. 5.Electron micrograph of a Vero cell at day 8 postinfection showing extensive vacuolation ofcytoplasm. Note that the viral material is being held by cytoplasmic strands (arrow). x6,600; bar = 1,500 nm.

day 10 (Fig. 2). At low magnification, this inclu-sion body appeared granular (Fig. 7A) andstained less intensely. At higher magnification,the nucleoprotein filaments were more looselypacked and had ill-defined outlines, comparedwith the first type of inclusion (Fig. 8A and B).The filaments measured about 14 nm in diameter(Fig. 7B). This type of inclusion, as visualizedby electron microscopy, showed peaks on days10 and 18 postinfection (Fig. 2).Nuclear changes. Nuclear changes became ap-

parent with the appearance of intranuclear fila-ments on about day 11. These changes beganwith the transformation of nucleoli into granularreticulated masses or several small clumps ofgranular material. The longer the incubationperiod, the more filaments were contained in theinfected nuclei. (Fig. 2). Each filament appearedto be made by a helical tube about 16 nm indiameter and a clear central core (Fig. 9). Theseintranuclear tubes were distinguishable fromtheir intracytoplasmic counterparts by their lackof an outer pentagonal coat (Fig. 9).

DISCUSSION

The results of the present experiments showthat CDV replicated in Vero cells throughout the43 days of the study. Synthesis of infectiousvirus was continuous; peak production on cer-tain days indicated that production was cyclical.The cells appeared to become free of virus aftervacuolation. The virus was continuously detect-ed in the supernatant fluid, although on certaindays, titers were low. No ultrastructural differ-ences were seen in the old Vero cell cultures, thevirus nucleoprotein present in them, or thestructure of the budding virus particles.

It has been reported that measles virus showsvarying degrees of persistence and latency afterprimary infection. Raine et al. (19), who havestudied long-term measles infection in culturesof hamster dorsal root ganglion, did not observecycles in the production of infectious measlesvirus or cytoplasmic nucleoprotein, but they didshow that synthesis of infectious measles viruscontinues until day 25 postinfection, as com-

VOL. 36, 1982

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FIG. 6. Electron micrograph of a Vero cell at day 9 postinfection showing a few dense bodies (arrows). Notethat there is no filamentous material in the cell. x11,800; bar = 1,000 nm.

pared with day 43, when, in the present study,observations were discontinued.The results of the present long-term tissue

culture study demonstrate how cells manage tosurvive CDV infection. This peculiar method ofshedding virus by vacuolation has not beenobserved for any other virus. It is difficult to saythat these cells become completely free of virus,but it is certain that they divide because the cellsheet becomes confluent again.Raine et al. (19) explained that the low titers in

the tissue culture fluid after day 26 postinfectionare due to the trapping of the virus particleswithin the interstices of the tissue. This phenom-enon was not observed for CDV. However, veryfew cells contained CDV nucleoprotein, and inonly a few of the cells were budding particlespresent.

Although a number of hypotheses have beenproposed to explain the mechanism of persist-ence of measles virus in tissue culture andanimal and human brain tissue (1, 3, 5), thesehypotheses are not supported by experimentaldata. The present ultrastructural studies, com-bined with viral assay, demonstrate that virusmultiplication slows down during a long incuba-

tion period, and a state of balance is achievedbetween virus multiplication and survival of thecell. The results of the viral assay of fresh Verocells did not indicate that the virus was growingmore slowly; therefore, it seemed that the virushad not changed its character to a slow type bythis time. It seems likely that the cells acquired amechanism of self-protection. There are variouspossibilities, although for tissue culture, one candiscount involvement of antibody or cell-mediat-ed immunity. It is possible that in tissue culture,the infected cells repeatedly free themselves ofvirus, divide, and are reinfected so that a cellpopulation is selected which is more resistant toreinfection by the virus.The second possibility involves the produc-

tion of interferon (10). It has been shown thatthis class of substances is produced by cells intissue culture or animals infected by almost anyanimal virus containing either DNA or RNA.Wong et al. (23) showed that primary Africangreen monkey cell cultures infected at a low orhigh multiplicity of infection produce small orlarge amounts of interferon, respectively, andthat after infection at a high multiplicity, inter-feron production continues as long as the cells

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FIG. 7. Electron micrograph of a Vero cell at day 7 postinfection showing type II CDV nucleoproteinfilaments in the cytoplasm. (A) x11,000; bar = 1,000 nm. (B) x21,000; bar = 500 nm.

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VOL. 36, 1982 LONG-TERM CDV INFECTION 317

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FIG. 9. Electron micrograph of a Vero cell at day 14 postinfection showing intranuclear filaments. Note theabsence of an outer pentagonal coat (arrows). x82,000; Bar = 250 nm.

survive. The authors suggested that persistentinfection of African green monkey cell culturesby rubella virus is associated with persistentactivity of the interferon system.Of the two types of filamentous inclusions

seen on various days, type I, which appearedfirst, ultrastructurally resembled in shape andsize the inclusions reported in previous studies(9, 12, 22).The second filamentous inclusion, type II,

which appeared on about day 7, has not beendescribed before. The filaments, although slight-ly smaller in diameter than those of type I, alsoappeared to be loosely packed. On the basis ofultrastructure, they can be identified as nucleo-protein filaments. It is quite possible that theCDV used in the present study was a mixture oftwo strains with different incubation periods.Recently, Allen et al. (2), using an experimentalhamster model, isolated large and small plaque-forming strains from Onderstepoort CDV, but asyet, these researchers have not demonstratedthe ultrastructural features of the strains.The intranuclear aggregates of filamentous

material which appeared late on about day 11

persisted throughout the entire subsequent peri-od of infection. The late appearance of intranu-clear filaments also occurs in measles virus, asdescribed by Raine et al. (19). It is important topoint out that when the number of intracytoplas-mic inclusion bodies and the viral titer weredecreasing, the number of nuclei containing in-tranuclear filaments were increasing. I havepreviously shown (15) that the intranuclear fila-ments lack the fuzzy "m" protein coat; there-fore, it appears that these filaments representincomplete nucleoprotein.

LITERATURE CITED

1. Adams, D. H., and T. M. Bell. 1976. The relationshipbetween measles virus infection and subacute sclerosingpanencephalitis (SSPE). Med. Hypotheses 2:55-57.

2. Allen, I.V., L. Cosby, and S. Pressdee. 1981. Experimentaldistemper in hamsters. Neuropathol. Appl. Neurobiol.7:245.

3. Byington, D. P., and K. P. Johnson. 1972. Experimentalsubacute sclerosing panencephalitis in the hamster: corre-lation of age with chronic inclusion-cell encephalitis. J.Infect. Dis. 126:18-26.

4. Byington, D. P., and K. P. Johnson. 1973. Subacutesclerosing panencephalitis (SSPE) agent in hamsters. II.The neuropathology of acute and chronic infections. Exp.

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LONG-TERM CDV INFECTION 319

Mol. Pathol. 18:345-356.5. Chen, T. T., I. Watanabe, W. Zeman, and J. Mealey. 1969.

Subacute sclerosing panencephalitis: propagation of mea-sles virus from brain biopsy in tissue culture. Science20:1193-1194.

6. Connolly, J., I. V. Allen, L. J. Hurwitz, and J. H. Millar.1967. Measles virus antibody and antigen in subacutesclerosing panencephalitis. Lancet i:542-544.

7. Dalton, A. J. 1955. Chrome-osmium fixative for electronmicroscopy. Anat. Rec. 121:281.

8. Horta-Barbosa, L., C. A. Fuccillo, J. L. Sever, and W.Zeman. 1969. Subacute sclerosing panencephalitis-isola-tion of measles virus from a brain biopsy. Nature (Lon-don) 221:974.

9. Imagawa, D. T. 1968. Relationships amoung measles,canine distemper and rinderpest viruses. Prog. Med.Virol. 10:160-193.

10. Isaacs, A., and J. Lindermann. 1957. Virus interference. I.The interferon. Proc. R. Soc. London B147:258-267.

11. Katz, M., S. Oyanagi, and H. Koprowski. 1969. Subacutesclerosing panencephalitis: structures resembling myxovi-rus nucleocapsids in cells cultured from brains. Nature(London) 222:888-890.

12. Koestner, A., and J. F. Long. 1970. Ultrastructure ofcanine distemper virus in explant tissue cultures of caninecerebellum. Lab. Invest. 23:196-201.

13. Kolar, O., and W. Zeman. 1967. Immunoelectrophoreticchanges of serum IgG during subacute inflammatory anddemyelinating diseases of the central nervous system. Z.Immunitaetsforsch. Allerg. Klin. Immunol. 134:267-275.

14. Narang, H. K. 1973. Virus-like particles in natural scrapieof the sheep. Res. Vet. Sci. 14:108-110.

15. Narang, H. K. 1981. Comparative morphology of measlesvirus and paramyxovirus-like tubules in multiple sclerosisusing ruthenium red stain. Neuropathol. Appl. Neurobiol.7:411-420.

16. Oyanagi, S., V. ter Meulen, M. Katz, and H. Koprowski.1971. Comparison of subacute sclerosing panencephalitisand measles viruses: an electron microscope study. J.Virol 7:176-187.

17. Oyanagi, S., V. ter Meulen, D. Muller, M. Katz, and H.Koprowski. 1970. Electron microscopic observations insubacute sclerosing panencephalitis brain cell cultures:their correlation with cytochemical and immunocytologi-cal findings. J. Virol. 6:370-379.

18. Payne, F. E., J. V. Baublis, and H. H. Itabashi. 1969.Isolation of measles virus from cell cultures of brain froma patient with subacute sclerosing panencephalitis. N.Engl. J. Med. 281:585-589.

19. Raine, C. S., A. L. A. Feldman, R. D. Sheppard, and M. B.Bornstein. 1971. Ultrastructural study of long term mea-sles infection in cultures of hamster dorsal-root ganglion.J. Virol. 8:318-329.

20. ter Meulen, V., D. Muller, M. Katz, M. Y. Kackell, and G.Joppich. 1970. Immunohistological, microscopical andneurochemical studies on encephalitides. IV. Subacutesclerosing (progressive) panencephalitis. Histochemicaland immunohistological findings in tissue cultures derivedfrom SSPE brain biopsies. Acta Neuropathol. 15:1-10.

21. Wilner, B. I. 1969. Myxoviruses, p. 61-69. In B. I. Wilner(ed.), A classification of the major groups of human andother animal viruses, 4th ed., Burgess Publishers, Minne-apolis, Minn.

22. Wisniewski, H., C. S. Raine, and W. J. Kay. 1972.Observations on viral demyelinating encephalomyelitis.Canine distemper. Lab. Invest. 26:589-599.

23. Wong, K. T., S. Baron, and T. G. Ward. 1967. Rubellavirus: role of interferon during infection of African greenmonkey kidney tissue cultures. J. Immunol. 99:1140-1149.

24. Zhdanov, V. M. 1975. Integration of viral genomes. Na-ture London 256:471-473.

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