Colorado tick fever virus: An electron microscopic study

VIKOLOGY

36, 28-40

Colorado FREDERICK

(1968)

Tick

Fever

A. MURPHY, -4ND

U.S.

Department Prevention

Virus:

an

Electron

Microscopic

PHILIP H. COLEMAN, ALYSE G. WILLIAM GARY, JR.

of Health, Education, and Environmental

Study Ii. HARRISOX,

and Welfare, Public Health Service, Bureau of Disease Control, ;Vational Communicable Disease Center, At Zanta, Georgia %%‘63

Accepted

February

13, 1968

Colorado tick fever (CTF) virus was observed by ultrathin section and negative contrast electron microscopy. In sections of infected cultured cells and of mouse brain, virus particles (75 rnp in diameter) with electroll dense cores were associated with intracytoplasmic granular matrices, arrays of intracytoplasmic filament,s, and fine kinky threads. Occasionally, virus particles were enveloped by membranes of cytoplasmic organelles. Intranuclear filaments in dense arrays were frequently found in infected cells. In negative contrast preparations the virus was round, 80 rnp in diameter, with regularly spaced surface projections suggestive of cubic symmetry. Partial removal of surface components permitted the observation of an inner capsid 50mr in diameter. CTF virus was directly compared with reoviruses, which in several ultrastructural characteristics it resembles.

membrane filtration (Koprowski and Cox, 1947). In the present investigation, infected cell cultures and brain tissue from nenborn mice were examined by ultrat’hin section and negative contrast electron microscopy in an attempt to characterize CTF virus ultrastructurally and to explore its mode and site of maturat’ion. Direct comparisons were made with reoviruses nhich, in several ultrastructural characteristics, resemble CTF virus.

INTRODUCTIOiV

Colorado tick fever (CT&‘) was recognized as a distinct’ acute, febrile disease of man as a result of evidence presented by Becker in 1930. Florio and his co-workers isolated the causative agent from blood of patients and from the tick Demacentor andersoni, and t’ransmitted the disease to human volunteers and hamst,ers(Florio et al., 1944, 1950). As a result of several invest’igations, especially those of Casals (1961), it has been concluded t’hat the virus of CTF is not! antigenically related to any ot,her known virus. Fulfillment of the criterion of biological t,ransmission between susceptible vertebrat,e hosts by hematophagous arthropods, has served t’o place CTF virus in the arbovirus family. This classification has been strengthened by demonstration of its sensitivity t’o sodium deoxycholate, heat, and pH condit,ions other than near neutrality and to its resistance to the metabolic inhibitors FUdR, BUdR, and actinomycin D (Trent, 1964; Trent and Scott, 1966). The virus has been entimat)edto be 35-50 rnp in size by collodion

MATERIALS

APr’D

METHODS

V+u.ses. The Florio st)rain of CTF virus, used as the prototype in this study, n-as kindly provided by Dr. Carl Eklund, Rocky :\lountain

I,aboratory,

Hamilton,

Rlont’ana.

The virus had been passed30 times in hamsters and 57 times in mice; the last 3 mouse passageswere made at terminal dilution. Seed virus was prepared as a 10% infected newborn mouse brain suspension; this maOerial was confirmed as being CTF by serum neutralization test’ing in mice by Dr. Eklund. In addition, CTF virus isolates of the follo\v28

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ing sources wre provided by Dr. Eklund: a human blood isolate in fourth newborn mouse brain passage; an isolate irOil Demacentor andersoni ticks in second newborn mouse brain passage; and an isolate from blood of a chipmunk in first newborn mouse brain passage. Another human blood isolate \vas provided by Dr. Charles C&her, iSationa1 Communicable Disease Center (SCYDC), ~Ulantn, Georgia, as the original blood and the first newborn mouse brain passage. Heovirus types 1, 2, and 3 were providctl b\- Dr. Paul Feorino, NCDC. Hemaggluti& of reovirw types 1, 2, and :3 n-ere not detected in CTlc virus stocks. h’.cperiwenfaZ anilwals. Litters of Z-day-old ICR strain mice were infected by intracerebra1 inoculation of 0.0% ml of a 1OW dilution of seed virus suspensions representing each of the CTF virus isolates described. Alice were free of signs of intercurrent infections and were kept in cages nith air filters. Brain tissue WIS collected at’ intervals before the appearance of clinical signs of disease and again \vhen animals were moribund (7-S days). cell dfzues. Baby ham&r kidney cells [BIiHZl ((‘-13)], obtained from t’he Amcrican Type Cult.ure Collect,ion (CCL lo), were grown :IS monolayers in a modified Eagle’s medium (Hnlonen et al., 1967) containing 10 % newborn calf serum. Maintenance medium contained 0.5 % fetal calf serum. Human amnion cells (FL line, I:ogh and 1 ,und, 19.57) were grown and maintained on thcl same media. Cell cultures infected ivith CTI; virus or reoviruses were incubated at 35” mtl harvested for ulkathin sectioning at several intervals urltil cytopat8hic effect was ~~IOUIIC~~ (7236 hours in BHX21 and FI, cells) ; preparaGons for negative cont’rast electron microscopy \\-we made from cell cultures she\\-ing advanced cytopathic effect. The latctrt \-irus of BHIC21 cells, described by Thomas et al. (1967), n-as observed in this ccl1 line, but \vas of no consequence in this study. I ‘lfrafh in section electron ~r~io~oscopy. Becalwe of the small size of infant mouse brains and the likelihood that the relat’ively large it~oculum volume delivered intracerebrally \\-ould o\-crride :IIIJ- specificity of infection

ELECTRON

?*IICROSCOPY

29

sites, whole brains, rather than specific parts were randomly cut int’o 1 mma blocks and processed for sectioning. Tissue was fixed for 60 minutes in 2.5 % glutaraldehyde, post,fixed for 30 minutes in 1% phosphat’ebuffered osmium t’et’roxide, dehydrated in a graded ethanol series, and embedded in an Aralditc-Epon mixture (Mollenhauer, 1964). Scraped cell monolayers were pellet’ed by centrifugation at 630 g for 5 minutes and thereafter processed as brain tissue except for a reduction of glut’araldehyde fixation t#ime to 20 minutes. Sections were cut with glass knives and st)ained with lead citrate (Reynolds, 1963). Calibration of magnifications was made from photographs of a 54,864 line per inch diffraction grating replica (Ladd Research Induskies, Inc., Burlington, Vermont) taken at each magnification step used. Negafive contrast electron ~nicroscopy. Carbon-coated grids prepared according to the technique of Simpson and Hauser (1966) were rendered hydrophilic by exposure to ult’raviolet irradiation. Very small quantities of infected mouse brain tissue were t’eased apart in large drops of 2 % sodium silicotungstate st’ain, pH 7.0 (Harrap and Juniper, 1966). Grids were float*ed on the drops and dried by touching to filter paper. Cell cult,ures tvere differentially centrifuged prior to negative staining. Low speed pellets (630 g, 5 minutes) and ultracentrifuge pellets (100,000 g, 3 hours) ncre dispersed in drops of stain and handled as described. nlagnification calibration was also performed at the kilovoltage and magnification st,eps used for phot’ographing negat,ive contrast specimens. Spraying and pseudoreplication t’echniyues were employed but did not yield further resolution of virus structure. RESULTS Thin-Section

Electron

Miwoscopy

In bot’h BHI221 and FI, cells a straightforward progression of cvcnts in the maturation of CTF virus was observed despite an asynchrony of infect’ion in cells. Most extensively studied was t)he prototype Florio strain CTli virus infection of BHK21 cells. However, since no significant variation between isolat,es occurred, other than involve-

30

FIG. 1. BHK21 cell infected velopment of the cytopathic granular matrices (A), virions filaments (D). X 24,000.

MURPHY,

ET

ilL.

with CTF virus (Florio strain) and harvested at a very early stage of deeffect visible by light microscopy. Infection is marked by formatiou of at margins of matrices (B), cytoplasmic filaments (C), and intranuclear

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FEVER

VIRUS:

ment of fewer cells with the direct isolate from human blood and the low passage isolates, findings relate to all similarly. The initial discernible change in cells was the appearance in t’he cytoplasm of uniformly granular areas of moderat’e electron density with definite yet unbounded margins. These areas varied in size from less t,han that’ of mitochondria, to extremes involving large port’ions of the cytoplasm. I\loderat>e numbers of elect’ron dense particles, 45 rnF in diameter, were found scattered throughout these granular matrices; these particles were indistinguishable from nucleoids of virus particles. The progression of infection was marked by the appearance of increasing numbers of virus particles at the periphery of the granular matrices (Fig. 1). These consisted of homogeneous or irregularly stippled round nucleoids, 4n rnp in diameter, surro:mded by an electron lucid

Fro. regularly particles

ELECTRON

MICROSCOPY

31

layer, considered to be the capsid of the virion. The total diameter of the CTF particles was 75 rnp (Fig. 2). In addition to this structure, an additional envelope was found around a small proportion of capsids in cist,ernae of t#he endoplasmic reticulum. This envelopment appeared to be a consequence of passage of the capsids through endoplasmic reticulum membranes (Figs. 3 and 5); the enveloped particles had a diameter of 90-95 rnp. Temporally and spatially related to the appearance of virions, were large numbers of multiple filaments which appeared in complex arrays (Figs. 1 and 4). Often filaments and granular matrices were contiguous and virus particles were associated intimately wit’h both structures. At a later stage t’here was a degeneration of organelles and a rarefaction of the cytoplasm, which permitted furt,her resolution

2. CTF virions accumulating in cytoplasm of BHK21 cell. Virions 73 mp in diameter contain irstippled or homogeneous electron dense nucleoids that are 4.5 rnr in diameter. Arrow indicates being enveloped. X 84,000. FIG. 3. Enveloped CTF virions accumulating in a cisterna of the endoplasmic reticulum of a BHK21 cell. 0111y a small proportion of virions in infect,ed cells was folmd to be enveloped. X 84,000.

32

MURPHY,

ET

AL.

FIG. 4. Rarefied cytoplasm of a BHKPl cell late in the course of infection. CTF ciated with cross-striated filaments (B) and fine kinky threads (C). These filaments the spindle microtubules associated with reovirus infection. X 84,000.

virions appear

(A) are assodifferent from

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FEVER

VIRUS:

of structures associated with virus maturation. Arrays of the tight,ly packed filaments in rarefied cells had cross-striations when sectioned in a favorable orientation. Interspersed among the filaments and virus particles were fine kinky threads (Fig. -1). These, in some cases, appeared to result from a breakdown of the granular matrices. All t,hc described structures \vere found throughout disthe cyt~oplasm; no exclusive pcrinuclear tribution \vas evident. Relatively few virus particles were found free in extracellular spaces even late in

FIG. 5. CTF virus infection of newborn dense particles considered viral nllcleoids Ellveloped particles are also present (D).

ELECTROS

~IICROSCOPY

33

infection of cell cultures. Masses of virions were, instead, contained in cells at advanced stages of degeneration and in cell debris, a finding indi&ivc of a predominance of “cell associat’ed” virus in CTF infectJions of cell cultures. Virus release from infected no evidence of cells was b!, cell dissolution; early ext,rusions of virus was found. In central nervous s\.stem tissue of II~Vborn mice infected with each of the isolates of CTE‘ virus, evidence of t’he same progression of viral maturation was found. Karly harvests of brain tissue revealed normal cell

mmlse brain. The cytoplasm of this (,4) within, and virions (B) aroruld X 65,000.

neutron contains electron a gramllar matrix ((7).

34

MURPHY,

architecture except’ for the presence of small intracytoplasmic uniformly granular areas without associated part,icles in a small number of cells. These granular matrices were idernical to those seen in CTF virusinfected cell cultures. Jiater harvests revealed larger numbers and increased size of matrices; associated with t’hesewere viral nucleoids and virus particles identical to t’hose seen in cell cultures (Fig. 5). Far fewer virus particles were found, however, and the striking cytopathic effect seen in cultured cells was not evident in neural cells. The envelopment of virus particles in host cell membrane was more frequent in mice than in cell cultures. Arrays of filaments indistinguishable from those seen in the cytoplasm were found in the nuclei of someinfected cells, both in cell cultures and in mouse brain (Fig. 6). These were characteristically found in closely parallel bundles and when sectioned favorably, appeared to be cross-striated. No other nuclear change occurred ot’her than margination of chromatin in some cells late in infection. Thin-Section Electron Microscopy of Reoviruses The morphology and mode of replication of CTF virus, as described, bears a striking resemblance to that of reoviruses (Dales, 1963; Anderson and Doane, 1966). In an attempt to make direct comparisons, reoviruses 1, 2, and 3 were inoculated into BHK21 cells and studied by electron microscopy. CTF virus and reovirus morphology and mode of replication were similar with the following exceptions: (1) enveloped reovirus virions were never encountered; (2) the crystalline patterns characteristic of high focal concentrations of reoviruses (Fig. 7) were never seen with CTF virus; (3) cytoplasmic matrices appeared more fibrous in reovirus-infected cells, more granular with CTF virus; (4) kinky threads were more prominent with reoviruses than CTF virus (Fig. 7); (5) arrays of filaments were more prominent in CTF virus infect’ions; (6) intranuclear filaments were never observed in reovirusinfected cells.

ET AL.

Negative Conkast Electron Microscopy CTF virus particles were observed in negatively stained preparations from both infected mouse brain tissue and, with better resolution, from infected cell cultures (Figs. S and 9). Virus particles were round or polygonal in outline with regularly spaced surface projections; their mean diamet,er was 80 rnp (range 73359 rnp) . Approximately 18-20 of t’he rather indistinct projections were counted around the periphery of favorably oriented particles, and reinforcement of surface projection images was obtained at n = lS, 19, and 20 by rotational analysis performed according to the technique of Markham et al. (1963). Quite prominent in all CTF virus preparations were delicate round structures, 50 rnp in diameter (Fig. 10). These were made up of subunits and were readily penetrated b.y stain. From observations of all graduations from complete coverage to complete loss of surface components or capsomeres from the 50 rnp particles, it was concluded that these represent an inner capsid of the CTF virion. Fully enveloped virus particles were not identified in negat,ive contrast preparations. Negative Contrast Electron Microscopy Reovimses

of

CTF virus particles were compared directly with reovirus type 2 by making similar negative stain preparations from ultracentrifuged BHK21 cell culture supernatants (Fig. 11 vs Fig. 9). Identical virion mean diameters (80 mp) were obtained n-it’h both viruses. However, a smaller inner capsid diameter was found with reoviruses, 45 rnp as compared with 50 rnp with CTF. A consistent difference between the amount of structural detail visible in the two viruses was evident. The capsomere structure of CTF virus appeared “ragged,” resulting in an inability to define surface capsomere patterns, a problem not encountered with reoviruses. DISCUSSION

In both its structural detail in negative contrast preparations and its mode and site of maturation as observed in ultrathin sections, CTF virus resemblesreoviruses. This

COLORADO

TICK

FEVER

VIRUS:

ELECTRON

1\lICROSCOPY

FIG. 6. Intranuclear array of filaments (il) with fine cross-striations in a BHKPl rell. Marginated chromat.in (B) and the nuclear membrane (C) are visible. X 63,000. FIG. 7. Reovirus type 3 in a BHK21 cell. This crystalline array of virions (A) is surrolmded by fine kinky threads (B). ‘\‘irion morphology appears quite similar t,o CTF virus. X 90,000.

FIG. appear

8. CTF virus in a negative cont.rast, preparation from a BHK21 cell culture. Surface projections regldarly spaced at the periphery of several part,icles. X 126,000. FIG. !I. CTF virus in a negative contrast preparation. Virion (A) diameter is 80 rn+ and inner rapsid (B) diameter is 50 rnp. X 320,000. FIG. 10. Group of inner capsids of CTF virus, somestill with remnants of outer components att arhed. x 191,000. 36

COLORADO

TICK

FEVER

\.IRUS:

ELECTRON

FIG. 11. Reovirus type 3 in a negative contrast prcparatioll ing stain (11) show double capside constrtlctioll; pctletratjioll inner cnpsid structure (B). This inner capsid is remarkably

MICROSCOPY

frown UHK21 cell c-ultlue. \.iriotls of the stain leaves a characteristic similar to that seen in CTF

37

excllldring of viriolls.

x 320,000.

\ras found to he true of the prototype I’lorio strain, as well as four independently identified low passageCTP isolates. ,4t least three ot*her ungrouped arhoviruses appear to have morphologic similarities to CTF virus an d reoviruses. Bluctongue virus morphology has been described as reoviruslike by Studdert et al. (196B), but further complexity of st,ructure involving multiple enveloping lavers has been reported b) Ritchic and Bowne (1967). Similar rcoviruslike symmetry has been described for African horse sickness virus by l’olson and Deeks (1963) and for Rift Valley fever virus by I,evett et al. (1963). In addition, the virus of epizootic diarrhea of infant, mice (EDIM) has recently been shown to resemble reoviruses by Adams and T\raft (1967) and by Banfield et al. (1967); no evidence was found of the presence of this virus in prcpnrations used in the present study. From observations of CTF virus in negative-contrast preparations the presence of cubic symmet’ry is strongly suggested.

The regularly spaced peripheral projections on many particles, the reinforcement of the images of these projections by rotation (Rlarkham et al., 1963), and the uniformity of virions favor consideration of cubic symmetry despite the inability to resolve capsomere patterns on virion surfaces. The structural similarit)y betel-een CTE’ and reoviruses may also serve as evidence of cubic symmetry; in part,icular, the remarkable similarity of inner capsids indicates a common structural design (Mayor and Jamison, lYt%i). If the similarity in numbers of peripheral projections (E-20), the similarity in rotational rcinforccment values (n = IS, 19, 20), the similarity in capsid diameter (SO mp), and the approximate similarity in capsomere size are considered together, then it is most likeI)- that CTF virus has the same number of capsomercs in its outer shell as reoviruses, namely 92. In sharp contrast to the ultrastructural aimilaritics between CTF and rcoviruscs, the biophgsical and biochemical properties of the viruses are quite different; CTF

38

MURPHY,

behaves like a typical arbovirus. Trent (1964) showed that CTF virus is extremely sensitive to heat, as are other arboviruses which have been studied: Tl/z at 37” = 21 minutes OS 153 minutes for reovirus (from Gomatos et al., 1962). Reoviruses are resistant to ether and other lipid solvents (Stanley, 1967), whereas CTF virus has been shown by Trent (1964) to be quite sensitive. Finally, reoviruses are not, inactivated by exposure to a wide pH range, whereas CTF virus infectivity is stable only between pH 7 and 8 (Trent and Scott, 1966). The ragged nature of the capsomeres of CTF virus is analogous to observat’ions with several Group A and B arboviruses (Osterrieth and Calberg-Bacq, 1966; Mussgay and Rott, 1964; Abdelwahab et al., 1964) where surface projections are irregular and symmetry unresolved. The property of lipid solvent lability, common to most arboviruses, including CTF, has been COW sidered to be related to phospholipids in virus surface components (Jlussgay and Rott, 1964). Such essential lipids may cause the ragged capsomere structure observed rather than t,he well resolved subunit pat-

ET

AL.

terns of many viruses without lipid con stituents (e.g., adenoviruses, reoviruses). The relationship of the membrane-like enveloping layer of CTF virus t’o its stability and infectivity remains unknown. The mode of replication of CTF virus is complex and only remotely similar to that of Group ,4 and B arboviruses (Morgan et al., 1961; Murphy et al., 1967). In particular, the close association of Group A and B arbovirus maturation n-it’h the endoplasmic reticulum differs from t,hat of CTF virus where capsids occur free in the cyt’oplasm rather than within membranous enclosures. The presence of a granular matrix closely related to virus particle maturation in CTF virus infections has not been found in Group A or B arbovirus infections. Jloreover, t’he consistent’ observations of intranuclear filaments in CTF virus infect’ions of cultured cells and in mouse brain cells may be unique among the arboviruses. Understanding of these will depend upon further informatlion of biochemical events of infect,ion. There never has been any assurance that arboviruses const’itute a homogeneous

FIG. 12. Chenuda virus in BHK21 cells. The granular matrix with virions at its periphery is similar to that seen in CTF virus infections. Negatively stained virions (right) have regularly spaced surface projections and an inner capsid structure, also similar to CTF virus. X 65,000 (section); X 320,000 (negative stain).

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family; the current classification scheme’s usefulness derives from an epidemiologicimmunologic basis rather than a physicochemical basis as is the case with ot’her virus groups. The observat’ions made here of a rather dist,inct’ morphology of CTF virus compared wit’h most arboviruses, may serve to cmphnsize the physical heterogeneit,v of agents in this large family. Chenuda, an arbovirus isolat’ed from Aqas I‘. I/etwanni ticks in Egypt (Taylor et al., 1966a,b) was also examined by negat,ive contrnstj and ultrathin sect’ion electron microscopy. Infect’ed BHK21 cells harvested at early signs of cyt’opathic effect contained virus particles and granular matrices similar to CTF virus (Fig. 12). Virions were SO mp in diameter in negative contrast preparations from ultracentrifuged &sue cukure supernntant fluid, and inner capsid forms ivere found in moderate numbers (diamctcr 50 mp; Fig. 12). REFERENCES AI~I)EI,\\.\H.YLL, K. S. E., ALMEID.I, J. l)., DCL\NI;, F. W., and MCLI~XN, 1). M. (1964). Powassan virus: morphology and cytopathology. Can. Med. ,tssoc. J. 90,1068-1072. AD.\MS, W. R., and KR.YFT, L. M. (1967). Electronmicroscopic study of the intestinal epithelium of mice infected with t,he agent of epizootic diarrhea of infant mice (EL)IhI virus). ilm. J. Pathol. 51, 39-60. ANDERSON, X., and DOANF;, F. W. (1966). An electron microscope study of reovirus type 2 in L cells. J. Palhol. Bacterial. 92, 433-339. BANFIELD, W. G., KXDIC, G., and BLACIC~I~ZLL, J. IT. (1967). Further observations on the virus of eplzootic diarrhea of infant mice. An elertron microscopic study. Proc. Electron Microscopy Sot. *4m. pp. 110-111. Clait,or Book Co., Baton Rouge, Loliisiana. Blsc~
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MICROSCOPY

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