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J. gen. Virol. (I972), x7, I85-195
Printed in Great Britain
Intra-erythrocytic Location o f Colorado Tick Fever Virus
By R. W. E M M O N S ,
L. S. O S H I R O ,
H. N. J O H N S O N
AND E. H. L E N N E T T E
Viral and Rickettsial Disease Laboratory, California State Department of Public Health,
Berkeley, California 947o4, U.S.A.
(Accepted I3 July I972)
SUMMARY
Although previous studies have shown that Colorado tick fever virus occurs
only transiently in the serum, but persists for prolonged periods in the blood cell
fraction of man and experimental animals, the exact nature of this cell-associated
viraemia was unknown. Using virus isolation, fluorescent antibody staining,
histological and electron-microscopic techniques, we have shown that persistent
viraemia is predominantly due to virus contained within erythrocytes. Erythrocyte
precursor cells are presumably infected in the bone marrow and are subsequently
released into the blood stream, persisting for prolonged periods despite the presence
of serum antibody. Virus either continues to replicate slowly or is stabilized and
remains viable within the erythrocyte. Colorado tick fever virus may be a useful
model virus for further study of the mechanism of blood cell infection, with
implications for other arboviruses or viruses believed to be involved in leukaemia
or other haematological disorders.
INTRODUCTION
Several studies reported in the literature describe the prolonged viraemia in man and
wildlife species infected with Colorado tick fever virus: ~7 days in man (Eklund, Kennedy &
Casey, I96I), 15 to 50 days in experimentally infected monkeys (Gerloff & Larson, ~959),
and similarly prolonged periods in other naturally or experimentally infected animals
(Burgdorfer, 1959, I96o; Burgdorfer & Eklund, 1959).
The association of this virus with blood cells was first noted in our laboratory in I96I.
The blood cells and plasma from heparinized blood of a chipmunk, Eutamias amoenus
(M-919), trapped in Modoc County, California, were tested separately, and both were
positive for virus at the time of capture, but at 17 and 38 days after capture only the cell
fraction yielded virus. At 60 days both plasma and blood cells were negative for virus.
A non-neuroadapted stock strain of the virus was prepared by subcutaneous passage of
blood cell suspension in adult mice. Virus persisted in the blood cells of infected mice to
38 days after inoculation, but was present in the serum for only a few days. Repeated
washing of the blood cells and removal of the buffy coat failed to reduce the virus titre in
the remaining red blood cell fraction (H. N. Johnson, unpublished studies).
Subsequent studies in man and experimentally infected animals confirmed that the virus
was associated largely with the erythrocyte fraction rather than the leucocyte fraction of the
blood and showed the value of fluorescent antibody staining to identify virus antigen in
blood cells (Emmons, I965, 1966, 1967; Emmons & Lennette, I966). Persistent viraemia
was demonstrated by virus isolation from washed blood cells and by fluorescent antibody
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R.W. EMMONS AND OTHERS
staining of blood smears from human cases. It was hypothesized that the virus infected
developing cells in the bone marrow, and that infection persisted through the various stages
of maturation and in mature blood cells released into the circulation (Emmons, 1965, 1967).
We now report evidence from infectivity and electron microscopic studies that the persistent
viraemia in Colorado tick fever is due predominantly to virus contained within erythrocytes.
METHODS
Colorado tick fever virus strains. The FLORIO strain was used to prepare serum for virus
identification by neutralization tests or fluorescent antibody staining. The 5th passage
non-neuroadapted M-919 strain, or virus strains isolated from the blood clots of human
cases, were used for experimental infections of cell cultures and animal hosts.
Virus titration and antibody determinations. Virus titrations were performed by standard
methods, using tenfold dilutions of stock viruses, plasma, or blood cell fractions to inoculate
suckling (I-tO 3-day-old) Swiss white mice (Rockefeller Foundation strain). All virus
isolates were identified by a standard neutralization test in mice, using antiserum to
Colorado tick fever virus prepared in hamsters, and by fluorescent antibody staining, using
the same hyperimmune hamster serum labelled with fluorescein isothiocyanate. Fluorescent
antibody staining was also used for identification of virus antigen in blood smears from
human cases or experimentally infected animals, as described previously (Emmons &
Lennette, I966). Specificity of the fluorescence was confirmed by positive and negative
controls in the staining procedure and by inhibition of the staining when conjugate was
absorbed with a 2o % suspension of infected mouse brain.
Standard methods for determining complement-fixing antibody, indirect fluorescent
antibody and neutralizing antibody titres (mouse test and plaque reduction test) were used
as described elsewhere (Emmons & Lennette, I966; Emmons et al. I969).
Cell culture methods, utilizing the B H K - z l line of hamster kidney cells, for comparative
electron microscopic studies of certain aspects of intracellular infection relating to the
present experiments, were described by Emmons et al. (1969) and Oshiro & Emmons (I 968).
Blood specimen preparation. Blood specimens from experimentally infected animals or
from human cases of Colorado tick fever were heparinized to prevent clotting of the blood.
The virus was previously shown to be unaffected by heparin (Emmons, 1965). When serum
was needed, separate blood samples without anticoagulant were obtained. After centrifuging
at 45o g for 5 min, the plasma was separated from cells and the cells were gently washed
three times with 5 to Io vol. of normal saline. The plasma was again centrifuged, to assure
removal of all cells. The blood fractions were titrated for virus content immediately if
possible, or after storage at - 7 o °C in flame-sealed glass ampoules. Serum or plasma
samples from experimental animals or human cases were held at 4 °C until all samples had
been collected, then were tested simultaneously for antibody titre.
Electron microscopic studies. Electron microscopic methods used for examination of
infected cell cultures have been described previously (Oshiro & Emmons, 1968); for
examination of human or mouse blood the following procedure was used. Infected cells
were concentrated by centrifuging 2 to 3 ml of heparinized blood at 8oo g for 2o min. After
removal of the plasma, 1.2 x 75 mm heparinized capillary tubes were filled ~ full with blood
from the reticulocyte layer of the previously centrifuged blood, then were flame-sealed
at one end and centrifuged at 8oo g for 2o rain. The section of each capillary tube
containing the upper I to 2 mm of the erythrocyte fraction plus the lowermost o.5 to I mm
of the buffy coat layer was then broken off, and immersed in toto in 2 % glutaraldehyde
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for several hours to allow penetration of the fixative. The packed, hardened column of
cells was pushed out of the capillary tube by inserting a smaller capillary tube into the lumen,
fixed in glutaraldehyde for an additional hour, and cut into three or four pieces, identified
according to position (buffy coat layer, interphase, and uppermost erythrocyte layer). The
cell preparations were washed in o.i M-phosphate buffer (pH 7"5) for several hours and
were then fixed in 1% osmium tetroxide, rinsed in phosphate buffer, dehydrated in a graded
series of ethanol concentrations, and embedded in Epon. Thin sections were cut and stained
with uranyl acetate and lead citrate and were examined in a Siemens Elmiskop I electron
microscope.
RESULTS
Viraemia and antibody in mice and man
Laboratory mice were infected with the M-919 strain of Colorado tick fever virus. At
each test day, heparinized blood samples from three mice were pooled, and virus titres in
the plasma and washed blood fractions were determined daily for I I days and then at
2- or 3-day intervals (Table I). The virus titre in the plasma was initially slightly greater
than that in the cells, but after 4 days was less; virus was not detectable in the plasma after
8 days. Virus was isolated from washed blood cells until day 48. Antibody was detected on
day 2i and thereafter, occurring simultaneously with viraemia for a total of 27 days.
Antibody titre end points were not determined in this experiment.
One human case of Colorado tick fever was studied weekly for viraemia and antibody
response (Table 2). The plasma yielded virus only on day 6 (low titre). In whole blood,
titration results were irregular after day 14, presumably due to the presence of antibody in
sufficient titre to partially neutralize the virus. Virus was consistently isolated from washed
blood cells at each bleeding. Tests after day 76 were not possible, since the patient withdrew
from the study, but viraemia presumably persisted longer. Although the patient suffered a
characteristic dengue-like, acute febrile illness, he was free of symptoms after the first
2 weeks and there was nothing unusual about the clinical course.
We have not had the opportunity to study other cases in such detail, but we attempted to
determine the approximate duration of cell-associated viraemia by obtaining lateconvalescent, heparinized blood samples from additional cases and processing them as
described above, to obtain washed blood cell fractions. Virus isolation was easily accomplished for several weeks after onset in such cases. The virus was isolated from blood and
virus antigen was demonstrated in blood smears from one case iot days after onset of
clinical illness. Virus antigen was demonstrated in blood cells by fluorescent antibody
staining in other cases at 96, 119, I2O, 123, 13o and 135 days after onset, although virus
could not be isolated from the washed blood cells. These cases had completely recovered
from clinical signs of illness when the late blood samples were obtained, and all had
developed significant levels of complement fixing, indirect fluorescent, and/or neutralizing
antibody.
Intra-erythrocytic location of virus
Evidence indicating that the virus is located intracellularly, predominantly in the erythrocytes, is summarized below. As reported previously, the erythrocyte fraction, from which
all detectable leucocytes were removed, had a virus titre equal to or greater than that of
whole blood or of the leucocyte fraction (Emmons, I967). Immune serum failed to neutralize
the virus in blood unless the erythrocytes were disrupted by freezing and thawing or by
lysis in distilled water.
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R. W . E M M O N S
AND
OTHERS
Table I. Viraemia and neutralizing antibody response in white Swiss mice following subcutaneous inoculation of 30 ooo suckling mouse i.c. L D 50 of Colorado tick fever virus, M-9 t 9
strain, stock virus
Days after
inoculation
I
2
3
4
5
6
7
8
9
lO
II
13
15
Titre of virus in blood fractions*
,
-~
Plasma
Washed cells
I "4
I "0
3"0
2"4
2"9
3"5
3'6
3"5
3"4
4"4
2'4
3"6
< I-O
3"5
< i 'o
3'7
o
2"4
o
o
o
o
18
o
2I
24
27
30
33
36
39
o
o
O
O
O
O
0
3"3
3"3
3"2
2-6
2"5
3"I
3"4
2"9
1"5
2"0
I "5
I '9
42
0
I'7
45
o
1.5
48
o
i "5
5I
54
57
o
o
o
o
o
o
8I
o
o
Plasma
neutralizing
antibodyt
Not tested
Not
tested
+
+
+
+
+
+
+
+
+
4+
+
+
+
* Log10 LD 50 of Colorado tick fever virus per I 'o ml of blood fraction; pooled blood samples from three
mice.
o No virus isolated.
--No antibody demonstrable.
4-Antibody demonstrable.
t Undiluted plasma tested against 5000 LD 50 of M-9I9 strain (suckling mouse intraperitoneal neutralization
test).
Infected b l o o d cells stained by the direct fluorescent a n t i b o d y m e t h o d (Fig. I) showed a n
intracellular location of virus antigen. F o r c o m p a r i s o n o f the staining characteristics,
infected B H K - z x cells are shown i n Fig. 2. The size a n d m o r p h o l o g y of the fluorescing b l o o d
cells were identical with that of erythrocytes. A n t i g e n was usually distributed i n a few large
clumps a n d n u m e r o u s finely particulate foci t h r o u g h o u t the cell. C o u n t e r s t a i n i n g of b l o o d
smears with Giemsa stain a n d r e - e x a m i n a t i o n u n d e r o r d i n a r y i l l u m i n a t i o n confirmed that
almost all fluorescing cells were erythrocytes. I n this procedure it was preferable to use
so % m e t h a n o l made u p i n a solution o f 5 % f o r m a l i n in n o r m a l saline as a fixative for the
b l o o d smear instead of acetone, since acetone-fixation disrupted the cell architecture a n d
resulted i n unsatisfactory G i e m s a staining. T h e staining was d i m m e r t h a n after acetone
fixation, b u t the fluorescing cells could still be easily located. I n rare instances a m o n o nuclear leucocyte appeared to also c o n t a i n antigen. The fluorescent cells were most a b u n d a n t
i n the reticulocyte layer of centrifuged blood. Infected cells located by fluorescence were
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Table 2. Colorado tick fever viraemia and antibody response in a patient naturally infected
by tick bite
Days after onset of symptoms
r
Test performed
Virus titre in plasma*
Whole blood smear, direct
F A stain
Virus titre, whole blood
Washed blood cells smear,
direct F A stain
Virus titre in washed blood cells
CF antibody titre
Indirect F A antibody titre
Plaque-reductionneutralizing
antibody titre
*
0
+
t
6
< I "7
+
14
2o
27
34
4t
48
55
62
69
0
0
0
0
0
0
0
0
0
0
+
+
+
+
+
+
+
+
+
-4-
4"6
+
3"6
+
I'7t
+
+
0
<I'Ot<I'O
+
+
t
76
0
0
0
0
0
+
+
+
+
+
~ 4'6
4 .2
3'7
4 .2
3"7
3"3
4"1
2"7
2"3
2"3
2'3
< t/4 < I[4
I[8
1/16 i / I 6 1/16 I/I6 I[I6 1/16 I[I6 I[I6
I/4 i[IO24 I/2O48 1/2048 1/2048 I[2048 i[I024 I[IO24 I/IO24 I[512 I[I024
< I / 4 1/32 1/256 I[256 I[256 1[256 1/512 I[512 1/256 I[512 1/512
Log10 LD 5o of Colorado tick fever virus per I-O ml of blood fraction.
N o virus isolated.
Colorado tick fever antigen present in blood cells.
Irregular titration: virus isolated only from io -~ or IO-s dilutions; undiluted blood negative.
Fig. ~. Fluorescent antibody staining of erythrocytes from patient with Colorado tick fever.
Fig. 2. Fluorescent antibody staining of BHK-2t cells 24 h after infection.
also re-examined by phase microscopy, by changing the substage condenser, and were
consistently seen to be erythrocytes. In some, small inclusions apparent by phase microscopy
coincided with concentrations of fluorescing antigen.
Attempts to stain the virus in unfixed erythrocytes in suspension by the fluorescent
antibody method, or to infect normal erythrocytes in vitro and then demonstrate antigen
by fluorescent antibody staining, were not successful, further indicating that virus antigen
was inside the erythrocyte, and not adherent to the cell surface. Disruption of the erythrocyte membrane was necessary before antibody could penetrate and combine with the virus
antigen.
Electron microscopic observation of blood cells from a human case (Figs. 3, 4) and from
experimentally infected mice (Figs. 5 to 7) revealed particles typical of Colorado tick fever
virus within erythrocytes and reticulocytes. Because of the rarity of infected cells, they were
only located after long search, and then most easily found in the reticulocyte layer. The virus
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R. W. E M M O N S A N D O T H E R S
Fig. 3. Two Colorado tick fever virus particles within a human erythrocyte.
Fig. 4. Higher magnification of a Colorado tick fever virus particle within a human erythrocyte,
Fig. 5. Colorado tick fever virus particles within a mouse erythrocyte,
Fig. 6. Fibres within a mouse erythrocyte are similar to the intracytoplasmic and intranuclear
fibres seen in tissue culture ceils infected with Colorado tick fever virus. A virus particle is also
shown within the same cell.
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Fig. 7. The fluorescent foci shown in Fig. I are presumably due to dusters of fibres such as those
illustrated in this figure. Mouse erythrocyte infected with Colorado tick fever virus.
Fig. 8. Intracytoplasmic fibres and virus particles within a BHK-2I cell infected with Colorado tick
fever virus.
Fig. 9. Intranuclear fibres within a KB cell infected with Colorado tick fever virus.
13
VlR 17
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R. W. E M M O N S A N D O T H E R S
Fig. IO. The antigenicity of the intranuclear fibres within a BHK-2I cell infected with Colorado tick
fever virus is illustrated by the tagging of fibres with ferritin-labelled antibody to Colorado tick
fever virus.
Fig. I I. Intracytoplasmic fibres tagged with ferritin-labelled antibody to Colorado tick fever virus.
BHK-2~ cell infected with Colorado tick fever virus.
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particles measured approximately 63 nm in outer diameter with a 50 nm core and were
identical in appearance to those seen in infected BHK-2I cells (Fig. 8). In addition, the
unique bundles of fibres described in infected cell cultures by Oshiro & Emmons (I968)
and by Murphy et al. 0968) and shown here for comparison (Figs 8, 9) were noted also in
mouse erythrocytes (Figs 6, 7)- These aggregates of virus-associated, electron-dense material
could be specifically tagged with ferritin-labelled Colorado tick fever virus antibody, as
shown in infected tissue culture cells (Figs IO, I I), clearly indicating their antigenic relationship to the virus. Because of the difficulty in locating infected blood cells by electron
microscopy, demonstration of ferritin tagging in them has not yet been accomplished. The
presence of these fibrillar aggregates, and fluorescing antigen, in the erythrocytes is evidence
that virus multiplication has occurred in the cells and that presence of virus particles was
not merely due to pinocytosis or to close adherence of virus particles to the cell surface.
DISCUSSION
The intra-erythrocytic location of Colorado tick fever virus has been shown by electron
microscopy, fluorescent antibody staining, phase microscopy, histochemical staining, and
virus titration methods. We presume that other blood cells (leucocytes and platelets) are
also infected initially, but being short-lived and few in relation to the erythrocytes, they are
not readily detected and do not persist in the convalescent phase of the disease. The
possibility that virus could also be associated with dislodged capillary endothelial cells,
macrophages, or other aberrant cells in the blood stream should also be considered. A
transient, moderate anaemia occurs in the disease but usually has not been looked for.
Haemolysis has not been recognized as a part of the haematological picture.
The prolonged duration of viraemia is apparently due to the presence of the virus intracellularly in the erythrocytes, protected from antibody or other host defence mechanisms.
Erythrocyte precursor cells in the bone marrow are presumably infected at early stages in
their development, but at least some of these infected cells continue to mature and are
subsequently released into the blood stream. The circulating cells are not all destroyed by
the virus or removed in the spleen, and some persist for prolonged periods, perhaps for
nearly their normal life-span. The virus either replicates slowly or is stabilized in some way
within the erythrocyte. Some metabolic functions persist in erythrocytes well into maturity,
perhaps sufficient to support virus replication to some extent. The virus is relatively stable.
We have re-isolated it from the blood clot of a human case after I6 months of storage at
4 °C. The prolonged persistence of viraemia has recently been confirmed by others (Earnest
et al. I97I ; G. Hughes, unpublished). There is no evidence thus far that chronic infection
of the bone marrow is a source of the persistent viraemia. Our experience with virus isolation
and fluorescent antibody staining studies on mice and three human cases, and reports by
others (Eklund & Kennedy, I96I ; Gerloff & Larson, 1959) indicate that virus infection of
the marrow occurs in the initial stage of infection, but does not persist. This point requires
further study however.
Many other studies, too numerous to review here, of experimental or natural virus and
rickettsial infections have shown blood cell association, usually with the leucocytes or
platelets (see reviews by Mims, I964; Gresser & Lang, I966). It is of special interest that
several of the viruses now assigned to the diplornavirus group, to which Colorado tick fever
belongs (Verwoerd, ~97o), share with this virus the properties of cell-associated viraemia
and formation of unusual crystalline or fibrous aggregations of virus-related material in
infected cells.
I3-2
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R. W. EMMONS AND OTHERS
Examples of virus-erythrocyte association are fewer. Adsorption of virus to erythrocyte
stroma, or some other undefined relationship to erythrocytes, has been shown or suggested
for lymphocytic choriomeningitis (Traub, 1938; Schwartzman, I944; Mims, T964), Rift
Valley fever (Mims, 1956); African swine fever (Sirbu, Ieremia & Bona, 1964), influenza
(Hamre, Appel & Loosli, I956), hog cholera (Powick, 1937), equine infectious anaemia
(Hyslop, I966), foot-and-mouth disease, fowl plague, Newcastle disease and mumps
(Epstein, Fonseca & DeRoberts, I95I ; Overman, I958), parvovirus infections (Margolis &
Kilham, I97O), bluetongue and epizootic haemorrhagic disease of deer viruses (G. L. Hoff,
personal communication), and hamster osteolytic virus (Portella, I964). Evidence of the
intra-erythrocytic location of virus has recently been reported for mammary tumour virus
(Nandi & Haslam, I97I), Rana pipiens virus (Bernard, Cooper & Mandell, 1968), gecko
virus (Stehbens & Johnston, 1966), Friend leukaemia and Rauscher viruses (Reilly &
Schloss, 1971), certain feline neoplasms (Gardner, 1971), and C-type viruses in certain
disorders of cats (Oshiro et al. 1972).
A number of implications and suggestions for future studies result from our findings on
Colorado tick fever. One practical result is that the presence of virus and virus antigen in
erythrocytes for prolonged periods after onset of the disease facilitate diagnosis by virus
isolation or fluorescent antibody staining at any stage of illness or convalescence. Although
no such instances are known to have occurred, the possibility of man-to-man transmission
of the virus via blood transfusion should be recognized. In the natural rodent hosts (chipmunks, field mice, ground squirrels) the prolonged viraemia probably facilitates host-tohost transmission of the virus by vector ticks and contributes to a more stable ecological
adaptation of the virus.
Colorado tick fever virus may serve as a useful and convenient model virus for exploring
further the mechanism of erythrocyte infection, with implications particularly for other
arboviruses, viruses believed to be involved in haemolytic or other haematopathologic
states, and various leukaemia viruses. The safety of Colorado tick fever virus for man should
not be assumed, however. Although generally producing only a benign, dengue-like illness,
it can cause severe disease, with haemorrhagic or meningo-encephalitic complications, and
convalescence is often prolonged even in the uncomplicated disease. The possibility is
intriguing that a virus which can replicate in bone marrow and blood cells might even be
an underlying or inciting cause of leukaemic or other haematological changes.
This study was supported in part by Grants AI-oi475 and AI-o82oo from the National
Institute of Allergy and Infectious Diseases, United States Public Health Service, Department of Health, Education, and Welfare.
We thank the following individuals for their excellent technical assistance in this study:
Mr Dale Dondero, Mr James Woodie, Mrs Veronica Devlin, Dr Osman Luis, Mr David
Banks, Mr Jerry Bartyzel, Mr William Burleson and Dr Wesley K. Ota.
The advice and suggestions of Dr Cedric Mims were also very helpful in planning some
of the experiments.
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