7-Ultrastructure of Felis catus whole fetus (Fcwf-4) cell

Abstract

About growth of FIPV in FcWf cell line under TEM

Full text

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Biological: Full-length
Ultrastructure of
Felis catus
whole fetus (Fcwf-4) cell
culture following infection with feline coronavirus
Amer Alazawy1, Siti-Suri Arshad1,*, Mohd-Hair Bejo1,
Abdul-Rahman Omar1, Tengku-Azmi Tengku Ibrahim2,
Saeed Sharif1, Faruku Bande1and Kamarudin Awang-Isa1
1
Department of Veterinary Pathology and Microbiology, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia and
2
Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
*To whom correspondence should be addressed. E-mail: suri@putra.upm.edu.my
Abstract Feline coronavirus (FCoV) consists of two biotypes based on their
growth in cell culture and their antigenicity. Infections with FCoV are
highly prevalent in the cat population worldwide. In this study, Felis
catus whole fetus (Fcwf-4)cell culture was infected with FCoV UPM11C/
08. Virus multiplication in cell culture was monitored and examined
under the transmission electron microscope. The virus particles revealed
the characteristic morphology of feline FCoV represented by envelope
viruses surrounded by peplomers. Virus attachment and entry into the
cell occurred 15 h post-infection (pi), and the myriad of virus particles
were observed both extracellularly and intracellularly after 48 h pi.
Thereafter, intracellular virus particles were observed urred 15 h post-infection ( pi), ed 15 h post-infection ( pi), ed 1 ((Fcwf-4)cel)-12 (rr)5i)(ru)16y and intracellftercoth0 (e)- (rus)-4T*[(Ther)17 (ea)25 (fter,)-489 (intr)21 (a)18 (cellular)-489 (virus)-481 (particli))-4her18 (e)-8 (J[(cell)-444 (occurr)17 (ed)-439 (15)-202 (h)-440 (pos)16 (t-infection)-449 (()-103 (pi),)-448 (and)-443 (the)-440 (myriad)-446 (of)-441 (vi)-7 (rus)-440 (particles)]TJ0 -1.202 TD[(w)22 (er)18 (e)-702 (observ)18 (ed)-70w (()-103 (pi),)-4_0 1and)-443zd_0 1anjred 1 ((Fcwv)1h43 (t-12 (r(pi16 -in (e1d)-cwfinf)1844303 (pi)(pos)16 and)-443ell)-1ellJa)24 (fter)-ter449-68 (coth0 (e)- (rus)-4T*[(Ther)17 (3 (p5 (fter,)-4-11o(e1d)-cwfinf)1844303 (pi)(pos8C3.s)-481 (particli))-4her18 (e)-8 (J[(cell)-444 (occurr)17)-597-43(Th15)-202 (4o))-7m0prr)ula89 (f)21 (a)1-44-1e(e)- (rus)-4,ticlul481rus)-4Trticli)intrparticf virus particles

ability in vitro. While serotype I grows poorly in
cell cultures, serotype II can grow well in many cell
line [12–14]. FECV is more tropic for mature apical
epithelium of the bowel, whereas FIPV infects
blood monocytes and spreads systemically [8,12,
15]. Most coronaviruses will grow only in cells
derived from the natural host animal or in cells
from a closely related species. FCoV serotype I
strain is difficult to grow in vitro; hence studies on
these viruses have been limited in view of their fas-
tidious growth in cell culture. On the other hand,
serotype II FCoV could be propagated more readily
in cell cultures. Growth of FCoV in a continuous
feline cell line of Felis catus whole fetus-4
(Fcwf-4), which possesses the properties of macro-
phages [16–18], was characterized by cytopathic
changes and giant cell formation. After binding
itself to specific receptor of infected cells, the virus
enters the latter by fusion either with the plasma
membrane or the endosome. The viral nucleocapsid
is released into the cytoplasm of infected cells to
become available for translation and transcription
[14,19].
FCoV has been described as large plemorphic
particles with numerous spike-like projections
extending from their envelope. Particle measure-
ments ranged from 75 to 150 nm in diameter.
Surface projections have been observed to vary in
size and shape, reported between 12 and 25 nm in
length [8,20,21]. Thus, the aim of this study was to
describe the physical properties of FCoV UPM11C/
08 isolates and the ultrastructure of Fcwf-4 cell line
following infection with this local isolate of FCoV.
Materials and methods
Virus
A prototype of local isolate FCoV designated as
UPM11C/08 was used in the study. The virus was
isolated from a cat presented at the University
Veterinary Hospital, Universiti Putra Malaysia
(UVH-UPM) with effusive-form FIP. Ascitic fluid
was obtained from the cat and screened for FCoV
with reverse transcriptase polymerase chain reac-
tion assay using primers targeting the untranslated
region of 3UTR [22]. The sample was adapted and
propagated in Fcwf-4 cell culture until character-
istic cytopathic effect (CPE) was observed and
stored at −20°C (Sanyo, Malaysia) for further purifi-
cation by sucrose gradient method. This isolate was
confirmed as type 1 FCoV (GenBank, HM 628778).
Cell culture
Fcwf-4 cell culture was obtained commercially
(ATCC CRL-2787) and maintained in Eagle’s
minimum essential medium, supplemented with
15% heat-inactivated fetal bovine serum (FBS)
(Gibco,UK), 100 IU of penicillin/ml, 100 µg of strep-
tomycin/ml and 2.5 µg of amphotericin/ml. Cultures
were maintained in a humidified incubator at 37°C
with 5% CO
2
(Galaxy, UK).
Virus inoculation
A total of 20 flasks (150 cm
2
) (Nunc, Denmark) con-
taining confluent monolayer of 3-day-old Fcwf-4
cells were infected with 100 µl of purified virus
stock for each flask, where five flasks were kept as
control receiving only saline. All infected and
control flasks were incubated at 37°C for 1 h to
allow virus adsorption before adding the mainten-
ance media containing 2% FBS. The cells were
further incubated and examined daily for CPE. For
transmission electron microscopy (TEM) analysis,
two infected flasks and one control flask were
removed consecutively at 6, 10, 15, 24 and 48 h
post-infection (pi).
Virus purification
The virus stock was purified by sucrose gradient for
the purpose of ultrastructural studies and negative
staining analysis. Briefly, infected culture exhibiting
80% CPE was freeze-thawed thrice, pooled and cen-
trifuged at 6000 × gfor 30 min (Hettich, Germany).
The supernatant was subjected to ultracentrifuga-
tion at 25 000 × gfor 3 h at 4°C (Beckman, USA).
The resultant pellets were resuspended in 1 ml of
Tris NaCl EDTA (TNE) buffer (0.05 M Tris, 0.001 M
EDTA, 0.15 M NaCl) and gently layered over a 20–
50% sucrose density gradient and centrifuged at
120 000 × gfor 8 h. The resultant virus band was
pooled and diluted with an equal volume of TNE
buffer and pelleted again by centrifugation at 120
000 × gfor 60 min. The purified virus was resus-
pended in the TNE buffer and stored at −70°C
(Rifco, Germany) until further use.
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Negative contrast electron microscopy
The purified virus was examined for negative con-
trast electron microscopy (NCEM) according to the
method of Gelderblom et al. [23]. Briefly, a drop
of purified virus was placed on a parafilm and
a carbon-coated formvar grid (Van Loenen
Instrument, The Netherland) was floated on the
virus drop for 7 min. The grid was removed and
excess liquid was blotted away and fixed with a
drop of 2% phosphotungstic acid (Sigma, USA) for
5 min. The grid was air-dried before examination
under TEM (Hitachi H7100, Japan) at an acceler-
ated voltage of 75 kV.
Transmission electron microscope
Control and infected Fcwf-4 cells were processed
for TEM according to the method of Hayat [24] with
some modification. Cell cultures were scraped from
the flasks, washed thrice with phosphate-buffered
saline and centrifuged at 6000 × gfor 30 min. The
pellets were fixed in 4% glutaraldehyde with 0.1 M
sodium cacodylate buffer for 4–6 h at 4°C, washed
with 0.1 M sodium cacodylate buffer, postfixed with
1% aqueous osmium tetraoxide for 2 h and again
rinsed in 0.1 M sodium cacodylate buffer. Following
addition of FBS, pellets were dehydrated in a
graded series of dilutions of acetone in distilled
water (35, 50, 75 and 95%) for 10 min each, fol-
lowed by three changes of absolute acetone for 15
min each. Pelleted samples were initially infiltrated
with a 50:50 mixture of resin and acetone and sub-
sequently embedded in resin and polymerized in an
oven at 60°C overnight (Memmert, Germany).
Ultrathin sections on a copper grid were stained
with uranyl acetate and lead citrate [25]. The prep-
arations were examined under TEM.
Results
Negative contrast electron microscopy
NCEM of purified virus revealed the characteristic
features of a coronavirus. The virion exhibited
slight pleomorphic feature, spherical to oval shape
with a diameter ranging from 75 to 150 nm. The
morphological features of FCoV UPM11C/08 iso-
lates were similar to those of other members of the
family Coronaviridae. Their corona demonstrated
the long petal-shaped surface projections up to 21
nm long (Fig. 1).
Transmission electron microscopy
Infected Fcwf-4 cell culture showed the presence of
typical FCoV particles. At 15 h pi, the virus particles
were observed extracellular and by 48 h pi, the
virus particles were found to be present at both
intracellular and extracellular. In the extracellular
compartment, the virus particles were spherical
measuring between 55 and 69 nm in diameter, with
spike-like electron-dense envelope. Numerous virus
particles were closely apposed to the host cell
plasma membrane (Fig. 2). At the same time, virus
particles were observed to penetrate the cell by
invaginating the plasma membrane. It carried into
the cytoplasm part of the cell membrane that it
invaginated to form vesicles (Fig. 3). Virus-like par-
ticles which morphologically resembled those of
extracellular virus particles were present in vacu-
oles of different sizes, with smaller vacuoles con-
taining 1–4 particles, while larger ones contained
5–24 virus particles (Fig. 4). In addition to the
rough spike-like electron-dense virus-like particles,
there were also particles which were larger in diam-
eter ranging from 69 to 149 nm with distinct smooth
electron-opaque surfaces (Fig. 5). The vacuole con-
taining these particles was distinctly more rounded,
firm with a thicker membrane. In many instances,
the vacuole membranes were seen to be no longer
intact leading to spillage of virus particles into the
cytoplasmic matrix (Fig. 6). In this context, the
virus particles were either in a form of aggregations
in the cytoplasmic matrix or occur singly in a
vesicle of the Golgi apparatus or within the endo-
plasmic reticulum (Fig. 7).
Discussion
This study reports a new insight into the ultrastruc-
ture of Fcwf-4 cell culture following an infection
with FCoV. The prototype FCoV UPM11C/08 was
isolated from ascitic fluid of a cat diagnosed as effu-
sive form of FIP. The purified FCoV possesses the
characteristic of coronavirus with the diameter and
peplomer sizes within the range of published data
[20,21,26]. Upon virus replication at 15 h pi, the
virus particles were detected extracellularly in
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Fig. 2. Micrograph shows Fcwf-4 cell culture infected with FCoV UPM11C/08 at 15 h pi. Virus particles are present at the extracellular
compartment and exhibit spherical shaped with spike-like electron-dense envelope (arrow). Virus particles are closely apposed to the plasma
membrane (arrowhead). Scale bar, 0.2 µm.
Fig. 1. NCEM of purified FCoV UPM11C/08 showing virion of spherical shapes. The virus particles are surrounded by knob-like spikes
envelope (arrow). Scale bar, 100 nm.
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abundance while at 48 h pi, the virus particles were
detected both at extracellular and intracellullar. In
many of the instances, the virus particles were
closely apposed to the plasma membrane, while
few particles were observed to invaginate the cell
membrane at different depths into the cytoplasmic
matrix. Deeply invaginated virus particles were
seen to carry along with it part of the cell mem-
brane that it invaginates with the absence of the
plasma membrane at the site of invagination.
Beesley and Hitchcock [21] and many other investi-
gators had reported similar observations but were
Fig. 3. Micrograph of Fcwf-4 cell culture infected with FCoV UPM11C/08 showing a virus particle invaginating host cell membrane. Note the
invaginated virus is coated with a trilaminar membrane (arrow). Scale bar, 0.2 µm.
Fig. 4. Micrograph shows small vacuoles containing 1–5 virus-like particles (arrows), while larger ones containing 20–24 virus-like particles
(arrowhead) following infection with FCoV UPM11C/08 isolate. Scale bar, 0.5 µm.
A. Alazawy et al. Ultrastructure of Felis catus whole fetus 5
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of the opinion that invagination of the virus par-
ticles could represent a route of secondary
infection.
The new insight on the virus particles referred to
in the present study was that virus-like particles
were present in vacuoles of two types. In addition,
the study also showed that the virus particles
appeared in two forms. Both types of vacuoles con-
tained a variable number of particles, ranging from
a single to numerous particles. The rough, spike-
like electron-dense virus particles present extracel-
lularly and those that invaginated the host cell
membrane appeared morphologically similar. At
this juncture, it is tempting to postulate the
Fig. 5. Micrograph showing the virus-like particles inside a vacuole following infection of FCoV UPM 11C/08 on Fcwf-4 cell culture. Note the
smooth electron-dense envelope virus-like particle (arrow) and distinct rounded, firm and thick vacuole membrane (arrowhead). Scale bar,
0.2 µm.
Fig. 6. Micrograph showing aggregation of virus particles within vacuoles following infection of FCoV UPM11C/08 isolate on Fcwf-4 cell
culture. Some parts of the vacuoles membrane were no longer intact and leading to spillage of virus-like particles into cytoplasmic matrix
(arrows). Scale bar, 1 µm.
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possibility that the extracellular particles and those
that invaginated the cell membrane were the same
viruses present within the vacuoles based on their
similar morphological features. However, contrary
to many published reports pertaining to the replica-
tion and budding of the FCoV particles [20,21,
27–29], no such proliferative growth involving the
vacuolar membrane was reported.
A smooth enveloped electron-opaque virus-like
particle detected in this study was found to
accumulate in a distinct rounded, firm and thicker
vacuole. Similarly, there is no evidence of replica-
tion and budding of virus from this vacuoles mem-
brane. We believed that the two morphologically
different virus-like particles observed in this study
are in fact FCoV as at this juncture of study we are
not able to show the transformation between these
two distinct particles. Probably, the sizes reflect
different maturation stages of the virus particles.
Further, the significance of the two types of vacu-
oles with structurally different virus particles con-
tained is also not known. In this context, it is again
tempting to postulate whether the artificial environ-
ment and the use of Fcwf-4 cell cultures could
induce the formation of two different vacuoles con-
taining two distinct particles. Somewhat similar
vacuoles containing virus particles have been noted
in reports by previous workers [14,20,28,30], but
these authors provided neither the structural details
of these vacuoles nor the virus particles they
contained.
In both types of vacuoles observed in the present
study, there were clear evidences to indicate the
vacuole membrane did not remain intact as spillage
of the virus was detected in the cytoplasmic matrix.
Thus, apart from the presence of virus particles
within vacuoles, the particles were also present
freely in the cytoplasmic matrix. When clusters of
the particles were present in the vicinity of the
Golgi complex, single particle was also present in
the vesicle of the Golgi apparatus.
Conclusion
It is concluded from the present study that follow-
ing the infection of FCoV in Fcwf-4 cell culture,
two different morphologically virus-like particles
were seen in their respective vacuoles. The vacu-
oles could be the site for virus replication although
no evidence of budding from the vacuolar mem-
brane was observed in this study. Rupture of vacu-
oles led to the spillage of the virus-like particles
into the cytoplasmic matrix.
Fig. 7. Micrograph showing virus-like particles in the vesicle of Golgi apparatus (arrow) and aggregations of extracellular virus-like particles
(arrowhead) following infection of FCoV UPM11C/08 isolate on Fcwf-4 cell culture. Scale bar, 1 µm.
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Funding
This work was supported by the Ministry of
Science, Technology and Innovation (MOSTI),
Malaysia, project number 02-01-04-SF0485:
Development of a rapid test for diagnosis of FCoV.
Acknowledgements
The authors would like to thank the staff of the Imaging Unit, Institute
of Bioscience, UPM.
References
1 Rohrbach B W, Legendre A M, Baldwin C A, Lein D H, Reed W M,
and Wilson R B (2001) Epidemiology of feline infectious peritonitis
among cats examined at veterinary medical teaching hospitals.
J. Am. Vet. Med. Assoc. 218: 1111–1115.
2 Pedersen N C (1995) An overview of feline enteric coronavirus and
infectious peritonitis virus infections. Feline Pract. 23:7–22.
3 Addie D D and Jarrett O (1992) A study of naturally occurring feline
coronavirus infections in kittens. Vet. Rec. 130: 133–137.
4 Pringle C (1999) Virus taxonomy–1999. Arch. Virol. 144: 421–429.
5 Cavanagh D (1997) Nidovirales: a new order comprising
Coronaviridae and Arteriviridae.Arch. Virol. 142: 629–633.
6 Holzworth J E (1963) Some important disorders of cats. Cornell Vet.
53: 157–160.
7 Wong W T, Cheng B Y, and Lee J Y S (1983) Feline infectious perito-
nitis –two case reports. Kajian Vet. Malaysia 15:30–35.
8 Pedersen N C, Boyle J F, Floyd K, Fudge A, and Barker J (1981) An
enteric coronavirus infection of cats and its relationship to feline
infectious peritonitis. Am. J. Vet. Res. 42: 368–377.
9 Ward B C and Pedersen N C (1969) Infectious peritonitis in cats.
J. Am. Vet. Med. Assoc. 1:26–35.
10 Addie D D, Toth S, Murray G D, and Jarrett O (1995) Risk of feline
infectious peritonitis in cats naturally infected with feline corona-
virus. Am. J. Vet. Res. 56: 429–434.
11 Arshad S-S, Lee W W, Hassan L, Kamarudin A I M, Siti-Farawahida A
W, and Cheng N A B Y (2004) Serological survey of catteries for cats
infected with feline coronavirus. J. Vet. Malaysia 17:19–22.
12 Pedersen N C, Black J W, Boyle J F, Evermann J F, McKeirnan A J,
and Ott R L (1984) Pathogenic differences between various feline
coronavirus isolates. Adv. Exp. Med. Biol. 173: 365–380.
13 Motokawa K, Hohdatsu T, Aizawa C, Koyama H, and Hashimoto H
(1995) Molecular cloning and sequence determination of the peplo-
mer protein gene of feline infectious peritonitis virus type I. Arch.
Virol. 140: 469–480.
14 Lai M and Cavanagh D (1997) The molecular biology of corona
virus. Adv. Virus. Res. 48:1–100.
15 Pedersen N C, Evermann J F, McKeirnan A J, and Ott R L (1984)
Pathogenicity studies of feline coronavirus isolates 79-1146 and
79-1683. Am. J. Vet. Res. 45: 2580–2585.
16 Holmes K V and Lai M (1996) Coronaviridae: the viruses and their
replication. Fields Virology, 3rd edn., pp: 1075–1093
(Lippincott-Raven Publishers, Philadelphia, PA).
17 Pedersen N C (1987) Virologic and immunologic aspects of feline
infectious peritonitis virus infection. Adv. Med. Biol. 218:
529–550.
18 Jacobse-Geels H E and Horzinek M C (1983) Expression of feline
infectious peritonitis coronavirus antigens on the surface of feline
macrophage-like cells. J. Gen. Virol. 64: 1859–1866.
19 Mohandas D V and Dales S (1991) Endosomal association of a
protein phosphatase with high dephosphorylating activity against a
coronavirus nucleocapsid protein. FEBS Lett. 282: 419–424.
20 Pedersen N C (1976) Morphologic and physical characteristics of
feline infectious peritonitis virus and its growth in autochthonous
peritoneal cell cultures. Am. J. Vet. Res. 37: 567–572.
21 Beesley J E and Hitchcock L J (1982) The ultrastructure of feline
infectious peritonitis virus in feline embryonic lung cells. J. Gen.
Virol. 59:23–28.
22 Sharif S, Arshad S-S, Bejo M-H, Omar A A, Zeenathul N, and Hafidz
M A (2009) Prevalence of feline coronavirus in two cat populations
in Malaysia. J. Feline Med. Surg. 11: 1031–1034.
23 Gelderblom H, Renz H, and Özel M (1991) Negative staining in diag-
nostic virology. Micron Microsc. Acta 22: 435–447.
24 Hayat M A (1986) Basic techniques for transmission electron
microscopy (Academic Press, New York).
25 Reynolds E S (1963) The use of lead citrate at high pH as an
electron-opaque stain in electron microscopy. J. Cell. Biol. 4:
208–212.
26 Hoshino Y and Scott F W (1980) Immunofluorescent and electron
microscopic studies of feline small intestinal organ cultures
infected with feline infectious peritonitis virus. Am. J. Vet. Res.
41: 672–681.
27 Weiss R C and Scott F W (1981) Pathogenesis of feline infectious
peritonitis: nature and development of viremia. Am. J. Vet. Res. 42:
382–390.
28 Ward J M (1970) Morphogenesis of a virus in cats with experimental
feline infectious peritonitis. Virology 41: 191–194.
29 Evermann J F, Heeney J L, McKeirnan A J, and O’Brien S J (1989)
Comparative features of a coronavirus isolated from a cheetah with
feline infectious peritonitis. Virus. Res. 13:15–27.
30 Klumperman J, Locker J K, Meijer A, Horzinek M C, Geuze H J, and
Rottier P J M (1994) Coronavirus M proteins accumulate in the Golgi
complex beyond the site of virion budding. J. Virol. 68: 6523–6534.
8J O U R N A L O F E L E C T R O N M I C RO S C O P Y , 2011
by guest on May 19, 2011jmicro.oxfordjournals.orgDownloaded from