J. gen. Virol. 0978), 39, 537-540
537
Printed in Great Britain
A Classification o f Feline Calicivirus Isolates Based
on Plaque Morphology
(Accepted 26 December 2977)
SUMMARY
Isolates of feline calicivirus (FCV) can be divided into four groups according
to plaque size under an agar overlay. All isolates classified as minute plaque, along
with certain other isolates, are sensitive to inhibitors present in agar.
Since the first isolation of a feline calicivirus (FCV) (Fastier, I957) it has become obvious
that these viruses are ubiquitous among cat populations throughout the world. One factor
of particular note in this group is marked inter-strain variation in certain characters: in
antigenicity (Povey, I974), pathogenicity (Hoover & Kahn, I973; Povey & Hale, ~974),
and apparently in tissue tropism (Wardley & Povey, I977). In this communication we
report on studies of two markers which reflect genetic variation: plaque size and sensitivity
to inhibitors in agar. Differences in plaque size among feline calicivirus isolates has been
reported before (Prydie, I973; Kalunda et al. I975) but there has been no previous account
of inhibition of FCV plaque formation by agar.
FCV strains were obtained from isolates made here and from other laboratories in
Europe and the U.S.A. Plaque assays were performed in 5 cm polystyrene plates containing
confluent monolayers of a feline embryo cell line, FEA (Jarrett et al. I973). The cells were
cultured in Eagle's MEM (Glasgow modification) with IO % foetal bovine serum and were
maintained at 37 °C in an atmosphere of 5 % CO2 in air. The growth medium was removed
and Ioo #1 of virus suspension, initially diluted in MEM, was inoculated into each plate.
Virus was adsorbed for 6o rain at 37 °C. The inoculum was then removed and the monolayers were washed in medium before application of the overlay. The agar overlay consisted of o'9 % bacto.agar (DIFCO) in Eagle's MEM with 5 % foetal bovine serum and
agarose overlays were identical except for the substitution of bacto-agar with agarose
(Miles Laboratories). Plates were maintained at 37 °C for 48 h before fixing and staining
with o'5 % crystal violet in IO % formalin. Plaque diameters were measured using a photographic enlarger at x 2 magnification. For each FCV strain the mean plaque diameter
was calculated from the measurement of between 50 and Ioo plaques.
Glasgow isolates were obtained from cats attending a veterinary clinic. Mouth swabs
were taken into MEM and the medium was divided into two: the first sample was used
for initial screening in which FCV isolation was recognized by the production of a
characteristic cytopathic effect in FEA monolayers. If positive for FCV, the second sample
was examined using an agar overlay as described above. Virus clones were produced by
three cycles of growing virus from single plaques in plates containing only one or two
plaques. After the third cloning, the virus suspension was dispensed in samples and stored
at - 7 o °C for future stock production. Stocks were made by infecting FEA monolayers
and harvesting the culture after 24 h. The suspension was ultrasonicated for 30 s, centrifuged at 2ooo rev/min for I5 min to remove cell debris and the supernatant fluid was
stored at - 7 o °C.
There was wide variation in plaque size under agar between FCV strains. Fig. I shows
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538
Agar
Aearose
1
2
3
4
Fig. i. Plaques produced by four FCV isolates. An agar overlay was used in the top row of plates
and an agarose overlay in the bottom row. I = FCV GIO [mean plaque diameter (m.p.d.) < I mm
agar, 2'4ram agarose]; z = KCD (m.p.d. I'7mm agar, 2.Imm agarose); 3 = FCV G2
(m.p.d. 2"5 mm agar, I"5 mm agarose); 4 = FCV GI (m.p.d. 3"6 mm agar, 2"3 mm agarose).
representative plates f r o m the assay o f four strains. Plaque sizes o f G I , G2 and F I I were
f o u n d to occur in a normal, or near normal, distribution whereas the frequency distribution
for plaques o f Gxo was positively skewed, most likely due to an ' i n f r a p l a q u e ' effect
(Lindenmann & Gifford, I963). These strains produced four distinct populations o f
plaque sizes (P < o ' o 0 and represent the four plaque size groups o f F C V (see Table I).
The minute plaque (MP), small plaque (SP) and large plaque (LP) groups are more or
less equally represented a m o n g the available isolates, but strains belonging to the fourth
group, producing 'extra-large' plaques (EP), are u n c o m m o n : we have now examined
34 F C V strains of which only three were classed as EP.
Next, agarose overlays were used to determine whether plaque size might be influenced
by inhibitory substances present in agar: agarose is the agar fraction free o f sulphated
polysaccharides which are k n o w n to inhibit virus multiplication in several virus-cell
systems (Takemoto, 1966). Agarose forms firmer gels than agar and so, unless inhibition
occurs with agar, the plaque size tends to decrease under agarose overlays. However, in
certain strains the plaque size was dramatically increased, e.g. G I o (see Fig. I). I f the
mean plaque diameter increased by twofold or more, the sensitivity to agar was scored
' + + ' (see Table I). The sign ' + ' indicates an increase in size, but less than twofold. As
can be seen from Table I, all o f the M P group were f o u n d to be sensitive to agar inhibitors
along with some o f the SP group. Only one strain o f the L P group, strain F P L , was
sensitive.
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539
Table ~. Plaque morphology of feline calicivirus isolates
Mean plaque diam.
(mm)
Sensitivity to
agar inhibitor
Plaque group
Strain
EP
GI
F P V 255
3"6
3"6
-
LP
F9
CFI
G2
FS
FPL
3" I
2- 9
2' 5
2'4
2"3
--+
SP
KCD
Ft~
FC
F5
I '7
t'7
i '4
i-3
+
+
MP
FI7
FJ
FRV
Fro
FI9
Gio
<
<
<
<
<
I
i
I
I
t
I
+ +
+
+
+
+
"~ ~-
To demonstrate further that the inhibition of these strains was associated with the
sulphated polysaccharide (polyanion) fraction of agar, a polycation, diethylaminoethyldextran (DEAE-dextran: Sigma Chemical Company), was added to the standard agar
overlay and the effect on plaque size was examined. This procedure was first described
by Liebhaber & Takemoto 096I) for EMC virus. As expected, strains showing an increased
plaque size with agarose produced larger plaques when DEAE-dextran was added to the
agar overlay. For example, the plaques of strain GIO, found to be of the MP type with
o to IOO/zg DEAE-dextran per ml of overlay, were converted to LP-size plaques with
500 to ~ooo #g/ml. Strain GI produced plaques of equal size with o to Iooo/zg DEAEdextran/ml in the overlay.
We are interested in finding out whether the mechanisms which underlie the differences
between FCV isolates which are apparent by the methods employed here, are relevant to
other differences, particularly pathogenicity. Kalunda et al. 0975) reported that in
neutralization tests using plaque reduction there was no correlation between plaque size
and serotype, and Prydie (I973) could find no correlation between plaque size and pathogenicity. However, our results suggest that a more strict classification based on plaque
morphology is required before any such comparisons can be made. For example, it has
been reported that different preparations of commerical agar may vary in their inhibitory
properties (Wallis et al. I962). A standardized assay method, such as the one detailed
here is required. Sub-classification based on sensitivity to agar inhibitors helps to further
characterize any new isolate. In addition, new isolates should be plaque cloned: during
this study we have encountered two isolates where a mixed plaque type was present on
primary isolation (EP/LP mixture and LP/MP mixture), and obviously an erroneous
result would be obtained if an attempt was made to describe and then classify such a
mixed population in terms of plaque size.
It may be relevant that in a study of another major virus of this genus, vesicular
exanthema virus of swine, it was recognized that there was variation in plaque morphology
both between and within antigenic types, and that a cloned LPF (large plaque former)
was pathogenic whereas a cloned MPF (minute plaque former) was not (McClain et al.
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540
Short communications
1958). Work is now in progress here to examine the pathogenicity of representative strains
from each FCV plaque type, and early results indicate a correlation between the MP
group and low pathogenicity, and the EP group with a high level of pathogenicity. If this
is found to be a general rule it may help to define the basis of variation in pathogenicity
among FCV strains.
E. Ormerod holds a Research Training Scholarship of the Wellcome Trust. We thank
Drs F. Btirki, E. Davies, E. A. Hoover and R. C. Povey for supplying virus stocks.
University of Glasgow
Department of VeterinaryPathology
Bearsden
Glasgow
Scotland
E. ORMEROD
O. JARRETT
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(Received 15 November 1977)
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