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J. gen. Virol. 0976), 3o, I-9
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Kelp Fly Virus
By P. D. S C O T T I * , A. J. G I B B S * AND N. G. W R I G L E Y t
*Research School of Biological Sciences, Australian
National University, Canberra, 26oi, Australia, and tNational Institute for
Medical Research, Mill Hill, London, NW7 tAA, U.K.
(Accepted I September I975)
SUMMARY
A virus from adult kelp flies (Chaetocoelopa sydneyensis) has been cultured in
larvae of the waxmoth (Galleria mellonella). This virus has isometric particles that
are 29_+ I nm in diameter and resemble reovirus particles in appearance. Some
chemical and physical properties of these particles have been determined. They
contain single-stranded RNA, but have a unique set of the properties of the particles reported for other picornaviruses, and differ from all in surviving for IO min
at 90 °C but not Ioo °C. The cryptogram of kelp fly virus is R/1:3"5/*:S/S:I/*.
INTRODUCTION
Adult kelp flies (Chaetocoelopa sydneyensis Schin.; Diptera, Phycodromidae) were
collected from rocks at Wapengo on the South Coast of New South Wales, Australia. The
larvae of these flies feed in kelp lying on beaches, and the adults congregate during winter
in large numbers on the spindrift-moistened undersurface )f overhanging rocks near the
high water level. A water extract of about to ~ apparently healthy flies collected from one
such cluster containing about 2 × IO5 individuals was examined in the electron microscope,
but no virus-like particles were found. The extract was then injected into grubs of the
greater waxmoth (Galleria mellonella), and after 4 days all the grubs had died. Extracts of
the dead grubs contained isometric virus-like particles, which were not found in extracts
of comparable uninoculated grubs. We have shown that these are the particles of a virus
which we call kelp fly virus (KFV) and which has subsequently been isolated from kelp
flies collected from several coast sites within 250 km of Canberra. In this paper we report
some properties of KFV, which seems to be distinct from any other previously described
virus.
METHODS
Virus. As stated above, kelp flies were tested for KFV by injecting water extracts of them
into the haemocoeles of Galleria mellonella grubs and subsequently the virus was bioassayed
or grown for study, by passaging in G. mellonella. Extracts of insects either for inoculum,
or for virus purification, were prepared by homogenizing the insects in 0"05 M-ammonium
acetate and carbon tetrachloride (about o.~ g insect/ml/ml). The emulsion was centrifuged
at 8oo0 g for 5 min and the aqueous phase collected. For this work penultimate instar grubs
were used, and were infected by injecting each with about 2. 5 #l of inoculum through a
proleg. For virus purification the inoculated grubs were kept for 3 days at 3o °C and
either used immediately or stored at - 2 o °C. In bioassay tests the grubs were kept for
4 days at 3o °C and the dead removed and recorded daily.
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2
P.D. SCOTTI~ A. J. GIBBS AND N. G. WRIGLEY
Virus purification. Extracts of infected grubs were prepared, as described above, except
that a few crystals of phenylthiocarbamide were added to suppress melanin formation.
Each extract was then rehomogenized several times with carbon tetrachloride to remove
lipid, and the virus particles sedimented either by centrifuging at t 2 o o o o g for I h or by
adding polyethylene glycol (tool. wt. 6oo0) to IO~o (w/v) and sodium chloride to 0"5 M and
centrifuging at 5ooog for 20 rain. Sedimented virus particles were resuspended in o'05
M-ammonium acetate, clarified by centrifuging at 8ooog for to rain, and the suspension
layered on a lO to 4o% (w/v) sucrose gradient and centrifuged for 2 h at 9oooog. The
light-scattering band was collected, and after dilution or dialysis the virus particles were
sedimented and concentrated as described above. The particles were further purified by
centrifuging in a caesium chloride gradient in neutral o.oI M-tris for 16 h at 9o0o0 g. The
light-scattering band was collected, dialyzed against o'o5 M-ammonium acetate, concentrated
as above, and stored at 4 °C.
Serology. Antisera were prepared by injecting rabbits intramuscularly twice at a fortnight's interval with virus preparations (about 2 mg virus/injection) emulsified in Freund's
complete adjuvant.
Serological testswere done either by the two dimensional diffusion-in-gel method (Mansi,
I958 ) or by titration in mixed liquids (Bawden 1956).
Stability tests. The stability of the virus at different temperatures was measured using
purified virus preparations diluted to an E2,o of I-o and then further diluted ioo-fold with
distilled water or neutral solutions of various salts. Portions were heated at different
temperatures for IO min in screw-cap glass tubes, cooled rapidly in tap water and then stored
in ice until tested for infectivity.
The acid stability of the virus particles was also measured using preparations diluted
similarly into o.i M-acetate buffer at various pH values. After 15 min at room temperature
the samples were mixed with 9 vol. of neutral o.2 M-tris buffer, and their infectivity tested.
The rate of inactivation of KFV particles by u.v. light was estimated using a purified
preparation at I E26o/ml (I cm path) which was then diluted I × Io-~-fold with 0"05 Mammonium acetate. 1"5 ml samples were irradiated for different periods in glass Petri
dishes 3o cm from a portable u.v. lamp (Mineralight I44W ). The samples were diluted with
3 vol. of buffer and their infectivity tested.
Biochemical analyses. KFV preparations were tested for nucleic acid by the orcinol and
diphenylamine methods (Schneider, 1957). Similar preparations were used to determine the
base composition of KFV nucleic acid by the acid hydrolysis and chromatography method
of Markham 0955), except that the chromatographic separation was done on 0.25 mm
thick layers of Machery and Nagel MN 3o0 cellulose using an isopropanol:water:HC1
(70: 24: 6) solvent.
For the estimation of amino acid composition of virus particles, preparations were
hydrolysed for 24 h in performic acid, and the resulting amino acids separated and assayed
in a Beckman amino acid analyser.
Biophysical analyses. The sedimentation coefficient (s20' o3 of KFV particles was estimated using a Beckman Model E ultracentrifuge or an M.S.E. Centriscan. Their density was
determined in CsC1 solutions also by centrifugation. Approx. o'o5 O.D. units of purified
virus particles were mixed with caesium chloride in o'ot M-tris and were centrifuged at
44ooo rev/min at 20 °C. The initial density of the CsC1 solution was measured in a Zeiss
refractometer at 25 °C and corrected to 2o °C using the relationship P20 = I38"o4/(I38"I I
(I/P25)- o'38) derived from published data (Dawson et al. I969). After centrifuging for t 6 h
the cells were scanned at 260 or 254 nm, and the densities of the peaks calculated using the
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Kelp Fly Virus
3
point on the gradient at which the initial density occurs, as a reference marker (the 'hinge
point method'), as described by lift, Voet & Vinograd (196I).
The KFV genome was extracted from particles suspended in o.I SSC by shaking twice
with phenol saturated with o. t SSC and then several times with ether. The strandedness of
the genome in o.I SSC was assessed by measuring its thermal hyperchromicity when heated
from 20 °C to 85 °C, at a rate of ~ °/min, using Escherichia coli D N A for comparison. The
size of the genome was estimated for us by Russell Regnery by comparing its rate of electrophoretic migration in I-5% agarose gels (Davey, 1973) with that of the Semliki Forest virus
and tobacco mosaic virus RNA genomes, which are 4"3 and 1.6 x lO 6 and 2.0 x lO 6 daltons
respectively.
The size and number of proteins in KFV particles was also assessed by comparing their
electrophoretic mobilities with those of various standard marker proteins in 5 to 15%
polyacrylamide gels (Weber & Osborn, I969).
Electron microscopy. Virus samples were mixed with 4% aqueous sodium silicotungstate,
dried on carbon-coated grids and examined in a Philips 3Ol electron microscope. The
magnification of the micrographs (about x 6o5oo) was calibrated using the 8-6 nm spacing
of fixed catalase crystals in separate micrographs.
RESULTS
Effect of temperature on yieM of KFV
The virus content of extracts of inoculated grubs kept at different temperatures was
estimated by serological titration which showed that after 3 days more virus was obtained
at 3o °C than at higher or lower temperatures (r5"5, 2o, 26, 34"5 °C) though the yield at
26 °C was similar to that at 30 °C; at 30 °C, I g of grubs yielded about 70 #g of virus.
Infectivity of KFV
The infectivity of a range of dilutions of KFV preparations were estimated as described
above.
KFV particles probably have a slightly greater mass than poliovirus particles (9 × lOG
daltons; Cooper et al. I97 I) because they have a larger RNA (see below); we assume a
particle mass of about I x io 7 daltons. One can assume by analogy with other viruses
(Gibbs & Harrison, 1975) that the extinction of KFV particles in 26o nm wavelength light
(I cm path length) should be about 7 0 . D . units/mg/ml. The infectivity experiments
indicated an LDs0 of approx. 2ooo particles/grub.
The physical properties of KFV particles
Purified preparations of KFV contained many isometric particles about 29_+ I n m in
diam., which were not found in preparations of uninfected grubs.
High resolution electron micrographs of these particles showed that they are strikingly
similar to the 'cores' of reovirus particles (Luftig et al. I972), though the latter were much
larger (52 nm diam.). Like the reovirus cores, the KFV particles had surface projections
apparently located on fivefold icosahedral axes (Fig. I). These projections, sometimes seen
end-on (Fig. I ; bz-b5), protruded some 8 nm from the virion surface with a hollow bell-like
shape about 8 nm diam. at the inner and increasing to about 12"5 nm at the outside end.
Separate brick-shaped subunits (Fig. I ; d4 and d5) were usually also found in the preparation and these also measured about 8 x I2 rim. However it is not clear what relation these
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4
v . D . SCOTTI, A. J. GIBBS AND N. G. W R I G L E Y
1
2
3
4
5
6
Fig. t. Selected particles of KFV at x 400000. Row a and frames bl and b4 show particles in fivefold orientation, frame b2 a threefold orientation (though this was relatively uncommon), and
rows c and d particles in twofold orientation, giving rise to our belief that their structure is icosahedral. In addition frames b2, b3, b4, and b5 show the proiecting subunits not merely at the periphery but also in end view in the particle surface. The large frame at lower right ( x 2ooooo) shows
the free brick-shaped subunits mentioned in the text.
have to the K F V particle, nor whether they originate from a missing outer layer o f
capsomers like that which is found in reovirus particles.
In the analytical centrifuge, K F V particles sedimented as a single c o m p o n e n t ; its mean
sedimentation coefficient (two estimates on each of three preparations) was 158S.
The apparent density (P20) o f K F V particles in CsC1 was ~.4z5 + 0.002 at p H 7, 1.43o at
p H 8 (only one determination), and 1.467 + o'oo5 at p H 9. The apparent density thus increased with p H over the range tested. Similar increases o f density in CsC1 have been
observed for bee acute paralysis virus (Newman et aL 1973) and for F M D V (Rowlands,
Sangar & Brown, t97~). This increase in density m a y result from a slow interaction between
the virus particles and the CsCI; in one experiment at p H 7, the density increased from
p = I'418 x2 h after the start o f centrifuging to a stable value o f 1.423 after ~7 h. This
result is similar to that for F M D V as reported by Rowlands et al. (~97~). Purified preparations o f K F V particles had u.v. absorption spectra typical o f nucleoproteins. They had a
clear peak in extinction at 259 n m wavelength and the mean E260/280 ratio of 2o preparations
was ~-65 (uncorrected for light-scattering).
The composition of K F V particles
Chemical tests showed that K F V particles contain R N A , and no detectable D N A , but
these tests do not indicate whether K F V R N A is single- or double-stranded. To distinguish
between these possibilities, R N A f r o m the particles was thermally denatured and its hyperchromicity measured (Fig. 2). There was a gradual increase in extinction of about 2o %
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Kelp Fly Virus
I
0'90
[
I
I
i
F
0'80
1
I
I
I
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g
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1
0"70
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J
f
J
0"62 20
I
I
30
40
I
I
50
60
Temperature (C)
I
I
70
80
Fig. 2. Effect of temperature on the optical density of KFV RNA and E. coli DNA,
, KFV RNA ; - - ,
E. coli DNA.
I
I
I
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I
4
KFV RNA o
T
x 3
RNA
O•TMV
1
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I
l0
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Distance migrated (cm)
I
30
Fig. 3- Relative positions of RNAs in agarose gels plotted against the tool. wt. of RNA.
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6
1'. D. SCOTTI, A. J. GIBBS AND N. G. WRIGLEY
1
I
I
I
I
I
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I
m
-e BSA
Catalase~ x'NNe. Fumarase
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KFVP
1
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arbonic
2 ~rase__
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0-5
Relative mobility
1.0
Fig. 4- The relative positions in I 2 ~oo polyacrylamide gels of KFV capsid proteins and
proteins of known tool. wt. plotted against tool. wt.
between 20 °C and 70 °C, which indicates that the RNA is principally single-stranded, with
about 40 % of its bases paired.
The size of the RNA extracted from KFV particles was estimated electrophoretically in
1.5 % agarose gels to be about 3"5 x io 6 daltons (Fig. 3).
The product obtained by acid hydrolysis of KFV particles or of extracted RNA gave four
u.v. absorbing spots when fractionated on thin cellulose layers. These spots had Rf values
close to those reported by Markham (~955) for guanine, adenine, cytidylic acid and uridylic acid, and when eluted were found to have the absorption spectra of these compounds.
The mean base ratio of four such determinations was: guanine I8.8 + 1.6; adenine 34"0 i I"3;
cytosine I8.6 ± o'9; uracil 28.6 + 0"5. The base ratios also provide further evidence that the
RNA is single-stranded.
KFV particles were chemically disrupted and the resulting separated proteins analysed
by electrophoresis in polyacrylamide gels. Two major proteins were found and these had
average estimated tool. wt. of 73 ooo + 7oo and 29 400 _+750 (mean of six analyses) (Fig. 4)The optical densities of these two proteins in stained gels were in the ratio I : 2 respectively.
Hence, assuming that these proteins fix stain in amounts determined solely by their mass,
they are present in KFV particles in a ~ :5 molar ratio.
Several minor protein species were also obtained even when the proteins had been
carboxymethylated before analysis and these were particularly well separated in the I2 %
polyacrylamide gels (Fig. 5). We do not know whether these minor species are artefacts,
minor components of the particles or absorbed contaminant proteins.
The amino acid composition (moles %) of the unfractionated proteins of KFV particles
was: ala, 8.1; arg, 3"2; asp, I8.6; cysteic acid, 1.7; glu, 8"9; gly, 8.o; his, 0.4; ile, 5"7; leu,
9"2; lys, 4"7; met sulphone, 2.2; phe, o'I5; pro, 7"5; set, 6.2; thr, 6.I; trp, not determined;
tyr, o'4; val, 8"9. These data were analysed by the FITMOL method (Gibbs & Mclntyre,
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Kelp Fly Virus
7
KFVP 1
KFVP 2
Top
Front
Migration
Fig. 5- Densitometer tracing of a typical i2 ~ polyacrylamide gel after staining with Coomassie
brilliant blue. KFV particles were disrupted by heating in a buffered solution of i ~ SDS and
0"5 ~ 2-mercaptoethanol and separated electrophoretically in the gel.
I97O). There was no clear evidence from this analysis that the protein contained an integral
number of amino acids between Ioo and looo residues. This suggests either that the protein
of the particle contains more than Iooo amino acids, or that it contains no amino acids
present as I to 3 molecules per polypeptide, or, more likely, that it is a protein mixture
whose constituents are not present in integral molar amounts.
Stability of KFV particles
K F V particles are unusually resistant to heat, and retained infectivity even after io min
at 9o °C though not after lo min at 98 °C or after autoclaving for IO min. Furthermore
there was no detectable loss of infectivity at 8o °C, though in some experiments there was
usually some loss at 9 ° °C, indicating a large temperature coefficient (Qx0 o) of inactivation.
Magnesium sulphate or sodium chloride had no detectable effect on the heat stability of
K F V (Table I); however infectivity tests on KFV heated in magnesium chloride solutions
at 80 °C showed that the salt decreased virus stability at least IoS-fold.
There was no detectable effect of pH on the infectivity and stability of K F V particles
between p H 3 and p H 7.
Ultraviolet irradiation of K F V preparations also indicated, like the dilution experiments,
a first-order inactivation and hence that the genome of K F V is probably not divided
between several particles.
DISCUSSION
The virus we have isolated appears to cause no symptoms in kelp flies. It could not be
detected by electron microscopy of an extract of the flies. The particles replicate in Galleria
mellonella larvae and were initially detected only in wax moth larvae injected with an extract
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+
I87
175
I6o
160
146
15o
Poliovirus
EMC
FMDV
H u m a n rhinovirus
~'42
1'40
I "33
I"43
1"34
1"36
1.35
1.34
I'34
1"33
1 "37
2"6
2.6
2.6
2.6
N.D.
N.D.
e. 2
>z
2-6
N.D.
c. 3'5
I
I
!
!
N.D.
N.D.
I
1
1
N.D.
I
StrandedSize
ness
20
24
24
25
I9
zi
I9
I9
G
34
27
26
29
3o
32
32
34
A
RNA
20
24
28
22
N.D.
N.D.
zI
18
18
N.D.
I9
C
26
25
22
25
3o
3o
31
29
U
5
5
7
7
N.D.
N.D.
N.D.
2
N.D.
N.D.
z
8
42, 32, 3z,
4o, 29, 29,
I8, I4,
> 35, 35, 30,
23,
25, I I
29,
lO
25, 8
42, 35, 28, 28, 26,
N.D.
N.D.
N.D.t
32, 3o
N.D.
N.D.
73, 29
Proteins
~ - ~
~
Number
Sizes× io -3
Some physico-chemical properties of picornavirus particles*
< 60 °C
< 60 °C
< 56 °C
5 ° °C
N.D.
N.D.
e. 5o °C
5o °C
c. 5o°C
N.D.
> 8 o °C
r
Thermal
inactivation
point
Inactivated
Unstable
Inactivated
Stable
N.D.
N.D.
Stable
Stable
Inactivated
N.D.
Stable
At pH3
Stability
~
N.D.
N.D.
N.D.
Stable
N.D.
N.D.
N.D.
Stable
N.D.
N.D.
Unstable
I n MgCI~
* T h e i n f o r m a t i o n in this Table is f r o m this paper a n d also f r o m Bailey & W o o d s (I974); Bellett (1967); B r o w n & Hull (I973); Brown, N e w m a n & Stott (I97o);
Burness, F o x & P a r d o e (1974); B u t t e r w o r t h (I973); N e w m a n e t al. (1973); R e i n g a n u m 0 9 7 3 ) ; R o w l a n d s , S a n g a r & B r o w n (1971); V a n d e W o u d e , Swaney &
B a c h r a c h (i 972) ; V a n d e n Berge & Boey6 (1972); Wallis, Melnick & R a p p (1965); Wildy (i 97 x).
N o t determined.
--
--]-
+
+
+
-
16o
I67
157
138
Bee acute paralysis
Cricket paralysis
Sacbrood
A r k a n s a s bee virus
(major c o m p o n e n t )
Bee virus X
Bee slow paralysis
-
I58
KFV
Virus
S
Accessory D e n s i t y
value particles
× 1o -G
T a b l e ~.
.z
;>
Z
G')
C3
.>
O
©
o'1
O0
Kelp Fly Virus
9
from kelp flies. N o similar particles were detected in uninfected grubs or from grubs injected with extracts of other insects. K F V has also been isolated on other occasions from
kelp flies obtained at different localities several months after the original collection. These
facts strongly suggest that the virus described in this paper does come from the kelp flies
and is not a virus carried latent in G. rnellonella grubs. As yet we have no information on
the ecology of KFV, though it seems to be most common in adult kelp flies in winter when
they are gregarious.
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