J. gen. Virol. (1982), 60, 115-123.
Printed in Great Britain
115
Key words: invertebrate RNA virus/calicivirus/virion disintegration
An Invertebrate Calici-like Virus: Evidence for Partial Virion
Disintegration in Host Excreta
By B. H I L L M A N ,
T. J. M O R R I S , * W. R. K E L L E N , 1 D. H O F F M A N 1
AND D. E. S C H L E G E L
Department of Ptant Pathology, University of California, Berkeley, and XStored Product
Insects Research Laboratory, AR, SEA, USDA, Fresno, California, U.S.A.
(Accepted 15 December 1981)
SUMMARY
A virus with morphological and physicochemical properties similar to those of the
vertebrate caliciviruses was isolated from navel orangeworms, Amyelois transitella
(Walker). Infected larvae contained two types of virus particles: a 185S, 38 nm
cupped particle (ACSVi) with a single major polypeptide of 70000 mol. wt. and a
165S, 28 nm smooth particle (ACSVii) with a single major polypeptide of 29000
tool. wt. Larval frass contained a heterogeneous population of virus particles.
Evidence is presented which suggests that the 38 nm particle degrades by proteolytic
digestion to produce predominantly 28 nm particles in frass. Virus particles contained
a single-stranded RNA of 36S (about 2.5 x 106 mol. wt.).
INTRODUCTION
The name 'calicivirus' has been adopted to describe a family of small RNA viruses which
have characteristic cup-shaped indentations on the surface of the virions (Schaffer, 1979).
Caliciviruses have been isolated from cats, swine, and several marine mammalian hosts. Based
on virus transmission studies and isolations from fish and a trematode, a calicivirus cycle
involving invertebrate parasites, fish and marine animals (with swine as an alternate host) has
been suggested (Smith et al., 1980a, b). In the former report the authors suggested that
California is a focus for calicivirus activity. KeUen & Hoffman (1981) have described a virus,
designated chronic stunt virus (ACSV), associated with only the granular haemocytes of
diseased navel orangeworms, Amyelois transitella (Walker), collected from almonds in
northern California. The virus was readily transmitted perorally, being lethal to neonate larvae
but attenuated in older larvae, causing retarded growth and delayed mortality. We report this
as the first insect pathogenic calici-like virus.
METHODS
Virus and host culture. Larvae were reared on an artificial bran diet in glass jars at 28 °C
as described by Kellen & Hoffman (1981). Infected colonies were maintained by
contaminating the diet with an inoculum consisting of crude extracts of infected larvae in
phosphate-bufered saline (PBS) when larvae reached the third instar. Larvae and frass were
separated from the diet by repeated straining through a fine mesh screen and were stored
frozen until used. Viruses used for standards were maintained and purified as described
previously (Morris et al., 1979). Cricket paralysis virus (CrPV) was isolated from diseased
crickets sent to the laboratory for diagnosis and was handled according to Reinganum et al.
(1970).
Virus purification. Samples were homogenized in 3 vol. (w/v)0.01 M-ammonium acetate at
pH 6-8 with a Polytron ® and clarified by centrifugation at 24000 g for 10 min at 4 °C. The
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116
B. HILLMAN AND OTHERS
virus was purified by precipitation with 8 % polyethylene glycol 6000 and ultracentrifugation
through 30% sucrose in the SW50.1 rotor (45000 rev/min for 90 min) as described
previously (Morris et al., 1979).
Virus analysis. Sucrose density gradient centrifugation for both purification and estimation
of sedimentation coefficients was performed in linear-log gradients in 0.01 M-ammonium
acetate according to Brakke & Van Pelt (1970). Isopycnic centrifugations in CsC1 or CSESO4
were as described by Morris et al. (1979) and some analyses were done in preformed
gradients (density 1.25 to 1.40 g/ml) centrifuged for 6 h at 8 °C in the SW50.1 rotor at
45000 rev/min (Burroughs & Brown, 1974). Separations were also done in preformed
potassium tartrate gradients (density 1.1 to 1-45 g/ml) under the same conditions. All
gradients were fractionated with an Isco Model 640 fractionator.
Virus nucleic acid. Virus nucleic acid was isolated by heating a purified suspension of virus
containing both types of particles for 2 rain at 60 °C in 0.1 M-tris-HC1, 0.002 M-Na 2 EDTA
pH 8, containing 2 % SDS and 1 mg/ml bentonite. The solution was then extracted by
vigorous shaking for 5 rain with 2 vol. phenol, containing 0.1% 8-hydroxyquinoline, adjusted
to pH 8 with NH4OH, and 1 vol. chloroform-pentanol (25 : 1, v/v). The emulsion was broken
by centrifugation and the aqueous phase was then ether-extracted. The nucleic acid was
subsequently concentrated by ethanol precipitation and stored at - 2 0 °C. Replicative form
nucleic acid (RF) was isolated from 0.8 g infected larvae according to Morris & Dodds
(1979). Following elution from the Cellex N 1 column, the RF-containing fraction was treated
with DNase at 20 gg/ml for 30 min at 30 °C, precipitated with ethanol and analysed by gel
electrophoresis.
Acrylamide gel electrophoresis. Virus particles were analysed by electrophoresis in 2.4 %
gels in a continuous buffer system of 0.003 M-tris, 0-02 M-glycine pH 8 as described
previously (Morris et al., 1979). Virus proteins were analysed in 12 % SDS slab gels according
to Laemmli (1970). Virus was disrupted by boiling for 1 min in 0 . 5 % SDS, 2.5%
mercaptoethanol, 0.01% bromophenol blue, and 5 % glycerol. Electrophoresis was performed
in an Isolab Model GS225 apparatus for 3 h at 150 V. The gels were stained with 0.05%
Coomassie Brilliant Blue in 50% methanol, 7% acetic acid, destained in the same solution,
and analysed with a Helena densitometer. Analysis of virus nucleic acid was in 2-4 % gels in
0.04M-tris, 0.02M-sodium acetate, 0.001M-Na 2 EDTA pH 7.4, and nucleic acid
composition was determined by in situ nuclease digestion (Morris & Dodds, 1979). Virus RF
was analysed by electrophoresis in a 5 % slab (Isolab) in the RNA buffer system. Following
electrophoresis for 6 h at 80 V, the gel was stained with 0.002% ethidium bromide and
destained with distilled water.
Electron microscopy. Virus suspensions were applied to parlodion-coated, carbonshadowed, 200 mesh copper grids and stained for 15 s with saturated uranyl acetate.
Observations were made in a Philips EM 300 electron microscope. Size estimations were
made from a carbon line block replica and with the use of internal standards.
RESULTS
When stunted larvae from chronically infected colonies were examined at 6 weeks
post-inoculation, approx. 500 larvae weighing a total of 2 g were required to yield 200 gg of
virus (100 ng per mg tissue). Frass obtained from the same colony (12 to 20 g) yielded up to 2
mg of virus (125 ng virus per mg frass). While yield was about the same for larvae and frass in
terms of virus per unit weight of tissue, the relative abundance of frass coupled with the benefit
of keeping virus-producing larvae alive made the use of frass for virus extraction desirable.
Consequently, much of the initial work on the virus (including antiserum production) was
performed on virus particles purified from frass. Such virus sedimented as a broad band
between 165S and 200S on sucrose gradients (Fig. l b). Unexpectedly, when sufficient
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A n invertebrate calici-like virus
117
(a)
0.5
E
r~
¢q
O
(b)
~0.5
t
Sedimentation
Fig. 1
t
mm
Fig. 2
Fig. 1. Sedimentation in linear-log sucrose gradients of virus preparations isolated from (a) infected
larvae and (b) larval frass. Centrifugation was at 38000 rev/min for 60 min in the SW50.1 rotor at
4 °C. The arrows mark the positions of standards of 58S, 95S, 115S and 200S centrifuged in sister
gradients.
Fig. 2. Electron micrographs of negatively stained virus. (a) Preparation of virus particles purified from
frass in various stages of disintegration; (b) 28 nm smooth virus particles from the 165S region of a
density gradient; (c) 38 nm cupped virus particles from the 185S region of the gradient; (d to f ) higher
magnification of cupped particles illustrating the two-, three- and fivefold axes of symmetry typical of
caliciviruses.
quantities of infected larvae were analysed, such p r e p a r a t i o n s c o n t a i n e d two discretely
sedimentable n u c l e o p r o t e i n species o f 165S a n d 185S (Fig. 1 a). Virus particles purified f r o m
frass consisted p r e d o m i n a n t l y o f s m o o t h 28 n m particles with some appearing larger a n d
partially degraded (Fig. 2 a). The 165S virus particles isolated from larvae a p p e a r e d similar to
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118
B. H I L L M A N A N D O T H E R S
E
!1.5
0.5
1.4 ,,~
E
1.3 g
d~
<
1.2
Sedimentation
Fig. 3. Isopycnic centrifugation in CsCI of a virus preparation purified from infected Iarvae. Gradients
with a mean density of 1.35 g/ml were centrifuged for 18 h at 4 °C in the SW50.1 rotor at 40000
rev/min. The arrow marks the position of the 38 nm cupped virus particles.
Table 1. Comparison of the properties o f Amyelois chronic stunt virus types i and ii (ACSV)
to San Miguel sea lion virus (SMSV), a mammalian picornavirus, and Trichoplusia ni
RNA virus (TR V)
Virus
t"
)k
Virus property
Virus size (nm)
Morphology
Sedimentation (S20,w)*
Density in CsCl (g/ml)
ACSVi
38 cm
Cupped
185S
~1.32
SMSV
38 nm
Cupped
183S
1.37
Rhinovirus
27 nm
Smooth
160S
1.44
TRV
35 nm
Fringed
200S
1.30
1.45
70
ACSVii
28 nm
Smooth
165S
Variable
(~1.40)
1-65
29
A260/280ratio
Major capsid proteins
(mol. wt. x 10-3)
1.48
70
1.70
32+
25+
8.5
1.40
67
36S
2.5
36S
2.5
36S
2-6
37S
2-6
34S
1.9
Virus RNA
s2o.w
Mol. wt. x 10-6
* Co-sedimenting with poliovirus (160S) or SMSV (183S) in 10 to 32% linear glycerol gradients yielded
values of 174S to 175S for ACSVii and 188S to 193S for ACSVi (F. L. Schaffer & M. Sorgel, personal
communication).
the 28 nm particles (Fig. 2b) isolated from frass, but the 185S virus particles were larger and
structurally distinctive (Fig. 2c). They had an estimated size o f 38 nm and possessed the
classical cupped substructure of vertebrate caliciviruses. Particles with characteristic two-,
three- and fivefold axes of s y m m e t r y (Fig. 2d, e, f respectively) were evident (Burroughs
et al., 1978). Spectral analysis o f the virus preparations indicated distinctive nucleoprotein
compositions (Table 1). These d a t a suggested the presence o f two distinct virus particles in
larvae.
Heterogeneity of the virus preparations
Some observations suggested that the 28 nm virus particles (ACSVii) might be a
degradation product o f the 38 nm cupped virus particles (ACSVi). Complex heterogeneity
was observed in virus preparations analysed on CsCl (Fig. 3). Nucleoprotein b a n d e d
throughout the gradients, from 1.28 to 1.40 g/ml, in both equilibrium runs for 18 h and in
preformed gradients centrifuged for 6 h. The 38 nm virus particles (ACSVi) b a n d e d in the
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(a)
(b)
A n invertebrate calici-like virus
(c)
119
(a)
0.2
r~
o
(b)
e~
<
0.2
Sedimentation
Fig. 4
Fig. 5
Fig. 4. Polyacrylamide gel electrophoretic analysis of virus preparations of: (a) 28 nm, I65S virus
particles; (b) larval preparation containing both virus particle types; (c) 38 nm, 185S virus particles.
Virus was electrophoresedin 2.4% gel in 0.003 M-tds-glycinepH 8 for 3 h at 150 V.
Fig. 5. Sedimentation in linear-log sucrose gradients of a virus preparation isolated from infected
larvae (a) before and (b) after treatment with a-chymotrypsin. Centrifugation conditions were as in
Fig. 1.
region of 1.32 g/ml, while the 28 nm virus particles (ACSVii) were heterodense throughout
the gradient with a discrete band at 1.40 g/ml. The density distribution pattern of virus was
unaltered in either Cs2SO 4 or potassium tartrate and seemed indicative of a gradual loss of
protein by the lighter cupped ACSVi. A precursor-product relationship was suggested
between ACSVi and ACSVii when virus preparations were analysed by electrophoresis on
2.4 % polyacrylamide gels. A complex series of electrophoretic variants was evident (Fig. 4),
with 38 nm virus particles producing a discrete population of four slow-moving electrophoretic Variants (track c) and 28 nm virus particles displaying a similar pattern of
faster-moving species (track a).
Conversion of A C S V i to A C S V i i by a-chymotrypsin
Yields Of ACSVi relative to ACSVii were increased by rapid extraction and purification of
virus from larvae. This implied a possible loss of ACSVi in tissue extracts. We attempted to
determine whether proteases would have an effect on the integrity of ACSVi. Treatment of
virus preparations with a-chymotrypsin at a substrate to enzyme ratio of 1:20 at 30 °C for
15 h resulted in complete conversion of electrophoretically slow particles ( A C S V i ) t o fast
particles (ACSVii) and of 185S to 165S particles when analysed in density gradients (Fig. 5).
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120
B. HILLMAN
AND
OTHERS
Fig. 6. Electron micrographs of negatively stained 1858 virus particles at (a) zero time, (b) 1 h, (c) 2 h
and (d) 4 h after incubation in ct-chymotrypsin.
e
a~
(¢)
t
68
t
40
t
30
t
Ig
Mobility --,
Fig. 7. Densitometer tracing of virus proteins analysed by electrophoresis in 12% acrylamide,
discontinuous, SDS slab gel: (a) purified preparation of virus from frass; (b) 1858 virus purified from
larvae; (c) 1658 virus produced by treatment with a-chymotrypsin. The arrows mark the locations of
protein standards of mol. wt. 68, 40, 30 and 18 (all x 103).
Electron microscopy showed a gradual loss of cupped structure in ACSVi after
a-chymotrypsin treatment with the production of intermediate stages and finally smooth 28
nm particles indistinguishable from ACSVii (Fig. 6).
Virus polypeptides
The analysis of virus proteins present in purified preparations of virus isolated from frass on
12 % slab gels revealed a complex polypeptide composition with two major species of mol. wt.
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A n invertebrate ealiei-like virus
(a)
121
(b)
e~
<
Sedimentation --,
Fig. 8
Fig. 9
Fig. 8. Sedimentation in linear-log sucrose gradients of RNA isolated from a virus preparation from
larvae and frass by phenol extraction. Centrifugation was in the SW50.1 rotor at 4 °C for 3.5 h at
45000 rev/min. The arrow marks the position of 33S RNA.
Fig. 9. Electrophoretic analysis of: (a) virus RNA in a 2.4% polyacrylamide gel (arrows mark the
positions of standards of mol. vet. 2,6 x 106, 2-0 x 106 and 1.1 x 106); (b) replicative form nucleic acid
(dsRNA) isolated from infected larvae in a 5 % polyacrylamide gel (arrows mark the positions of
standards of mol. wt. 5.2 x 106, 4.0 x 106 and 2-2 x 106).
55 x 103 and 29 x 103 and minor proteins o f mol. wt. 23 x 103 and
R a p i d l y purified 185S virus had only one m a j o r protein (mol. wt. 70 x
other minor species (Fig. 7b) while 165S virus produced by treatment
possessed a single major 29 x 103 tool. wt. protein and only traces o f the
16 x 103 (Fig. 7a).
10 3) with at least six
with w c h y m o t r y p s i n
other species.
Virus nucleic acid
Nucleic acid analysis from purified virus was attempted only on the mixed population
obtained from larval frass because of the difficulty in obtaining high yields o f virus from
larvae. Only a single major species of R N A with a sedimentation coefficient o f 36S (Fig. 8)
was obtained from such virus. This preparation produced a single m a j o r b a n d when analysed
on 2 . 4 % acrylamide gels and was sensitive to R N a s e but not D N a s e digestion. It had an
apparent mol. wt. of 2.5 x 10 6 (Fig. 9 a). The double-stranded R N A replicative nucleic acid
( R F ) isolated from infected larvae electrophoresed as a single m a j o r species o f mol. wt.
approx. 5 x 10 6 (Fig. 9b). This value confirms a single-stranded virion R N A tool. wt. of 2-5 x
106 .
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122
B. HILLMAN
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DISCUSSION
A virus disease causing severe stunting and death in navel orangeworms has been shown to
be caused by a virus with striking morphological and physicochemical similarity to
mammalian caliciviruses. A second type of virus particle, lacking the calicivirus morphology,
was also associated with infected larvae and was the predominant virus particle in frass from
infected hosts. Virus preparations from infected hosts displayed marked density and
electrophoretic heterogeneity indicative of in vivo degradation. The heterogeneity, complex
polypeptide patterns, and in vitro conversion of 38 nm virus particles (ACSVi) to 28 nm virus
particles (ACSVii) by treatment with a-chymotrypsin supports the concept that the calici-like
virus (ACSVi) is converted to the smaller virus (ACSVii) by natural proteolytic action on
release from the host into the gut lumen. Both forms of the virus have been shown to be
infectious. Preliminary specific infectivity studies have demonstrated no significant difference
between the two virus particles (B. Hillman et al., unpublished results).
Considerable progress has been made recently on the classification of small RNA viruses of
invertebrates (Longworth, 1978). A number of invertebrate viruses which are similar to
vertebrate picornaviruses have been identified and these are being considered for inclusion
within the Picornaviridae family (Matthews, 1980). Although a superficial similarity between
one group of insect viruses (the Nudaurelia fl capensis group) and caliciviruses has been
recognized (Reinganum et al., 1978), this group has not been considered in a recent review on
caliciviruses because of significant differences in morphology and genome size (Schaffer,
1979). The virus of navel orangeworms (ACSVi) that we report here has an obvious
ealicivirus morphology with appropriate physicochemical similarity (Table 1) to be considered
for inclusion within the newly proposed Caliciviridae family (Burroughs & Brown, 1974;
Schaffer et al., 1980). The only major discrepancy between ACSVi and mammalian
caliciviruses is the light density of the invertebrate virus. This could reflect a difference in
caesium uptake among caliciviruses (Schaffer, 1979) and will have to be explored further.
Although preliminary serological studies have failed to indicate any close relationship to
mammalian caliciviruses (F. L. Schaffer, personal communication), this work and an
evaluation of replicative strategy will have to be pursued before the virus can be seriously
considered the first invertebrate member of the Caliciviridae.
An interesting feature of this invertebrate virus is its apparent natural degradation to 28 nm
picorna-like virus particles on release from the host in frass. This phenomenon seems similar
to reports on the identification of putative human caliciviruses that appear partially degraded
in faecal extracts (Curry & Roberts, 1980). Our results indicate that identification of
picornavirus and calicivirus infections by electron microscopy of faecal material could easily
lead to erroneous results.
We wish to thank Drs F. L. Schafferand F. Brownfor helpfuldiscussions.This work was supported
in part by a USDA-SEA/AR cooperative agreement no. 58-9AH2-9-478 entitled 'Determination of
Extraneous Viral Contaminantsin BaculovirusPreparations'.
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