Download Purification and characterization of the infectious hypodermal and

Journal of General Virology(1990), 71, 2657--2664. Printedin Great Britain
2657
Purification and characterization of the infectious hypodermal and
haematopoietic necrosis virus of penaeid shrimps
Jean-Robert Bonami, 1. Bronwen Trumper, 2 Jocelyne Marl, 1 Michel Brehelin 1
and Donald V. Lightner z
~ERPAM, Laboratoire de Pathologie ComparOe, INRA-CNRS, Universit~ des Sciences et Techniques du Languedoc,
Place E. Bataillon, 34095 Montpellier Cedex 5, France and 2Department of Veterinary Science, University of Arizona,
Tucson, Arizona 85721, U.S.A.
Infectious hypodermal and haematopoietic necrosis
(IHHN) is one of the most important viral diseases of
cultured penaeid shrimps and is potentially a limiting
factor in the development of farming projects for some
species of these shrimps. Although the I H H N agent
was recognized early as being viral in origin, attempts
to characterize it were inconclusive because of difficulties in obtaining sufficient amounts of purified virions to
permit its characterization. Recent improvements of
purification procedures have allowed the physico-
chemical characterization of this virus. Purified
I H H N V is a non-enveloped icosahedral particle averaging 22 nm in diameter, exhibiting a mean buoyant
density of 1.40 g/ml in CsCI. The genome is a single
molecule of ssDNA with an estimated size of 4.1 kb by
molecule length measurement in transmission electron
microscopy. As determined by S D S - P A G E , the particle contains four polypeptides with Mrs of 74K, 47K,
39K and 37.5K, respectively. From its characteristics,
this virus could be a member of the Parvoviridae family.
Introduction
in the discipline of marine invertebrate virology, very
few viruses have been characterized. They are two
reoviruses (Bonami, 1980; Mari, 1987; Mari & Bonami,
1988a) and a parvovirus (Mari & Bonami, 1988b) from
the crabs Macropipus depurator and Carcinus mediterraneus, one reovirus from the oyster Crassostrea virginica
(Meyers, 1979; Winton et al., 1987), one iridovirus
(Devauchelle et al., 1977) from the annelid worm Nereis
diversicolor and one baculovirus (Summers, 1977) from
the shrimp Penaeus duorarum. However, only some
characteristics of the marine shrimp baculovirus have
been reported, and a report about the isolation,
purification and characterization of I H H N V (directly
contradictory with our findings) has been recently
published (Lu et al., 1989).
We report here our results on the purification, ultrastructure and the physicochemical properties of
IHHNV.
Infectious hypodermal and haematopoietic necrosis
(IHHN) disease (Lightner et al., 1983a) has been found
in cultured penaeid shrimps in the Americas and Asia
(Lightner, 1985, 1988) and in French Polynesia (Lightner
et al., 1983b, 1990a). The gross signs of the disease and
the histological diagnostic procedures for it have been
extensively described (Lightner et al., 1983a, 1990a;
Lightner, 1985, 1988). The international spread of
I H N N disease into new regions with asymptomatic
carriers underscores the importance of I H H N as one of
the major diseases of cultured penaeid shrimps (Lightner
et al., 1983b, 1990b). The demonstration of virus-like
particles in sections of infected tissue by electron
microscopy (Lightner et al., 1983a) and the ability to
reproduce the disease experimentally using bioassays
indicated that the aetiological agent was a virus (Bell &
Lightner, 1984); the agent was named infectious hypodermal and haematopoietic necrosis virus (IHHNV).
However, attempts to isolate an I H N N V have been
unsuccessful or have yielded amounts of viral particles
insufficient to permit its characterization. Recently,
purification procedures have been improved, allowing us
to obtain the higher concentrations of purified virions
necessary to complete the characterization of the I H H N
agent.
Methods
Experimental animals. Although the small juvenile stages of
P. stylirostrisare mostseverelyaffectedby IHHN disease,we usedas a
source of IHHNV a single 10 kg batch of large size (30 to 50 g)
P. vannamei obtained from the Ocean Institute (Oahu, Hawaii) i n
December 1986.Theseshrimps werestoredat -70 °C until processed.
Infection by IHHNV in this batch of shrimps was confirmedby direct
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J.-R. Bonami and others
histological examination of Davidson's preserved specimens and by
bioassay tests for the virus using 0.05 to 0-1 g P. stylirostris as the
indicator for virus presence (Lightner et al., 1983b).
Bioassay studies. A sample of eight frozen gnathothoraces ('heads')
from the adult P. vannamei population from the Oceanic Institute were
bioassayed with 0.05 to 0.1 g juvenile P. stylirostris (the indicator
shrimp for presence or absence of IHHNV). In the bioassay, 50 to 55
indicator shrimps were stocked into each of six separate 40 litre glass
aquaria. Finely minced 'heads' of P. vannamei were fed to the indicator
shrimps in three of the aquaria (exposed group) on days 1, 3, 5, 8, 11, 14
and 17 of the bioassay. The remaining three tanks, which were
physically isolated from the other tanks, served as negative control
tanks. Shrimps in all six tanks were fed twice daily on an artificial dry
pelleted ration at approximately 10~ of their total biomass. Water
temperature was maintained between 24°C and 28°C with a room air
conditioner. Salinity was maintained at 20 to 25%°.
Sampling for histopathological diagnosis of IHHN was scheduled for
days 0, 14, 21, 28, 42 and 60 of the bioassay and as moribund
individuals were observed. However, actual sampling was done on days
0, 17, 21, 23, 25, 26 and 28 of the bioassay (Table 1). Two or three
shrimps per replicate tank were taken at each scheduled sampling time.
The bioassay was terminated on day 28 because IHHN-positive
indicator shrimps were found in day 17 and day 21 samples, and
because all the indicator shrimps had died by day 26.
Infectivity studies. Using similar bioassay procedures, semi-purified
and purified preparations of IHHNV from sucrose and CsCI gradients
were tested for infectivity and for their ability to produce IHHN
disease in juvenile P. stylirostris. In these studies various fractions that
were found to contain virus according to their absorption spectra at
260 nm (sucrose gradients), or because they were found to contain 20 to
22nm diameter virus-like particles (from CsCI gradients) when
examined by transmission electron microscopy (TEM), were injected
into the tail muscle of 0.5 to 2 g juvenile P. stylirostris in five separate
experiments (Table 2). Samples of obviously ill shrimps were taken as
they were observed in these studies for histopathological confirmation
of IHHN, or samples were taken according to scheduled sampling
schemes (Table 2).
Virus purification. Only heads (gnathothorax with the carapace
removed) were used for virus extraction and all buffers used were
filtered through 0.22 ~tm membranes to remove particulate contaminants and bacteria. After homogenization in TN buffer (0.02 M-TrisHCI, 0.4 M-NaC1 pH 7.4) the suspension was clarified in three steps at
2000, 5000 and 15000 r.p.m, in runs of 10min, 10min and 30rain,
respectively. The resulting supernatant was pelleted for 3-5 h at
145000g then resuspended in TN buffer, shaken with activated
charcoal, filtered on a Celite-235 bed and extracted three or four times
in freon (l,l,2-trichloro-l,2,2-tritiuoroethane). The final suspension
was pelleted at 145000g for 3.5 h and then resuspended in a small
volume of buffer before being layered on a 15 to 40~ (w/w) sucrose
gradient and centrifuged for 3 h at 140000 g. Fractions (1 ml) from the
sucrose gradient were collected with an Autodensiflow fraction
collector and the absorbance at 254 nm was determined and recorded
using an Isco UA5 u.v. monitor. Fractions containing virions were
pooled, diluted in TN buffer and centrifuged for 3 h at 140000g.
Pellets, resuspended in TN buffer, were layered on a preformed 25 to
45~ (w/w) CsC1 gradient in TN buffer and isopycnically centrifuged
for 16 h at 145000g. The band of virus was collected using the same
method as indicated for the sucrose gradient, diluted in TN buffer,
pelleted for 1.5 h at 250000g and resuspended in a small volume of
buffer.
Virus density. Because of the refractive index of TN buffer, we used
CsC1 in 0.1 x TE (10 mM-Tris-HC1, 1 mM-EDTA pH 7.4) buffer which
does not interfere with the refractive index values obtained. Ultrafiltered (0.22 p.m) CsC1 preformed gradients (20 to 40% w/w and 28 to
48% w/w in 0-1 x TE) were used for isopycnic centrifugations of empty
and full particles, respectively, in runs of 17.5 h. Fractions (0.5 ml) were
collected, absorbance at 254 nm was recorded with an Isco UA5
monitor and the refractive index of each fraction was measured using
an Abb~ refractometer.
Electron microscopy. Purified virus suspensions were negatively
contrasted on carbon-collodium-coated 200-mesh copper-palladium
grids using ultrafiltered (0.22 ~tm) 2 ~ sodium phosphotungstate (PTA)
in doubled distilled water pH 7. Molecules of extracted nucleic acid
were spread on carbon-coated grids according to the procedure of
Delain & Brack (1974) and contrasted with platinum-palladium
rotational shadowing. Observations were performed using an Hitachi
HU 11-C electron microscope.
Tobacco mosaic virus (TMV) was used as an internal standard for
particle size reference and qbX174 DNA was used for length
measurement of IHHNV nucleic acid molecules. The length was
measured using a Numonics digitizer connected to a microcomputer.
Spectrophotometry. Spectra from 200 to 320 nm were recorded for
both purified virus suspensions and extracted nucleic acid; concentrations of proteins and nucleic acid were estimated according to the
procedures of Layne (1957) and Maniatis et al. (1982).
SDS-PAGE. 10~ polyacrylamide vertical gel slabs were run using a
phosphate buffer (0.1 M-Na2HPO4-NaH2PO 4 pH 7) containing 0-1
SDS. The run duration was 3 h at a constant 150 V. Samples were
treated with a dissociating medium (1~ SDS, 5M-urea, 0.1~
2-mercaptoethanol) and heated to 100 °C for 3 min. Mr markers were
phosphorylase B, albumin, ovalbumin, carbonic anhydrase, trypsin
inhibitor and ct-lactalbumin, which have Mrs of 94K, 67K, 43K, 30K,
20-1K and 14-4K, respectively. Bromophenol blue and sucrose were
added to the samples and Mr markers before electrophoresis. After
washing in fixative solution the gels were stained with Coomassie blue
or with silver stain (Ag-stain 'Daiichi'; Daiichi Pure Chemicals). The
Mr of the polypeptides was estimated by measurement of the
electrophoretic mobilities according to the method of Weber & Osborn
(1969).
Nucleic acid extraction. Purified virus suspensions were treated with
pre-activated proteinase K at a final concentration of 50 ~tg/ml for 0.5 h
at 37 °C, followed with Sarkosyl (final concentration 0.5 ~) for 1.0 h at
65 °C. Nucleic acid was extracted twice with buffer-equilibrated
phenol (Maniatis et al., 1982), once with a 1:1 mix of equilibrated
phenol and chloroform and twice with chloroform-isoamyl alcohol
(24:1). The aqueous phase was then adjusted to 0.3 M-sodium acetate
pH 5.2, two volumes of absolute ethanol were added and the nucleic
acid was allowed to precipitate overnight at - 30 °C. The nucleic acid
was then pelleted, dried and resuspended in TNE buffer (10 mM-TrisHC1, 100 mM-NaCI, 1 mM-EDTA, pH 8).
Agarosegelelectrophoresis. Electrophoretic separation of nucleic acid
segments was performed in 0-7% agarose gels in TAE buffer
(0.04M-Tris-acetate, 0-001 M-EDTA pH 8) run at 100V for 2h.
Ethidium bromide, sometimes incorporated into the gel (0.5 ktg/ml) or
used as stain after the electrophoresis, was used to stain nucleic acid
bands. Phage 2 HindlII and EcoRI-HindlII DNA digests were used as
Mr markers. Before each run, samples and markers were heated to
60 °C but separate samples were treated by heating (3 min at 100 °C)
with RNase, DNase I or nuclease S1, according to the method of
Maniatis et al. (1982).
Gels containing methylmercuric hydroxide (Maniatis et al., 1982)
were used to determine, in association with RNase treatment, the
nature of the nucleic acid. For these experiments all nucleic acid
extractions were performed using RNase-free conditions.
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I H H N V purification and characterization
2659
Results
Bioassay studies
The results of the bioassay run on the adult P. vannamei
using juvenile P. stylirostris as the indicator for the
presence or absence o f I H H N V confirmed that the
P. vannamei were infected with I H H N V . Severe I H H N
infections were present in the indicator shrimps by day
17 of a planned 60 day study and no exposed indicator
shrimps survived to day 26 o f the bioassay. I n contrast,
I H H N was not observed in samples o f control (unexposed) indicator shrimps, nor were significant mortalities observed in the control groups (Table 1).
Isolation and purification of I H H N virions
The quantity o f purified virions obtained f r o m between
five and seven gnathothoraces from 30 to 50 g adult
shrimps was estimated by measuring the absorbance at
260 and 280 n m (Layne, 1957); virus yield from this
quantity o f shrimp tissue was estimated at 75 ~tg to
200 ~tg, depending on the degree o f infection in the
animals sampled. The degree of purity o f the isolated
I H H N V preparations was determined by T E M and gel
electrophoresis. T E M of negatively contrasted purified
I H H N V preparations showed the absence o f cell
c o n t a m i n a n t s (Fig. l a). Likewise, S D S - P A G E o f the
virus preparations showed no bands other t h a n those
expected to be present in small viruses (Fig. 3).
Fig, 1. Purified IHHNV. (a) Large amount of full particles; bar
marker represents 200 nm. (b) Full and empty particles at higher
magnification from a non-fully purified virus suspension, small cell
debris is associated with the particles. Note the angular shape of
virions. Bar marker represents 40 nm. Negative stain (PTA).
1,6
Infectivity studies
I H H N disease was induced experimentally using semipurified (from sucrose gradients) and purified I H H N V
(from CsC1 gradients) in artificially exposed juvenile P.
stylirostris. I H H N disease was confirmed histologically
in those shrimps indicated as positive for the disease in
Table 2.
1.5
1.4
El
Table 1. Results of the bioassay run on adult Penaeus
vannamei with juvenile P. stylirostris serving as the
e~
t~
<
indicator shrimp
1.3
Day of bioassay
(number IHHN-positive/number sampled)
Treatment
0
17
21
23
25
26
28
Control 1
Control 2
Control 3
Exposed 1
Exposed 2
Exposed 3
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
2/2
3/3
2/2
0/2
0/2
0/2
1/1
1/2
2/2
1/1
1/1
-
3/3
3/3
1/1
0/3
0/3
0/3
NS
us
NS
0/2
1/1
* Ns, No sample was taken because no indicator shrimp survived to
day 28 of the bioassay.
1.2
1
10
5
Fraction number
Fig. 2. CsCI equilibrium centrifugation of full IHHNV particles. A is
given at 254 nm.
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J.-R. Bonami and others
Buoyant density of virions
1
2
F o r full particles, using a 28 to 48% (w/w) p r e f o r m e d
CsC1 g r a d i e n t in 0.1 × T E buffer, the d e n s i t y o f I H H N V
was found to be b e t w e e n 1-390 a n d 1.420, w i t h a m e a n
value o f 1.405 at the s u m m i t o f the r e c o r d e d p e a k (Fig. 2).
F o r e m p t y particles, using a p r e f o r m e d 20 to 4 0 % (w/w)
CsCI g r a d i e n t , the d e n s i t y h a d a m e a n value o f 1.298 (not
shown).
Size and structure of purified I H H N V
In n e g a t i v e l y s t a i n e d virus suspensions, full a n d e m p t y
p a r t i c l e s were clearly o b s e r v e d (Fig. 1 a a n d b). U s i n g
T M V ( d i a m e t e r 18 n m ) as an i n t e r n a l size m a r k e r , the
virions e x h i b i t e d a m e a n d i a m e t e r o f 22 nm, w i t h a n
a v e r a g e o f 20 a n d 23 n m for side to side a n d p o i n t to
point, respectively. T h e i n n e r c o m p o n e n t o f e m p t y
p a r t i c l e s s h o w e d an a v e r a g e d i a m e t e r o f 13 nm. D u e to
the presence o f five- a n d six-sided particles in the
p r e p a r a t i o n s , we c o n c l u d e t h a t this virus is i c o s a h e d r a l
in s h a p e (Fig. 1 b).
Polypeptides of l H H N V
I H H N V p o l y p e p t i d e s were i n v e s t i g a t e d by S D S - P A G E
in a 10~o gel. A total o f four p o l y p e p t i d e s were o b s e r v e d
b o t h in C o o m a s s i e blue- a n d silver-stained gels (Fig. 3).
F r o m different gel e l e c t r o p h o r e s i s p r e p a r a t i o n s , we h a v e
e s t i m a t e d the Mr o f the four p o l y p e p t i d e s to be 74K,
4 7 K , 39K a n d 37-5K, r e s p e c t i v e l y (Fig. 4).
Fig. 3. Silver-stained SDS-PAGE of IHHNV polypeptides. Lane 1,
IHHNV polypeptides; lane 2, protein markers. Pointers indicate
positions of IHHNV polypeptides. For sizes see Fig. 4 and text.
single b a n d was found from p h e n o l - e x t r a c t e d viral
nucleic a c i d run in a r e g u l a r 0-7% agarose gel or in
methylmercuric hydroxide conditions.
A t t e m p t s to c h a r a c t e r i z e the nucleic a c i d show t h a t it
is R N a s e - r e s i s t a n t in the c o n d i t i o n s w h e r e this e n z y m e is
active a g a i n s t r i b o s o m a l R N A (not shown) a n d it is
Nature and structure of the 1HHNV nucleic acid
U s i n g purified virions in d i s s o c i a t i n g m e d i u m h e a t e d at
60 °C for 3 m i n a n d run in a n S D S - 0 . 7 % agarose gel, only
one nucleic a c i d b a n d was present. Likewise, only a
Table 2. Results of infectivity studies with semi-purified and purified preparations of lHHNV using
juvenile Penaeus stylirostris as the indicator for infectious virus
Days post-injection (number IHHN-positive/number examined)
Study
1
2
Gradient
Sucrose
Sucrose
3
Sucrose
4
CsCI
5
CsCI
Fraction/band
Control gradient
Peak 1
Peak 2
Saline control
Band
Pellet
Control
Peak
Diffuse band
Sharp band
Sharp band
1
2
3
0/1
0/1
0/1
1/2
0/2
0/3
0/3
.
1/3
.
0/2
1/2
.
.
.
.
.
2/2
0/2
.
.
0/4
1/4
.
0/1
.
4
6
7
-
1/1
-
3/3
1/1
0/1
2/2
2/2
2/2
3/3
1/1
2/2
0/4
2/4
.
.
i/2
.
0/1
.
9
0/1
2/2
2/2
0/11
5/5
4/6
.
.
.
.
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11
12
0/1
1/1
1/1
0/4
3/4
.
1/1
.
~-
3/3
16
IHHNV purification and characterization
100
I
I
I
I
I
1
I
I
I
50
L
~
20
I
1
I
I
I
I
I
I
I
2661
I
IIII
15
Migration (cm)
Fig. 4. Determination of the Mr of IHHNV polypeptides (arrows);
protein markers, arrow heads.
1
2
3
4
5
6
~21-2
-5.1
~4.9
~4.2
3.5
~2.0
"-1.9
-1.7
"-1-3
Fig. 5. Agarose gel electrophoresis of IHHNV ssDNA. Lane 1,
markers (2-HindlII); lane 2, IHHNV DNA; lane 3, heated (100 °C,
3 min) IHHNV DNA; lane 4, IHHNV DNA; lane 5, SI nucleasetreated viral DNA; lane 6, markers (2-HindllI-EcoRI). Sizes of
markers are indicated in kb.
sensitive to nuclease S 1, suggesting that the nucleic acid
is a ssDNA (Fig. 5). Moreover, the characteristics of the
nucleic acid when run in non-denaturating gels, and the
absence of hyperchromicity at 260 nm when it is gently
heated, confirm that the nucleic acid of I H H N V is
ssDNA. When co-electrophoresed with dsDNA markers
in a 0.7~ gel, the size of the ssDNA in the band can be
estimated at about 3-75 kb (Fig. 5). Moreover, a smear
was clearly shown along the migration area of the gels in
all our nucleic acid preparations. We initially hypothesized that this smear was due to freezing the virions
before extraction, underlining the fragility of this
Fig. 6. Isolated ~X174 RF DNA molecules (a, b) compared with
IHHNV ssDNA molecules (c, d, e) obtained from the electroeluted
3.75kb band. Platinum palladium shadowing; TEM. Bar marker
represents 0-5 gm.
ssDNA, giving numerous small pieces of different sizes.
But as the material making up the smear is sensitive to
RNase I and nuclease S1, the results suggest strongly the
presence of ssRNA molecules in our preparations. This
is confirmed by the absorbance spectrum of the total
extracted nucleic acid which shows a 280/260 ratio of
close to 2, indicating the presence of RNA.
TEM examination of total nucleic acid extract spread
on a carbon-coated grid and rotationally shadowed,
demonstrated supercoiled molecules associated with
circular-like and different sized linear molecules. The
3.75 kb band (Fig. 5) was removed from the gel,
electroeluted, the ethidium bromide was extracted and
the D N A recovered by cold alcohol precipitation before
being spread on a carbon-coated grid and shadowed (Fig.
6). Compared with ~X174 replicative form (RF) D N A
circular molecules which are double-stranded, the pictures confirm the single-stranded nature of the I H H N V
DNA, which shows a smaller diameter. By length
measurement, an average value of 1.36 gm (with a range
from 1-26 to 1.40 gm) was obtained for I H H N V ssDNA
molecules; the average value for (I)X174 RF D N A was
1.78 gm (from 1-75 to 1.81 gin). As ~X174 RF D N A has
a size of 5-386 kbp, we can estimate the molecular size of
the I H H N V ssDNA at about 4-1 kb. Since one base is
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J.-R. Bonami and others
equivalent to an Mr of approximately 330, the Mr of
I H H N V ssDNA can be estimated at 1350K.
Compared with the size (3.75 kb) determined by
agarose gel electrophoresis, this last value (4-1 kb) may
be closer to a true estimation because error is introduced
into the estimation when double-stranded markers are
compared to single-stranded viral DNA.
Discussion
Although the presence of virions in IHHNV-diseased
shrimps was suggested by the observation in TEM of
negatively stained particles in different laboratories
several years ago (J.-R. Bonami, unpublished results;
Lightner, 1985), only recently have improvements in the
purification procedures resulted in sufficient yields of
I H H N V to permit characterization.
The physicochemical characteristics of purified
IHHNV, i.e. its small size of about 21 nm, its density in a
CsC1 gradient of 1-40, the presence of polypeptides
showing Mr ranging from 74K to 37.5K and a single
genomic segment of ssDNA of 4.1 kb, indicate that this
agent is closely related to the Parvoviridae family
(Matthews, 1982).
Despite Mr differences between I H H N V polypeptides
and those reported for parvoviruses such as rat virus,
H-I, bovine parvovirus, minute virus of mice, adenoassociated virus types 1, 2 and 3 (Rose, 1974; Berns et al.,
1986), the I H H N V polypeptides are much closer in Mr to
those of the Parvoviridae than those of the Picornaviridae.
The picornavirus polypeptides have MrS ranging from
35K to 6.5K, which are usually less than the smallest of
the I H H N V polypeptides; the exception to this is the
VP0 polypeptide (36.5K to 41K) which is found as a trace
of a fifth chain in picornaviruses such as poliovirus,
encephalomyocarditis virus, foot-and-mouth disease
virus, human rhinovirus type 14 and murine encephalomyelitis virus (Rueckert, 1976, 1986). On the other hand,
the presence of four polypeptides in I H H N V could be
related to similar findings in insect parvoviruses (densovirus; DNV) such as DNV from Galleria mellonella
(Tijssen & Kurstak, 1979) and Pseudoplusia includens
picarnavirus (Chao et al., 1985) or in a parvo-like virus
(PC84) of crustacea (Mari & Bonami, 1988b).
In the earliest paper on IHHNV, Lightner et al.
(1983a) suggested that I H H N V was either a picornavirus or a parvovirus. Later, following more ultrastructural and histochemical studies of IHHNV-infected
shrimp tissues and some attempts to isolate and
characterize the virus, one of us (D. V. Lightner) had
tentatively classified I H H N V with the picornaviruses
(Lightner, 1985, 1988; Lightner et al., 1990a). This
conclusion was based on the size of the virion in purified
preparations (20 nm) and in sections (17 to 24 nm) and
upon the morphological characteristics of infected cells.
Eosinophilic intranuclear Cowdry type A inclusion
bodies are the principal diagnostic feature used to
diagnose I H H N disease (Bell & Lightner, 1984).
Although these prominent intranuclear inclusions
present in shrimps with I H H N are evidence of nuclear
involvement, the inclusion body in such nuclei is
Feulgen-negative and virions have not been observed
within such nuclei. Rather, cytoplasmic masses of
virions, sometimes in paracrystalline arrays, have been
found in affected tissues of P. stylirostris with I H H N
(Lightner et al., 1983a; Lightner, 1985, 1988). Comparable ultrastructural studies of P. vannamei (or of other
penaeids) with I H H N infections have. not been done.
Hence, because of its small size, the absence of Feulgenpositive intranuclear inclusion bodies and the presence
of virus masses and arrays in the cytoplasm, I H H N V was
tentatively placed with the picornaviruses.
It was only after several nucleic acid extractions from
purified virions isolated from different batches of
P. stylirostris and P. vannarnei that a more accurate
characterization of I H H N V was achieved. Demonstration of the D N A nature of the I H H N V nucleic acid
indicates that I H H N V is more closely related to the
Parvoviridae than to the Picornaviridae. However, the
significance of the presence of ssRNA in our preparations along with larger amounts of ssDNA has not yet
been elucidated. However, because a smear and not a
band is equally in evidence using fully denaturing gels
(containing methylmercuric hydroxide) and RNase-free
extraction protocols, we do not believe that this ssRNA
can be attributed to a contaminating picorna-like virus.
It appears to be more probable that they are different size
fragments of messenger R N A adsorbed or shared by the
I H H N V particles.
Finally, the infectivity tests using semi-purified and
purified virions demonstrate, by successfully reproducing the histopathological symptoms, mortalities and
similarly sized particles, that the isolated virus is the
causative agent of I H H N disease.
A report about isolation and characterization of
I H H N V has been recently published (Lu et al., 1989).
However, the reported results are directly contradictory
to our findings. We believe that Lu et al. (1989) did not
isolate IHHNV, but characterized a normal shrimp
macromolecule. We base this opinion on the following:
(i) Lu et al. (1989) report the 'virus' particles shown in
Fig. 2 of their paper to be 19 nm in diameter yet,
according to the magnification bars in Fig. 1, the 'virus'
particles are closer to 12 nm in diameter; (ii) Lu et al.
(1989) provide no evidence of empty capsids in Fig. 2 or
elsewhere in the manuscript, this is noteworthy as they
are found in most virus preparations; (iii) the shape of
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I H H N V purification and characterization
the particles in Fig. 2 is more star-like than icosahedral;
(iv) the diameter (12 nm) and star-like morphology of the
'virus' particles are consistent with that of hexameric
haemocyanin molecules (respiratory pigment in
crustacea) as reported by van Bruggen (1986) and B ijlholt
& van Bruggen (1986).
Concerning the presence of RNA in the preparations
of Lu et al. (1989), it should be pointed out that
haemocyanin is a glycoprotein which gives a positive
periodic acid-Schiff (PAS) reaction and thus it may give
a false positive reaction for RNA with the orcinol
method. Only SDS-PAGE analyses of these isolates
would aid the characterization of these 'virus' particles.
Moreover, in contrast to our own results, the authors
report 'inconsistent' attempts to infect shrimps with the
'CsCl-banded virus preparation' obtained.
In invertebrate virology the Parvoviridae family, and
particularly the genus Densovirus, is very well represented, especially in insects (Matthews, 1982) in which
extensive studies have been done. Conversely, in marine
invertebrates and more particularly in marine crustacea,
small-sized non-enveloped viruses have been described
only on the basis of TEM observations of infected tissues
and/or negatively stained isolated particles, and these
have been called picorna-like (Bonami, 1977, 1980;
Johnson, 1978; Kuris et al., 1979; Foster et al., 1981;
Lightner et al., 1983a) or parvo-like viruses (Lightner &
Redman, 1985), depending on their location in the cell.
Except for the PC84 virus of the crab Carcinus
mediterraneus, which was demonstrated to be a DNAcontaining virus (Marl & Bonami, 1988 b), no other small
DNA-containing viruses have been isolated, purified
and characterized in the crustacea.
The findings of the study reported here indicate that
IHHNV has cubic symmetry with an icosahedral shape,
an average diameter of 22 nm in purified preparations
and a density of 1.40 for whole virions and 1.28 to 1.30 for
empty particles. The nucleic acid of I H H N V is ssDNA
with an Mr of approximately 1350K and a size of 4.1 kb
as determined by length measurement of molecules. Its
capsid is composed of four polypeptides with Mrs of 74K,
47K, 39K and 37.5K, respectively. These characteristics
are consistent with those of the family Parvoviridae
(Matthews, 1982) and thus we have concluded that
IHHNV is a member of this family of viruses.
This work was funded in part by grants from the Groupement de
Cooperation Scientifique sur les Bases Biologiques de l'Aquaculture
and by grants from the U.S.D.A. Marine Shrimp Farming Consortium,
Sea Grant (U.S. Department of Commerce) and the State of Hawaii
Aquaculture Development Program.
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