Download A new nodavirus is associated with covert mortality disease of shrimp

Journal of General Virology (2014), 95, 2700–2709
DOI 10.1099/vir.0.070078-0
A new nodavirus is associated with covert mortality
disease of shrimp
Qingli Zhang,1,2 Qun Liu,1 Shuang Liu,1 Haolin Yang,1 Sun Liu,1
Luoluo Zhu,1 Bing Yang,1,2 Jiting Jin,1 Lixue Ding,1 Xiuhua Wang,1,2
Yan Liang,1,2 Qintao Wang1 and Jie Huang1,2
Correspondence
Qingli Zhang
[email protected]
Jie Huang
[email protected]
Received 16 July 2014
Accepted 8 September 2014
1
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture,
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
106 Nanjing Road, Qingdao 266071, PR China
2
National Laboratory for Marine Science and Technology, Qingdao 266071, PR China
A new nodavirus, named covert mortality nodavirus (CMNV), is associated with covert mortality
disease of shrimp which has caused serious loss in China since 2009. Histopathological
examination of shrimp suffering the disease revealed coagulative necrosis of striated muscle
similar to typical histopathology features of infectious myonecrosis virus (IMNV), Penaeus
vannamei nodavirus (PvNV) and Macrobrachium rosenbergii nodavirus (MrNV). However, shrimp
suffering this disease tested negative for IMNV, MrNV and PvNV by reverse transcription (RT)PCR. Additionally, eosinophilic inclusions were found in epithelium of the tubules in the
hepatopancreas and lymphoid organ, and mass karyopyknotic nuclei existed in the muscle and
lymphoid organ. The tubular epithelium of the hepatopancreas showed significant atrophy. A
cDNA library was constructed from total RNA of infected shrimp. Sequencing and alignment
analysis showed that one clone with an 1185 bp insert (designated CMNV-7) shared 54 , 53 and
39 % identity with the amino acid sequences of RNA-dependent RNA polymerase from Flock
House virus, black beetle virus and MrNV. The results of fluorescence in situ hybridization showed
that the hepatopancreas, striated muscle and lymphoid organ were positively reacting tissues. The
mean size of negative-stained virus particles was 32 nm. In addition, a nested RT-PCR assay was
developed for CMNV, and the RT-PCR detection results revealed that Fenneropenaeus
chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were
CMNV-positive.
INTRODUCTION
A major disease that occurred in the shrimp farming
industries in China before 2009 was generally named ‘covert
mortality disease’ due to the most moribund shrimp hiding
on the bottom in deep water rather than swimming to the
surface or in shallow water, as did shrimp suffering from
white spot disease (Zhang, 2004; Xing, 2004; Song &
Zhuang, 2006; Xu & Ji, 2009; Gu, 2012; Huang, 2012). The
shrimp suffering the disease exhibited clinical signs
including hepatopancreatic atrophy and necrosis, empty
stomach and guts, soft shell, slow growth, and in many cases
abdominal muscle whitening and necrosis (Zhang, 2004;
Huang, 2012). The farmers noted that dead shrimp could be
found in the diseased population every day and mortality
increased during 60–80 days post-stocking with a cumulative mortality up to 80 %. Early mortality syndrome (EMS)/
The GenBank/EMBL/DDBJ accession number for the RNA-dependent
RNA polymerase gene sequence is KM112247.
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acute hepatopancreas necrosis disease (AHPND) that
emerged in the south of China in 2010 (Lightner et al.,
2012) was once thought to represent severe cases of covert
mortality disease as its clinical signs were similar to that of
covert mortality disease to some degree.
Infection with several RNA viruses has been found to
cause typical muscle necrosis in cultured shrimp. Infectious
myonecrosis virus (IMNV) and Penaeus vannamei nodavirus (PvNV) infect Litopenaeus vannamei and result in
muscle necrosis (Poulos et al., 2006; Tang et al., 2007; Flegel,
2012). Macrobrachium rosenbergii nodavirus (MrNV) can
infect freshwater prawns and L. vannamei (Qian et al.,
2003; Bonami et al., 2005; Senapin et al., 2011, 2013;
NaveenKumar et al., 2013), causing muscle necrosis in tail
muscles. However, IMNV, MrNV and PvNV were not
detectable in shrimp suffering from covert mortality disease
using relevant reverse transcription (RT)-PCR methods. As
has been observed for IMNV, MrNV and PvNV, histological
examination of shrimp revealed coagulative necrosis of
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CMNV causes covert mortality disease of shrimp
the skeletal muscle and pyknosis was found in the muscle.
In addition, eosinophilic inclusions were identified in the
hepatopancreas and lymphoid organ. The histological characteristic suggested the disease might be caused by a virus.
In the present study, a new nodavirus, tentatively named
covert mortality nodavirus (CMNV), was identified as
the aetiological agent of covert mortality disease through
construction of a cDNA library and sequencing, virus
extraction, fluorescence in situ hybridization (FISH),
histopathology, transmission electron microscopy (TEM),
and challenge testing.
(a)
(b)
RESULTS
Observation of clinical signs of farmed shrimp
The farmed shrimp of L. vannamei suffering from covert
mortality disease exhibited obvious clinical signs, including
hepatopancreatic atrophy with colour fading, empty stomach and guts, soft shell, and slow growth, and in many cases
were accompanied by uneven slightly whitish muscle lesion
areas in the abdominal segments or slightly pale body (Fig.
1a, b). The moribund shrimp sank to the bottom of deep
water and were rarely found in shallow water or swimming
under the surface. Farmers monitor the bottom of ponds for
dead shrimp by throwing out and drawing back a
convenient iron-made tetrahedroid with a net at the bottom
triangle and a drawstring at the top. Dead shrimp were also
often observed on the bottom downstream of aerators.
Moribund and dead shrimp could be found every day in
diseased ponds. The mortality began 1 month post-stocking
and increased after 60–80 days post-stocking, accompanied
by an increase of nitrite nitrogen (NO22-N) with a
cumulative mortality up to 80 %.
Histopathology of CMNV infection
Histological examination showed that muscle fibres composing the whitish muscle lesions had muscle fragmentation
tending towards coagulative, muscular lysis, and myonecrosis (Fig. 2a). Multifocal myonecrosis in the striated muscle
was accompanied by haemocytic infiltration and karyopyknosis of haemocytes (Fig. 2b). Vacuolation in the cytoplasm
of hepatopancreocytes and eosinophilic inclusions were
observed within the tubular epithelium of the hepatopancreas (Fig. 2c, d). In some cases, swollen nucleoli could be
found in hepatopancreocytes. Inclusions and nuclear
pyknosis were observed in the lymphoid spheroids (Fig. 2e,
f). Additionally, specimens of Fenneropenaeus chinensis and
Marsupenaeus japonicus with whitish abdominal muscle
collected from diseased ponds exhibited the same gross
histopathological characteristics.
TEM of virus particles
TEM of ultrathin sections of the hepatopancreas of
CMNV-infected shrimp revealed the presence of spherical
http://ijs.sgmjournals.org
Fig. 1. Clinical signs of L. vannamei suffering from covert mortality
disease: (a) from the artificial infection experiment and (b) from a
farmed pond. Black arrows show whitening muscle of the
abdominal segment. White arrows indicate hepatopancreas
atrophy and colour fading. The framed triangles show the healthy
shrimp, the black triangles indicate the shrimp suffering covert
mortality disease and the white triangles indicate the hepatopancreas of healthy shrimp.
unenveloped virus-like particles with a diameter of
~24.9±1.8 nm (n521) (Fig. 3a, b). Unenveloped viruslike particles were also observed with TEM of negativestained grids coated with the inoculation extracted from
diseased shrimp for challenge tests and purified virions
obtained by sucrose-gradient centrifugation (Fig. 3c, d).
Small spherical particles were also observed by TEM.
The larger virus-like particles were 32.1±5.5 nm (n537)
in diameter and the smaller spherical particles were
19.0±1.9 nm (n515) in diameter (Fig. 3c, d).
Sequencing and phylogenetic analyses
In total, 326 clones and 221 sequences were obtained
through the cDNA library constructed with the anchor
random primers from the total RNA of the hepatopancreas
of diseased shrimp. A BLAST search showed the amino acid
sequence of a 1185 bp fragment clone insert from the
cDNA library (designated CMNV-7; GenBank accession
number KM112247) shared 54, 53 and 39 % identity with
the amino acid sequences of RNA-dependent RNA
polymerase from Flock House virus (FHV), black beetle
virus (BBV) and MrNV, respectively (Table 1). Nucleotide
alignment by BLAST showed no significant similarity in
GenBank when the nucleotide fragment of CMNV-7 was
submitted. The resulting phylogenetic tree inference
revealed that the deduced amino acid sequence of the
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Q. Zhang and others
(b)
(a)
10 µm
20 µm
(d)
(c)
20 µm
10 µm
(f)
(e)
50 µm
10 µm
RNA-dependent RNA polymerase gene of CMNV
(CMNV-7) clustered in the genus Alphanodavirus, which
includes MrNV, PvNV, Nodamura virus, Boolarra virus,
FHV, BBV, Drosophila melanogaster American nodavirus
and Pariacoto virus (Fig. 4). Bootstrap values indicated that
the node support of the CMNV-7 isolate was 98 %,
strongly suggesting that the isolate was a novel virus within
the genus Alphanodavirus. The isolate was therefore
tentatively named CMNV.
Confirmation of the causative pathogen by
experimental challenge
To further identify whether CMNV was the aetiological
agent, experimental infections were conducted. The results
of the challenge test showed that cumulative mortality of
shrimp in the 0.22 mm filtered injection, unfiltered
injection and per os infection groups was 100, 100 and
84.85±2.14 %, respectively, by day 10 post-injection. In
contrast, there was no mortality of shrimp in the control
group injected with PBS (Fig. 5). Further, histopathological
examination showed striated muscle necrosis and the
presence of karyopyknosis, and inclusions in the hepatopancreas and lymphoid organ. All of the observations were
almost identical to those in clinical specimens. In addition,
RT-PCR analysis indicated that all shrimp from the
infected group were strongly positive for CMNV, whereas
shrimp from the control group were negative.
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Fig. 2. Histopathology of L. vannamei suffering
from covert mortality disease. Black arrows
show the karyopyknotic nuclei and white
arrows show the light purple inclusions. (a, b)
Haematoxylin and eosin (HE) staining of
abdominal muscle necrosis. The muscle
showed fragmentation tending towards coagulative and dissolving necrosis (black triangles). The framed triangles show the normal
skeletal muscle. (c, d) HE staining of atrophied
and necrotic hepatopancreatic epithelium.
Note that the inclusions in the hepatopancreatic tubular epithelium are one of the typical
histopathologic characteristics of CMNV infection. (e, f) HE staining of the lymphoid organ; (f)
shows the detail of the frame in (e). Note the
lymphoid organ spheroids. Bar, 20 (a), 10 (b),
20 (c), 10 (d), 50 (e) and 10 (f) mm.
FISH of CMNV
A 165 bp PCR-amplified product from CMNV-7 was
labelled with fluorescein and used as a probe to detect
CMNV by in situ hybridization in sections of infected L.
vannamei from the experimental infection. The results
showed that fluorescent signals were evident in the
hepatopancreas, lymphoid organ and striated muscle of
shrimp of the filtered injection group (Fig. 6). No reaction
was observed in tissues prepared from uninfected shrimp
(data not shown). The probe reacted most intensely with
the inclusions in the tubular epithelium and myoepithelial
cells surrounding the hepatopancreatic tubules (Fig. 6a–c).
For striated muscle, a few inclusion-like bodies showed a
very strong fluorescent signal. Diffusely distributed fluorescence was also observed within the muscle (Fig. 6d–f).
The probe also reacted intensely with the inclusions of the
lymphoid organ cells (Fig. 6g–i).
In addition, when the probe was used to detect CMNV in
shrimp that were collected from diseased ponds characterized with obvious whitish abdominal muscles, similar
fluorescent signals could also be found in the lymphoid
organ, striated muscle and hepatopancreas of those shrimp.
CMNV RT-PCR assay
A nested RT-PCR assay was developed for highly sensitive
detection of CMNV. The amplification result showed that
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Journal of General Virology 95
CMNV causes covert mortality disease of shrimp
(a)
(c)
(b)
Fig. 3. Transmission electron micrographs of
ultrathin section of the hepatopancreas and
negative-stained CMNV virions. (a) Viral inclusion (big black arrow) and CMNV particles
(white arrows) in the inclusion can be observed
in the ultrathin section of the hepatopancreas.
(b) Magnified micrograph of the virus particles
in the ultrathin section of the hepatopancreas.
The white arrows show the virus particles
assembled in the inclusion. (c) Virus-like
particles collected from sucrose-gradient fractionation. The size of the virus-like particles is
not homogeneous. The larger virus-like particles
(black arrows) are ~32 nm in diameter and the
smaller virus-like particles (white arrows) are
~19 nm in diameter. (d) Magnified micrograph
of the virus-like particles. The black arrows
indicate the larger virus-like particles and the
white arrows indicate the smaller virus-like
particles. Bar, 100 (a) and 50 (c, d) nm.
(d)
Table 1. Percentage similarity of the amino acid sequence of RNA-dependent RNA polymerase of CMNV compared with other
nodaviruses
Virus
Alphanodaviruses
Covert mortality nodavirus
Flock House virus
Black beetle virus
Macrobrachium rosenbergii nodavirus (China strain)
Macrobrachium rosenbergii nodavirus (Australia strain)
Macrobrachium rosenbergii nodavirus (Malaysia strain)
Penaeus vannamei nodavirus
Drosophila melanogaster American nodavirus
Nodamura virus
Boolarra virus
Pariacoto virus
Betanodaviruses
Striped jack nervous necrosis virus
Tiger puffer nervous necrosis virus
Atlantic halibut nodavirus
Golden pompano nervous necrosis virus
Atlantic cod nodavirus
Japanese flounder nervous necrosis virus
Dragon grouper nervous necrosis virus
Barfin flounder nervous necrosis virus
Redspotted grouper nervous necrosis virus
http://ijs.sgmjournals.org
Abbreviation
GenBank accession number
Sequence similarity
to CMNV (%)
CMNV
FHV
BBV
MrNV-CN
MrNV-AU
MrNV-MY
PvNV
DmANV
NoV
BoV
PaV
AIL48199
NP_689444
YP_053043
AAQ54758
AEY63648
AEQ39078
YP_004207810
ACU32794
NP_077730
NP_689439
NP_620109
100
54
53
39
39
39
37
48
44
46
35
SJNNV
TPNNV
AHNV
GPNNV
ACNV
JFNNV
DGNNV
BFNNV
RGNNV
NP_599247
YP_003288759
AAY34458
ACX54065
ABR23192
ACN58225
AAU85148
YP_003288756
ACX69744
33
32
32
32
32
31
31
31
31
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Q. Zhang and others
NoV
99
PvNV
MrNV-AU
95
94
MrNV-CN
89
88
MrNV-MY
CMNV
Alphanodavirus
BoV
98
FHV
100
BBV
75
57
DmANV
PaV
31
93
DGNNV
GPNNV
83
RGNNV
70
JFNNV
SJNNV
100
Betanodavirus
TPNNV
AHNV
93
BFNNV
60
71
ACNV
Fig. 4. Phylogenetic tree based on the deduced amino acid sequence of the CMNV-7 clone with RNA-dependent RNA
polymerases from other nodaviruses (for virus abbreviations, see Table 1). The tree was reconstructed by the neighbour-joining
method using MEGA5.0 and the numbers indicate percentage bootstrap support from replicates.
120
Cumulative mortality (%)
100
80
60
Per os infection
Unfiltered group
Filtered group
Control
40
20
0
0
24
48
72 96 120 144 168 192 216 240
Time after challenge (h)
Fig. 5. Cumulative mortality curves of L. vannamei in the
experimental infection. One group of healthy shrimp was challenged via per os infection (Per os infection). Three groups of
healthy shrimp were infected with a dilution of intramuscular
injection with unfiltered tissue homogenate (Unfiltered group),
injection with a diluted, 0.22 mm filtered tissue homogenate
(Filtered group) and injection with normal saline (Control),
respectively. Cumulative mortalities of shrimp are shown as means
of data from two replicates for each experimental group (each
replicate included 22 individuals).
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the first step of the PCR produced a 618 bp amplicon and
the second step of the RT-PCR produced a 165 bp
amplicon. The RNA samples extracted from purified virus,
and shrimp in the filtered group and unfiltered group were
all strongly CMNV-positive after the first step of the RTPCR (Fig. 7, lanes 1–3). The RNA samples extracted from
the healthy shrimp were CMNV-negative after the first and
second step of the RT-PCR. Specificity analysis against the
extracted nucleic acid samples from shrimp infected with
yellow head virus (YHV), MrNV, white spot syndrome
virus (WSSV) and Taura syndrome virus (TSV) indicated
that the nested RT-PCR was specific for CMNV.
When the newly developed nest RT-PCR was used to
amplify RNA from the hepatopancreas and muscle of
different shrimp for epidemiological investigation, some
specimens of L. vannamei, F. chinensis and M. japonicus with
gross signs of atrophied hepatopancreas, soft shell, slow
growth or whitish abdominal muscle were CMNV-positive.
DISCUSSION
The name ‘covert mortality disease’ (or ‘bottom death’)
was broadly used in China before 2009 to describe the
disease in which mortality was hidden under deep water in
shrimp farms (Xing, 2004; Zhang, 2004). It was suggested
that environmental factors, such as NO22-N, high temperature, etc., caused the disease (Xu & Ji, 2009). AHPND,
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CMNV causes covert mortality disease of shrimp
(a)
(b)
20 mm
(c)
20 mm
(d)
20 mm
(e)
20 mm
(f)
20 mm
(g)
20 mm
(h)
20 mm
(i)
20 mm
for which the causative agent was demonstrated to be
virulent strains of Vibrio parahaemolyticus (FAO, 2013;
Tran et al., 2013), hit the shrimp farming industries in 2010
in China (NACA, 2012). Many cases of EMS/AHPND were
still called covert mortality disease, as the new syndrome
looks like a severe case of covert mortality disease.
However, the present study reveals that the covert
mortality disease was caused by a suspected viral infection,
with typical histopathology of inclusions and pyknosis in
the tubular epithelium of the hepatopancreas, muscle and
lymphoid spheroids, which does not match the histopathology of infection with any known viruses. Phylogenetic
analysis of the sequenced clone insert in the cDNA libraries
from diseased shrimp revealed a new nodavirus belonging
to the genus Alphanodavirus. FISH with the probe
amplified from the fragment showed consistent signals
with the inclusions observed in the haematoxylin and eosin
(HE)-stained histopathology in the hepatopancreas,
lymphoid organ and muscle. The challenge test, which
http://ijs.sgmjournals.org
20 mm
Fig. 6. FISH using the CMNV-7 165 bp probe
on histological sections of L. vannamei
infected with CMNV: (a–c) hepatopancreas,
(d–f) muscles and (g–i) lymphoid organ. (a, e,
g) Fluorescein fluorescence; (b, f, h) tissues
stained by Evans blue; (c, d, i) merged images.
Green fluorescence in (c, d) indicates specific
binding of the CMNV-7 165 bp probe to viral
nucleic acids in infected cells; brown or yellow
signal indicates background. In (c), green
fluorescent inclusions were observed in the
tubular epithelium of the hepatopancreas and
the myoepithelial cells surrounding the hepatopancreatic tubules. In (d), Green fluorescence was very strong and diffusely
distributed within the necrotic muscle, and
can also be seen in the inclusions of necrotic
muscle. In (i), green fluorescent inclusions
were observed in the lymphoid spheroids.
Bar, 20 mm.
proved that both intramuscular injection with bacteriumfree filtered inoculum of tissue homogenates prepared
from the shrimp suffering from covert mortality disease
and per os infection could successfully pass on the disease
to healthy L. vannamei, indicated a virus-like infectious
agent as the most probable cause of the disease. The
challenged shrimp L. vannamei became infected and
exhibited the signs and lesions associated with the disease.
TEM examination of ultrathin sections of the lesions of
diseased shrimp and negative staining of virus suspensions
indicated particles with a mean diameter of 32 nm
(measured from the negative staining of purified virions;
a mean diameter of 25 nm was measured with ultrathin
sections). These results fulfil Rivers’ postulates for the
demonstration of a viral aetiology (Rivers, 1937). Based on
the findings of the current study, the aetiological agent of
the disease has been designated CMNV.
In this study, TEM showed that the size of CMNV virus
particles is larger than those of MrNV and PvNV; however,
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Q. Zhang and others
M
1
2
3
4
5
6
7
8
9 10 11 12
750 bp
500 bp
250 bp
100 bp
Fig. 7. RT-PCR detection of the presence of CMNV in the
experimentally infected L. vannamei, and the spontaneously
infected F. chinensis and M. japonicas collected from diseased
ponds. M, DL2000 molecular mass marker. Lanes 1–6: result of
the first step of RT-PCR; lanes 7–12: result of the second step of
RT-PCR. Lanes 1 and 7, 2 and 8, and 3 and 9: amplification
results of cDNAs from purified virus, and shrimp of the filtered
group and unfiltered group, respectively. Lanes 4 and 10 and 5
and 11: amplification results of cDNAs from F. chinensis and M.
japonicas, respectively. Lanes 6 and 12: negative controls.
it is in the diameter range of 32–33 nm, which is the typical
size of alphanodaviruses (Thiéry et al., 2012). In addition
to the major virus-like particles with a mean diameter of
32 nm, smaller particles were found in the virus preparations with a mean diameter of 19 nm. Given the size of the
smaller particles (i.e. 19 nm) and the tissue of viral
isolation (i.e. the hepatopancreas), it is highly likely that
a densovirus-like hepatopancreatic parvovirus (HPV) or
perhaps even infectious hypodermal and haematopoietic
necrosis virus (IHHNV) has been co-isolated with CMNV.
Nevertheless, the smaller particles were negative for HPV
and IHHNV when they were analysed by PCR methods
recommended by the World Organisation for Animal
Health (OIE, 2012) (data not shown). Extra small virus
particles of 14–16 nm diameter were also found in preparations of MrNV, which causes whitish muscle disease in
M. rosenbergii (Qian et al., 2003). The present study may
provide the second instance for the nodavirus-associated
satellite virus recognized by the International Committee on
Taxonomy of Viruses (Briddon et al., 2012).
The bootstrap values of cluster nodes of CMNV compared
with insect and crustacean nodaviruses were 98 and 94 %,
respectively. The fact that CMNV-7 shared the higher
bootstrap value with some of the insect nodaviruses rather
than the crustacean nodaviruses might be attributed to
the phylogenetic analysis, which was based on the partial
sequence of the RNA-dependent RNA polymerase gene of
CMNV (CMNV-7).
The clinical signs of CMNV infection have some similarities with the other two crustacean nodaviruses (MrNV and
PvNV) and IMNV (abdominal muscle whitening) in terms
of muscle lesions; however, these four viruses differ in
virulence. MrNV can cause 100 % mortality in the infected
post-larval and juvenile M. rosenbergii (Arcier et al., 1999;
Sahul Hameed et al., 2004; NaveenKumar et al., 2013).
PvNV does not cause mortality in L. vannamei in
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laboratory infections and the survival was reduced from a
mean of 74 % in 2003 (before PvNV was detected) to 50 %
after the appearance of PvNV in 2004 (Tang et al., 2007).
IMNV causes 20 % mortality in laboratory-infected L.
vannamei (Tang et al., 2005). Typically, the disease caused
by IMNV progresses slowly, with low mortality rates that
persist throughout the growing season, and the infection
can cause 40–60 % mortality in affected ponds (Nunes
et al., 2004). In contrast, our result showed that CMNV
cumulative mortality of L. vannamei in the per os infection
group is 84.85±2.14 % by day 10 post-injection and the
median lethal time (LT50) is 107 h. In the CMNV-infected
shrimp ponds, a slowly developing mortality was found
during daily management and cumulative mortality of L.
vannamei may be variable up to 80–90 %. High CMNVrelated mortality indicates that CMNV possesses higher
virulence than PvNV and IMNV.
In addition, the host range of MrNV, PvNV, IMNV and
CMNV appears to be different. MrNV was isolated originally from M. rosenbergii (Arcier et al., 1999) and several
species of marine penaeid shrimp (Fennerpenaeus indicus,
M. japonicus and P. monodon) have been identified as
reservoir hosts (Sudhakaran et al., 2006; Mello et al., 2011).
PvNV appears to have a limited host range; it was isolated
originally from L. vannamei (Tang et al., 2007) and did not
infect M. rosenbergii in a 4 week injection bioassay as
determined by RT-PCR analysis (Tang et al., 2011). IMNV
can infect L. vannamei, Litopenaeus stylirostris and P.
monodon, and it caused moderate mortality (20 %) in
infected L. vannamei and 0 % mortality in L. stylirostris or
P. monodon over a period of 4 weeks (Tang et al., 2005).
Our results from histopathology and nested RT-PCR
showed that CMNV can infect L. vannamei, F. chinensis
and M. japonicus, which indicated that the host range of
CMNV is wide in cultured shrimp species.
The target tissues of CMNV are different from those of
MrNV, PvNV, IMNV. Until now, there was no evidence or
report that demonstrated that MrNV, PvNV and IMNV
can cause hepatopancreatic atrophy and necrosis. In our
study, the shrimp with clinical signs collected from both
CMNV-infected ponds and from the experimental infection showed that CMNV caused hepatopancreatic atrophy
and necrosis. FISH of the hepatopancreas of CMNVinfected shrimp revealed that the CMNV infected the
tubular epithelium of the hepatopancreas. Early studies on
tissue tropism of fish nodavirus indicated that nodavirus
was detected primarily in nervous tissues (Nguyen et al.,
1997; Skliris & Richards, 1999; Grove et al., 2003; Nopadon
et al., 2009), whereas recent studies have shown that RNA
and viral proteins of nodavirus are present in the liver and
non-nervous organs, with the highest persistence of viral
antigens in the liver (Lopez-Jimena et al., 2011, 2012).
Virus detection in the liver may support epitheliotrophism
of nodaviruses (Lopez-Jimena et al., 2012). In addition,
the obvious fluorescent signal in myoepithelial cells
surrounding the hepatopancreatic tubules indicated
that myoepithelial cells were also targeted cells and the
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CMNV causes covert mortality disease of shrimp
destruction of myoepithelial cells might result in the atrophy
of hepatopancreatic tubules.
Additionally, similar to PvNV to some degree, CMNVrelated mortality may be sporadic in ponds, but the
mortality will be aggravated by environmental stressors,
such as NO22-N and high temperature. When the air
temperature is .30 uC and water temperature is .28 uC,
survival in CMNV-infected ponds decreases sharply.
Higher mortality can be found after 60–80 days poststocking during the summer crop (Zhang, 2004; Xing,
2004; Song & Zhuang, 2006; Xu & Ji, 2009; Gu, 2012).
In conclusion, through isolation, reinfection and histopathology, we revealed that CMNV, a new nodavirus, is
the causative agent of shrimp covert mortality disease.
Additionally, we developed a FISH and nested RT-PCR
method for the detection of CMNV. The results of the
current study indicate that CMNV is a new and highly
virulent nodavirus. These findings underscore the need for
professionals versed in aquatic animal health and farmers
in the shrimp aquaculture industries to pay close attention
and take measures to prevent disease outbreaks and
economic losses caused by CMNV.
METHODS
Shrimp. Penaeid shrimp exhibiting signs of whitish abdominal muscle
and hepatopancreatic atrophy were collected for virus isolation from
farms in Fujian, Shandong and Hebei Provinces in China during 2010–
2013. All farms were suffering from an outbreak of covert mortality
disease. For RT-PCR tests, L. vannamei, F. chinensis and M. japonicus
were sampled from shrimp farms from the coastal provinces of China
during 2012 and 2013. Apparently healthy subadult L. vannamei
shrimp (10 g mean weight) for challenge testing were purchased from a
shrimp farm in Changyi, Shandong Province. The shrimp were tested
and were demonstrated to be free of WSSV, YHV, TSV, IHHNV,
Enterocytozoon hepatopenaei and AHPNS by PCR or RT-PCR methods
recommended by the World Organisation for Animal Health (OIE,
2012; Flegel & Lo, 2013; Suebsing et al., 2013).
Virus isolation. Virus isolation was conducted according to the
method reported by Bonami et al. (2005) with a minor revision.
Sampled cephalothoraxes and whitish abdominal muscle were homogenized in TN buffer (20 mM Tris/HCl, 400 mM NaCl, pH 7.4) and
clarified at 1400 g for 15 min. The supernatant was clarified for a
second time at 10 000 g for 25 min. The final supernatant was then
centrifuged at 130 000 g for 4 h (Ultracentrifuge CP100WX; Hitachi).
After resuspension, pellets were twice Freon (1,1,2-trichloro-2,2,1trifluoroethane)-extracted and then pelleted for 4 h at 160 000 g. After
resuspension in TN buffer, the final pellet was layered onto a 20–50 %
(w/w) sucrose gradient and centrifuged at 160 000 g for 3 h.
ultrathin sectioning, the fixed tissues were secondarily fixed with 1 %
osmium tetroxide for 2 h, and then embedded in Spurr’s resin and
stained with uranyl acetate and lead citrate. Ultrathin sections were
prepared on collodion-coated grids by the Equipment Center of the
Medical College of Qingdao University. A suspension of the virus
from the isolation was dropped on the collodion-coated grids and
then negatively stained with 2 % phosphotungstic acid (pH 7.0). All
grids were examined in a JEOL JEM-1200 electron microscope
operating at 80–100 kV.
Challenge test. Healthy L. vannamei shrimp (10 g mean weight)
were cultured temporarily in indoor tanks for 5 days before they were
used for challenge tests. For the challenge study, the shrimp were
divided into an unfiltered injection group, a filtered injection group
and a control group. Each group included two replicates and each
replicate included 22 individuals. Cephalothoraxes and whitish
abdominal muscle were homogenized and clarified as described
above. The final supernatant was clarified for a second time at
10 000 g for 25 min and divided into two parts in two tubes. The
supernatant in the first tube was injected directly into healthy shrimp
in the lateral area of the fourth abdominal segment. The supernatant
in the second tube was filtered through a 0.22 mm membrane and
then centrifuged at 130 000 g for 4 h. The precipitation in the second
tube was resuspended as viral inoculum and injected directly into
healthy shrimp in the lateral area of the fourth abdominal segment.
The control group shrimp were injected with normal saline. For per os
infection, two challenge groups of healthy shrimp, 22 individuals of L.
vannamei per group, were fed minced CMNV-infected tissues
(9 mm3) at 10 % of total body weight after 24 h starvation.
Thereafter, the shrimp were maintained with pellet feeds for 10 days.
At the end of the bioassay, shrimp were sampled for nested RT-PCR
analysis. The healthy shrimp were raised similar to the control group.
Construction of a cDNA library and sequencing. Total RNA was
extracted from the cephalothoraxes of challenged shrimp by using a
QIAamp viral RNA kit (Qiagen) following the manufacturer’s
protocol. The cDNA was synthesized using an anchor random
primer (P17N8: 59-GTTTCCCAGTAGGTCTCNNNNNNNN-39) and
dsDNA was synthesized by PCR amplification according to the
method of Hang et al. (2012). The amplification products were
examined by agarose gel electrophoresis and then purified with a
PurElute GX DNA Gel Extraction and Cleanup kit (EdgeBio). A rapid
ligation amplicon pyrosequencing library for the purified, anchored,
random RT-PCR products was prepared by using a GS FLX Titanium
Rapid Library Preparation kit (Roche 454 Life Sciences) (Hang et al.,
2012). Clones containing the insert were sequenced and the amino
acid sequenced was deduced.
Phylogenetic analysis. The sequence of CMNV (CMNV-7) was
submitted for a BLAST (National Center for Biotechnology Information)
search and highly similar matches were included in the dataset for
phylogenetic analysis. A total of 20 RNA-dependent RNA polymerase
sequences (Table 1) were aligned with CLUSTAL_W as implemented in
MEGA5.0 (Tamura et al., 2011) using the default settings. The alignment
file was checked visually for alignment gaps and missing data. A
phylogenetic tree was then reconstructed by the neighbour-joining
method with bootstrap analysis (1000 replicates) using MEGA5.0.
Histopathological sections. Samples of the cephalothoraxes and
whitish abdominal muscle were fixed in Davidson’s alcohol formalin
acetic acid fixative (Bell & Lightner, 1988) for 24 h and then changed
to 70 % ethanol. Paraffin sections were prepared and stained with HE
according to the procedures of Bell & Lightner (1988).
TEM. Ultrathin sections of the hepatopancreas from infected shrimp
and virus preparations were analysed using TEM. Small pieces of the
hepatopancreas in ~1 mm3 of infected shrimp were fixed in 2.5 %
glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 4 uC. Before
http://ijs.sgmjournals.org
Probe labelling and FISH. A pair of primers designed from the
cDNA clone was used to label the fluorescent probe. PCR reaction
mixture (25 ml) contained 10 ng CMNV-7 plasmid, 10 mM Tris/HCl
(pH 8.3), 50 mM KCl, 4 mM MgCl2, 2 mM dATP/dCTP/dGTP,
1.5 mM dTTP, 0.5 mM Fluorescein-12-dUTP, 0.4 mM primers
(CMNV-7Probe-F: 59-GGCGATGACGGCTTGA-39 and CMNV7Probe-R: 59-GGCGGTGAGATGGATTTT-39) and 2.5 U Taq DNA
polymerase (PCR Fluorescein Labelling Mix; Roche Applied Science).
The PCR was carried out according to the protocol supported by the
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Q. Zhang and others
PCR Fluorescein Labelling Mix. Following PCR, the fluorescentlabelled DNA probe was precipitated with ethanol, resuspended in
water and stored at –20 uC.
Isolation and mapping of telomeric pentanucleotide (TAACC)n
repeats of the Pacific whiteleg shrimp, Penaeus vannamei, using
fluorescence in situ hybridization. Mar Biotechnol (NY) 8, 467–480.
Tissues originating from CMNV-infected ponds or experimental
challenge tests were fixed with RNA-friendly fixative (Hasson et al.,
1997) for 24 h and then transferred into 70 % ethanol for storage. The
fixed tissues were embedded in paraffin blocks and sectioned (6 mm
thick) in accordance with standard methods (Lightner, 1996). The
sections were then subjected to in situ hybridization assays according
to the protocol supported by the PCR Fluorescein Labelling Mix with
minor modifications adopted by Alcivar-Warren et al. (2006) and
Panphut et al. (2011). The PCR amplification product with
Fluorescein-12-dUTP and nucleic acid from healthy shrimp were
used in negative control hybridization. After incubation, the specimens were washed and viewed under a confocal microscope. The
cytoplasm was stained by Evans blue.
Arcier, J. M., Herman, F., Lightner, D. V., Redman, R. M., Mari,
J. & Bonami, J. R. (1999). A viral disease associated with mortalities
Nested RT-PCR. Total RNA was extracted from haemolymph and
cephalothoraxes of shrimp using RNAiso Plus (TaKaRa). The extracted
RNA was denatured initially at 65 uC for 5 min and then cooled on ice
for 2 min. The denatured RNA was then reverse transcribed using
SuperScript III Reverse Transcriptase (Invitrogen) at 50 uC for 60 min
and then denatured at 70 uC for 15 min. The reaction mixture contained
2 ml cDNA, 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2,
1.5 mM dNTP, 0.4 mM primers (CMNV-7F1: 59-AAATACGGCGATGACG-39 and CMNV-7R1: 59-ACGAAGTGCCCACAGAC-39) and
2.5 U TaKaRa EX Taq DNA polymerase (TaKaRa). The PCR was
performed at 94 uC for 4 min, followed by 35 cycles of 94 uC for 30 s,
45 uC for 30 s and 72 uC for 40 s, ending with 72 uC for 7 min. In the
first step, the RT-PCR amplified a 619 bp amplicon from the viral
genome. For the second step of the PCR, the PCR mixtures were the
same as that described above, except different template and primers
(CMNV-7F2: 59-CACAACCGAGTCAAACC-39 and CMNV-7R2: 59GCGTAAACAGCGAAGG-39) were included in the reactions. The
amplification was performed with the following cycling parameters:
initial denaturation at 94 uC for 4 min, followed by 30 cycles of 94 uC for
20 s, 50 uC for 20 s and 72 uC for 20 s, and a final extension at 72 uC for
7 min. A 165 bp amplicon was amplified by the second step of the PCR.
To test the specificity of the RT-PCR, nucleic acids from shrimp
infected with YHV, MrNV, WSSV and TSV were used as template in
the RT-PCR. PCR products (5 ml) were analysed in a 1.5 % agarose
gel containing GeneFinder (Bio-V).
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Bonami, J. R., Shi, Z., Qian, D. & Sri Widada, J. (2005). White tail
disease of the giant freshwater prawn, Macrobrachium rosenbergii:
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ACKNOWLEDGEMENTS
Hasson, K. W., Hasson, J., Aubert, H., Redman, R. M. & Lightner, D. V.
(1997). A new RNA-friendly fixative for the preservation of penaeid
The authors would like to thank Dr Andrew D. Winters (Michigan
State University) for his generous help in the grammatical correction of
the manuscript. The authors also thank Dr Leigh Owens (James Cook
University), Dr Timothy W. Flegel (Mahidol University) and Dr
Donald V. Lightner (University of Arizona) for their advice on
histopathology. This work was supported by the projects under the
Special Fund for Agro-scientific Research in the Public Interest (grant
201103034), China Agriculture Research System (CARS-47), the
Special Scientific Research Funds for Central Non-profit Institutes,
Chinese Academy of Fishery Sciences (2013A0601 and 2014A06XK01),
the Construction Programme for ‘Taishan Scholarship’ of Shandong
Province of China, and the Programme for Chinese Outstanding
Talents in Agricultural Scientific Research.
shrimp samples for virological detection using cDNA genomic
probes. J Virol Methods 66, 227–236.
Huang, J. (2012). Experience in EMS/AHPNS from China. In:
Report of the Asia Pacific Emergency Regional Consultation on the
Emerging Shrimp Disease: Early Mortality Syndrome (EMS)/Acute
Hepatopancreatic Necrosis Syndrome (AHPNS), pp. 21–23. Edited by
NACA Bangkok: Network of Aquaculture Centres in Asia-Pacific.
Lightner, D. V. (1996). A Handbook of Shrimp Pathology and
Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. Baton
Rouge, LA: World Aquaculture Society.
Lightner, D. V., Redman, R. M., Pantoja, C. R., Noble, B. L. & Tran, L. H.
(2012). Early mortality syndrome affects shrimp in Asia. Glob.
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