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Journal of General Virology (2014), 95, 2700–2709 DOI 10.1099/vir.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 1
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture,
Qingli Zhang Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
zhangql@ysfri.ac.cn 106 Nanjing Road, Qingdao 266071, PR China
Jie Huang 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
Received 16 July 2014 chinensis, Litopenaeus vannamei and Marsupenaeus japonicus suffering from this disease were
Accepted 8 September 2014 CMNV-positive.

INTRODUCTION acute hepatopancreas necrosis disease (AHPND) that

emerged in the south of China in 2010 (Lightner et al.,
A major disease that occurred in the shrimp farming
2012) was once thought to represent severe cases of covert
industries in China before 2009 was generally named ‘covert
mortality disease as its clinical signs were similar to that of
mortality disease’ due to the most moribund shrimp hiding
covert mortality disease to some degree.
on the bottom in deep water rather than swimming to the
surface or in shallow water, as did shrimp suffering from Infection with several RNA viruses has been found to
white spot disease (Zhang, 2004; Xing, 2004; Song & cause typical muscle necrosis in cultured shrimp. Infectious
Zhuang, 2006; Xu & Ji, 2009; Gu, 2012; Huang, 2012). The myonecrosis virus (IMNV) and Penaeus vannamei noda-
shrimp suffering the disease exhibited clinical signs virus (PvNV) infect Litopenaeus vannamei and result in
including hepatopancreatic atrophy and necrosis, empty muscle necrosis (Poulos et al., 2006; Tang et al., 2007; Flegel,
stomach and guts, soft shell, slow growth, and in many cases 2012). Macrobrachium rosenbergii nodavirus (MrNV) can
abdominal muscle whitening and necrosis (Zhang, 2004; infect freshwater prawns and L. vannamei (Qian et al.,
Huang, 2012). The farmers noted that dead shrimp could be 2003; Bonami et al., 2005; Senapin et al., 2011, 2013;
found in the diseased population every day and mortality NaveenKumar et al., 2013), causing muscle necrosis in tail
increased during 60–80 days post-stocking with a cumulat- muscles. However, IMNV, MrNV and PvNV were not
ive mortality up to 80 %. Early mortality syndrome (EMS)/ detectable in shrimp suffering from covert mortality disease
using relevant reverse transcription (RT)-PCR methods. As
The GenBank/EMBL/DDBJ accession number for the RNA-dependent has been observed for IMNV, MrNV and PvNV, histological
RNA polymerase gene sequence is KM112247. 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. (a)
In addition, eosinophilic inclusions were identified in the
hepatopancreas and lymphoid organ. The histological char-
acteristic 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), (b)
histopathology, transmission electron microscopy (TEM),
and challenge testing.


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 stom-
ach and guts, soft shell, and slow growth, and in many cases Fig. 1. Clinical signs of L. vannamei suffering from covert mortality
were accompanied by uneven slightly whitish muscle lesion disease: (a) from the artificial infection experiment and (b) from a
areas in the abdominal segments or slightly pale body (Fig. farmed pond. Black arrows show whitening muscle of the
1a, b). The moribund shrimp sank to the bottom of deep abdominal segment. White arrows indicate hepatopancreas
water and were rarely found in shallow water or swimming atrophy and colour fading. The framed triangles show the healthy
under the surface. Farmers monitor the bottom of ponds for shrimp, the black triangles indicate the shrimp suffering covert
dead shrimp by throwing out and drawing back a mortality disease and the white triangles indicate the hepatopan-
convenient iron-made tetrahedroid with a net at the bottom creas of healthy shrimp.
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 unenveloped virus-like particles with a diameter of
and increased after 60–80 days post-stocking, accompanied ~24.9±1.8 nm (n521) (Fig. 3a, b). Unenveloped virus-
by an increase of nitrite nitrogen (NO22-N) with a like particles were also observed with TEM of negative-
cumulative mortality up to 80 %. stained grids coated with the inoculation extracted from
diseased shrimp for challenge tests and purified virions
obtained by sucrose-gradient centrifugation (Fig. 3c, d).
Histopathology of CMNV infection Small spherical particles were also observed by TEM.
Histological examination showed that muscle fibres com- The larger virus-like particles were 32.1±5.5 nm (n537)
posing the whitish muscle lesions had muscle fragmentation in diameter and the smaller spherical particles were
tending towards coagulative, muscular lysis, and myonecro- 19.0±1.9 nm (n515) in diameter (Fig. 3c, d).
sis (Fig. 2a). Multifocal myonecrosis in the striated muscle
was accompanied by haemocytic infiltration and karyopy- Sequencing and phylogenetic analyses
knosis of haemocytes (Fig. 2b). Vacuolation in the cytoplasm
of hepatopancreocytes and eosinophilic inclusions were In total, 326 clones and 221 sequences were obtained
observed within the tubular epithelium of the hepatopan- through the cDNA library constructed with the anchor
creas (Fig. 2c, d). In some cases, swollen nucleoli could be random primers from the total RNA of the hepatopancreas
found in hepatopancreocytes. Inclusions and nuclear of diseased shrimp. A BLAST search showed the amino acid
pyknosis were observed in the lymphoid spheroids (Fig. 2e, sequence of a 1185 bp fragment clone insert from the
f). Additionally, specimens of Fenneropenaeus chinensis and cDNA library (designated CMNV-7; GenBank accession
Marsupenaeus japonicus with whitish abdominal muscle number KM112247) shared 54, 53 and 39 % identity with
collected from diseased ponds exhibited the same gross the amino acid sequences of RNA-dependent RNA
histopathological characteristics. polymerase from Flock House virus (FHV), black beetle
virus (BBV) and MrNV, respectively (Table 1). Nucleotide
alignment by BLAST showed no significant similarity in
TEM of virus particles
GenBank when the nucleotide fragment of CMNV-7 was
TEM of ultrathin sections of the hepatopancreas of submitted. The resulting phylogenetic tree inference
CMNV-infected shrimp revealed the presence of spherical revealed that the deduced amino acid sequence of the
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Q. Zhang and others

(a) (b)

20 µm 10 µm

(c) (d)

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 coa-
20 µm 10 µm gulative and dissolving necrosis (black trian-
gles). The framed triangles show the normal
(e) (f)
skeletal muscle. (c, d) HE staining of atrophied
and necrotic hepatopancreatic epithelium.
Note that the inclusions in the hepatopancrea-
tic tubular epithelium are one of the typical
histopathologic characteristics of CMNV infec-
tion. (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),
50 µm 10 µm
20 (c), 10 (d), 50 (e) and 10 (f) mm.

RNA-dependent RNA polymerase gene of CMNV FISH of CMNV

(CMNV-7) clustered in the genus Alphanodavirus, which
A 165 bp PCR-amplified product from CMNV-7 was
includes MrNV, PvNV, Nodamura virus, Boolarra virus,
labelled with fluorescein and used as a probe to detect
FHV, BBV, Drosophila melanogaster American nodavirus
CMNV by in situ hybridization in sections of infected L.
and Pariacoto virus (Fig. 4). Bootstrap values indicated that
vannamei from the experimental infection. The results
the node support of the CMNV-7 isolate was 98 %,
showed that fluorescent signals were evident in the
strongly suggesting that the isolate was a novel virus within
hepatopancreas, lymphoid organ and striated muscle of
the genus Alphanodavirus. The isolate was therefore
shrimp of the filtered injection group (Fig. 6). No reaction
tentatively named CMNV.
was observed in tissues prepared from uninfected shrimp
(data not shown). The probe reacted most intensely with
Confirmation of the causative pathogen by the inclusions in the tubular epithelium and myoepithelial
experimental challenge cells surrounding the hepatopancreatic tubules (Fig. 6a–c).
For striated muscle, a few inclusion-like bodies showed a
To further identify whether CMNV was the aetiological
very strong fluorescent signal. Diffusely distributed fluor-
agent, experimental infections were conducted. The results
escence was also observed within the muscle (Fig. 6d–f).
of the challenge test showed that cumulative mortality of
The probe also reacted intensely with the inclusions of the
shrimp in the 0.22 mm filtered injection, unfiltered
lymphoid organ cells (Fig. 6g–i).
injection and per os infection groups was 100, 100 and
84.85±2.14 %, respectively, by day 10 post-injection. In In addition, when the probe was used to detect CMNV in
contrast, there was no mortality of shrimp in the control shrimp that were collected from diseased ponds character-
group injected with PBS (Fig. 5). Further, histopathological ized with obvious whitish abdominal muscles, similar
examination showed striated muscle necrosis and the fluorescent signals could also be found in the lymphoid
presence of karyopyknosis, and inclusions in the hepato- organ, striated muscle and hepatopancreas of those shrimp.
pancreas 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 A nested RT-PCR assay was developed for highly sensitive
shrimp from the control group were negative. detection of CMNV. The amplification result showed that
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CMNV causes covert mortality disease of shrimp

(a) (b)

Fig. 3. Transmission electron micrographs of

ultrathin section of the hepatopancreas and
negative-stained CMNV virions. (a) Viral inclu-
sion (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
(c) (d)
assembled in the inclusion. (c) Virus-like
particles collected from sucrose-gradient frac-
tionation. 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.

Table 1. Percentage similarity of the amino acid sequence of RNA-dependent RNA polymerase of CMNV compared with other

Virus Abbreviation GenBank accession number Sequence similarity

to CMNV (%)

Covert mortality nodavirus CMNV AIL48199 100
Flock House virus FHV NP_689444 54
Black beetle virus BBV YP_053043 53
Macrobrachium rosenbergii nodavirus (China strain) MrNV-CN AAQ54758 39
Macrobrachium rosenbergii nodavirus (Australia strain) MrNV-AU AEY63648 39
Macrobrachium rosenbergii nodavirus (Malaysia strain) MrNV-MY AEQ39078 39
Penaeus vannamei nodavirus PvNV YP_004207810 37
Drosophila melanogaster American nodavirus DmANV ACU32794 48
Nodamura virus NoV NP_077730 44
Boolarra virus BoV NP_689439 46
Pariacoto virus PaV NP_620109 35
Striped jack nervous necrosis virus SJNNV NP_599247 33
Tiger puffer nervous necrosis virus TPNNV YP_003288759 32
Atlantic halibut nodavirus AHNV AAY34458 32
Golden pompano nervous necrosis virus GPNNV ACX54065 32
Atlantic cod nodavirus ACNV ABR23192 32
Japanese flounder nervous necrosis virus JFNNV ACN58225 31
Dragon grouper nervous necrosis virus DGNNV AAU85148 31
Barfin flounder nervous necrosis virus BFNNV YP_003288756 31
Redspotted grouper nervous necrosis virus RGNNV ACX69744 31

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Q. Zhang and others

94 89 MrNV-CN
88 MrNV-MY
CMNV Alphanodavirus
75 BBV
57 DmANV
SJNNV Betanodavirus

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.

the first step of the PCR produced a 618 bp amplicon and

120 the second step of the RT-PCR produced a 165 bp
amplicon. The RNA samples extracted from purified virus,
100 and shrimp in the filtered group and unfiltered group were
all strongly CMNV-positive after the first step of the RT-
Cumulative mortality (%)

80 PCR (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
Per os infection yellow head virus (YHV), MrNV, white spot syndrome
40 Unfiltered group virus (WSSV) and Taura syndrome virus (TSV) indicated
Filtered group that the nested RT-PCR was specific for CMNV.
20 Control
When the newly developed nest RT-PCR was used to
amplify RNA from the hepatopancreas and muscle of
0 24 48 72 96 120 144 168 192 216 240
different shrimp for epidemiological investigation, some
Time after challenge (h) 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.
Fig. 5. Cumulative mortality curves of L. vannamei in the
experimental infection. One group of healthy shrimp was chal-
lenged via per os infection (Per os infection). Three groups of DISCUSSION
healthy shrimp were infected with a dilution of intramuscular
injection with unfiltered tissue homogenate (Unfiltered group), The name ‘covert mortality disease’ (or ‘bottom death’)
injection with a diluted, 0.22 mm filtered tissue homogenate was broadly used in China before 2009 to describe the
(Filtered group) and injection with normal saline (Control), disease in which mortality was hidden under deep water in
respectively. Cumulative mortalities of shrimp are shown as means shrimp farms (Xing, 2004; Zhang, 2004). It was suggested
of data from two replicates for each experimental group (each that environmental factors, such as NO22-N, high tempe-
replicate included 22 individuals). rature, etc., caused the disease (Xu & Ji, 2009). AHPND,
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CMNV causes covert mortality disease of shrimp

(a) (b)

20 mm 20 mm

(c) (d)

Fig. 6. FISH using the CMNV-7 165 bp probe

20 mm 20 mm
on histological sections of L. vannamei
(e) (f) 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
20 mm 20 mm
fluorescent inclusions were observed in the
tubular epithelium of the hepatopancreas and
(g) (h) (i)
the myoepithelial cells surrounding the hepa-
topancreatic tubules. In (d), Green fluor-
escence 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.
20 mm 20 mm 20 mm
Bar, 20 mm.

for which the causative agent was demonstrated to be proved that both intramuscular injection with bacterium-
virulent strains of Vibrio parahaemolyticus (FAO, 2013; free filtered inoculum of tissue homogenates prepared
Tran et al., 2013), hit the shrimp farming industries in 2010 from the shrimp suffering from covert mortality disease
in China (NACA, 2012). Many cases of EMS/AHPND were and per os infection could successfully pass on the disease
still called covert mortality disease, as the new syndrome to healthy L. vannamei, indicated a virus-like infectious
looks like a severe case of covert mortality disease. agent as the most probable cause of the disease. The
However, the present study reveals that the covert challenged shrimp L. vannamei became infected and
mortality disease was caused by a suspected viral infection, exhibited the signs and lesions associated with the disease.
with typical histopathology of inclusions and pyknosis in TEM examination of ultrathin sections of the lesions of
the tubular epithelium of the hepatopancreas, muscle and diseased shrimp and negative staining of virus suspensions
lymphoid spheroids, which does not match the histopatho- indicated particles with a mean diameter of 32 nm
logy of infection with any known viruses. Phylogenetic (measured from the negative staining of purified virions;
analysis of the sequenced clone insert in the cDNA libraries a mean diameter of 25 nm was measured with ultrathin
from diseased shrimp revealed a new nodavirus belonging sections). These results fulfil Rivers’ postulates for the
to the genus Alphanodavirus. FISH with the probe demonstration of a viral aetiology (Rivers, 1937). Based on
amplified from the fragment showed consistent signals the findings of the current study, the aetiological agent of
the disease has been designated CMNV.
with the inclusions observed in the haematoxylin and eosin
(HE)-stained histopathology in the hepatopancreas, In this study, TEM showed that the size of CMNV virus
lymphoid organ and muscle. The challenge test, which 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 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).
750 bp IMNV causes 20 % mortality in laboratory-infected L.
500 bp
vannamei (Tang et al., 2005). Typically, the disease caused
250 bp
by IMNV progresses slowly, with low mortality rates that
100 bp 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
Fig. 7. RT-PCR detection of the presence of CMNV in the cumulative mortality of L. vannamei in the per os infection
experimentally infected L. vannamei, and the spontaneously group is 84.85±2.14 % by day 10 post-injection and the
infected F. chinensis and M. japonicas collected from diseased median lethal time (LT50) is 107 h. In the CMNV-infected
ponds. M, DL2000 molecular mass marker. Lanes 1–6: result of shrimp ponds, a slowly developing mortality was found
the first step of RT-PCR; lanes 7–12: result of the second step of during daily management and cumulative mortality of L.
RT-PCR. Lanes 1 and 7, 2 and 8, and 3 and 9: amplification vannamei may be variable up to 80–90 %. High CMNV-
results of cDNAs from purified virus, and shrimp of the filtered related mortality indicates that CMNV possesses higher
group and unfiltered group, respectively. Lanes 4 and 10 and 5 virulence than PvNV and IMNV.
and 11: amplification results of cDNAs from F. chinensis and M.
japonicas, respectively. Lanes 6 and 12: negative controls.
In addition, the host range of MrNV, PvNV, IMNV and
CMNV appears to be different. MrNV was isolated origi-
nally from M. rosenbergii (Arcier et al., 1999) and several
species of marine penaeid shrimp (Fennerpenaeus indicus,
it is in the diameter range of 32–33 nm, which is the typical
M. japonicus and P. monodon) have been identified as
size of alphanodaviruses (Thiéry et al., 2012). In addition
reservoir hosts (Sudhakaran et al., 2006; Mello et al., 2011).
to the major virus-like particles with a mean diameter of
PvNV appears to have a limited host range; it was isolated
32 nm, smaller particles were found in the virus prepara-
originally from L. vannamei (Tang et al., 2007) and did not
tions with a mean diameter of 19 nm. Given the size of the
infect M. rosenbergii in a 4 week injection bioassay as
smaller particles (i.e. 19 nm) and the tissue of viral
determined by RT-PCR analysis (Tang et al., 2011). IMNV
isolation (i.e. the hepatopancreas), it is highly likely that
can infect L. vannamei, Litopenaeus stylirostris and P.
a densovirus-like hepatopancreatic parvovirus (HPV) or
monodon, and it caused moderate mortality (20 %) in
perhaps even infectious hypodermal and haematopoietic
infected L. vannamei and 0 % mortality in L. stylirostris or
necrosis virus (IHHNV) has been co-isolated with CMNV.
P. monodon over a period of 4 weeks (Tang et al., 2005).
Nevertheless, the smaller particles were negative for HPV
Our results from histopathology and nested RT-PCR
and IHHNV when they were analysed by PCR methods
showed that CMNV can infect L. vannamei, F. chinensis
recommended by the World Organisation for Animal
and M. japonicus, which indicated that the host range of
Health (OIE, 2012) (data not shown). Extra small virus
CMNV is wide in cultured shrimp species.
particles of 14–16 nm diameter were also found in prepara-
tions of MrNV, which causes whitish muscle disease in The target tissues of CMNV are different from those of
M. rosenbergii (Qian et al., 2003). The present study may MrNV, PvNV, IMNV. Until now, there was no evidence or
provide the second instance for the nodavirus-associated report that demonstrated that MrNV, PvNV and IMNV
satellite virus recognized by the International Committee on can cause hepatopancreatic atrophy and necrosis. In our
Taxonomy of Viruses (Briddon et al., 2012). study, the shrimp with clinical signs collected from both
CMNV-infected ponds and from the experimental infec-
The bootstrap values of cluster nodes of CMNV compared
tion showed that CMNV caused hepatopancreatic atrophy
with insect and crustacean nodaviruses were 98 and 94 %,
and necrosis. FISH of the hepatopancreas of CMNV-
respectively. The fact that CMNV-7 shared the higher
infected shrimp revealed that the CMNV infected the
bootstrap value with some of the insect nodaviruses rather
tubular epithelium of the hepatopancreas. Early studies on
than the crustacean nodaviruses might be attributed to
tissue tropism of fish nodavirus indicated that nodavirus
the phylogenetic analysis, which was based on the partial
was detected primarily in nervous tissues (Nguyen et al.,
sequence of the RNA-dependent RNA polymerase gene of
1997; Skliris & Richards, 1999; Grove et al., 2003; Nopadon
et al., 2009), whereas recent studies have shown that RNA
The clinical signs of CMNV infection have some similar- and viral proteins of nodavirus are present in the liver and
ities with the other two crustacean nodaviruses (MrNV and non-nervous organs, with the highest persistence of viral
PvNV) and IMNV (abdominal muscle whitening) in terms antigens in the liver (Lopez-Jimena et al., 2011, 2012).
of muscle lesions; however, these four viruses differ in Virus detection in the liver may support epitheliotrophism
virulence. MrNV can cause 100 % mortality in the infected of nodaviruses (Lopez-Jimena et al., 2012). In addition,
post-larval and juvenile M. rosenbergii (Arcier et al., 1999; the obvious fluorescent signal in myoepithelial cells
Sahul Hameed et al., 2004; NaveenKumar et al., 2013). surrounding the hepatopancreatic tubules indicated
PvNV does not cause mortality in L. vannamei in 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 ultrathin sectioning, the fixed tissues were secondarily fixed with 1 %
of hepatopancreatic tubules. osmium tetroxide for 2 h, and then embedded in Spurr’s resin and
stained with uranyl acetate and lead citrate. Ultrathin sections were
Additionally, similar to PvNV to some degree, CMNV- prepared on collodion-coated grids by the Equipment Center of the
related mortality may be sporadic in ponds, but the Medical College of Qingdao University. A suspension of the virus
mortality will be aggravated by environmental stressors, from the isolation was dropped on the collodion-coated grids and
then negatively stained with 2 % phosphotungstic acid (pH 7.0). All
such as NO22-N and high temperature. When the air grids were examined in a JEOL JEM-1200 electron microscope
temperature is .30 uC and water temperature is .28 uC, operating at 80–100 kV.
survival in CMNV-infected ponds decreases sharply.
Higher mortality can be found after 60–80 days post- Challenge test. Healthy L. vannamei shrimp (10 g mean weight)
stocking during the summer crop (Zhang, 2004; Xing, were cultured temporarily in indoor tanks for 5 days before they were
2004; Song & Zhuang, 2006; Xu & Ji, 2009; Gu, 2012). used for challenge tests. For the challenge study, the shrimp were
divided into an unfiltered injection group, a filtered injection group
In conclusion, through isolation, reinfection and his- and a control group. Each group included two replicates and each
topathology, we revealed that CMNV, a new nodavirus, is replicate included 22 individuals. Cephalothoraxes and whitish
the causative agent of shrimp covert mortality disease. abdominal muscle were homogenized and clarified as described
above. The final supernatant was clarified for a second time at
Additionally, we developed a FISH and nested RT-PCR 10 000 g for 25 min and divided into two parts in two tubes. The
method for the detection of CMNV. The results of the supernatant in the first tube was injected directly into healthy shrimp
current study indicate that CMNV is a new and highly in the lateral area of the fourth abdominal segment. The supernatant
virulent nodavirus. These findings underscore the need for in the second tube was filtered through a 0.22 mm membrane and
professionals versed in aquatic animal health and farmers then centrifuged at 130 000 g for 4 h. The precipitation in the second
in the shrimp aquaculture industries to pay close attention tube was resuspended as viral inoculum and injected directly into
healthy shrimp in the lateral area of the fourth abdominal segment.
and take measures to prevent disease outbreaks and
The control group shrimp were injected with normal saline. For per os
economic losses caused by CMNV. 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.
METHODS Thereafter, the shrimp were maintained with pellet feeds for 10 days.
At the end of the bioassay, shrimp were sampled for nested RT-PCR
Shrimp. Penaeid shrimp exhibiting signs of whitish abdominal muscle analysis. The healthy shrimp were raised similar to the control group.
and hepatopancreatic atrophy were collected for virus isolation from
farms in Fujian, Shandong and Hebei Provinces in China during 2010– Construction of a cDNA library and sequencing. Total RNA was
2013. All farms were suffering from an outbreak of covert mortality extracted from the cephalothoraxes of challenged shrimp by using a
disease. For RT-PCR tests, L. vannamei, F. chinensis and M. japonicus QIAamp viral RNA kit (Qiagen) following the manufacturer’s
were sampled from shrimp farms from the coastal provinces of China protocol. The cDNA was synthesized using an anchor random
during 2012 and 2013. Apparently healthy subadult L. vannamei primer (P17N8: 59-GTTTCCCAGTAGGTCTCNNNNNNNN-39) and
shrimp (10 g mean weight) for challenge testing were purchased from a dsDNA was synthesized by PCR amplification according to the
shrimp farm in Changyi, Shandong Province. The shrimp were tested method of Hang et al. (2012). The amplification products were
and were demonstrated to be free of WSSV, YHV, TSV, IHHNV, examined by agarose gel electrophoresis and then purified with a
Enterocytozoon hepatopenaei and AHPNS by PCR or RT-PCR methods PurElute GX DNA Gel Extraction and Cleanup kit (EdgeBio). A rapid
recommended by the World Organisation for Animal Health (OIE, ligation amplicon pyrosequencing library for the purified, anchored,
2012; Flegel & Lo, 2013; Suebsing et al., 2013). random RT-PCR products was prepared by using a GS FLX Titanium
Rapid Library Preparation kit (Roche 454 Life Sciences) (Hang et al.,
Virus isolation. Virus isolation was conducted according to the 2012). Clones containing the insert were sequenced and the amino
method reported by Bonami et al. (2005) with a minor revision. acid sequenced was deduced.
Sampled cephalothoraxes and whitish abdominal muscle were homo-
genized in TN buffer (20 mM Tris/HCl, 400 mM NaCl, pH 7.4) and Phylogenetic analysis. The sequence of CMNV (CMNV-7) was
clarified at 1400 g for 15 min. The supernatant was clarified for a submitted for a BLAST (National Center for Biotechnology Information)
second time at 10 000 g for 25 min. The final supernatant was then search and highly similar matches were included in the dataset for
centrifuged at 130 000 g for 4 h (Ultracentrifuge CP100WX; Hitachi). phylogenetic analysis. A total of 20 RNA-dependent RNA polymerase
After resuspension, pellets were twice Freon (1,1,2-trichloro-2,2,1- sequences (Table 1) were aligned with CLUSTAL_W as implemented in
trifluoroethane)-extracted and then pelleted for 4 h at 160 000 g. After MEGA5.0 (Tamura et al., 2011) using the default settings. The alignment
resuspension in TN buffer, the final pellet was layered onto a 20–50 % file was checked visually for alignment gaps and missing data. A
(w/w) sucrose gradient and centrifuged at 160 000 g for 3 h. 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 Probe labelling and FISH. A pair of primers designed from the
acetic acid fixative (Bell & Lightner, 1988) for 24 h and then changed cDNA clone was used to label the fluorescent probe. PCR reaction
to 70 % ethanol. Paraffin sections were prepared and stained with HE mixture (25 ml) contained 10 ng CMNV-7 plasmid, 10 mM Tris/HCl
according to the procedures of Bell & Lightner (1988). (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
TEM. Ultrathin sections of the hepatopancreas from infected shrimp (CMNV-7Probe-F: 59-GGCGATGACGGCTTGA-39 and CMNV-
and virus preparations were analysed using TEM. Small pieces of the 7Probe-R: 59-GGCGGTGAGATGGATTTT-39) and 2.5 U Taq DNA
hepatopancreas in ~1 mm3 of infected shrimp were fixed in 2.5 % polymerase (PCR Fluorescein Labelling Mix; Roche Applied Science).
glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 4 uC. Before 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 fluorescent- Isolation and mapping of telomeric pentanucleotide (TAACC)n
labelled DNA probe was precipitated with ethanol, resuspended in repeats of the Pacific whiteleg shrimp, Penaeus vannamei, using
water and stored at –20 uC. fluorescence in situ hybridization. Mar Biotechnol (NY) 8, 467–480.
Tissues originating from CMNV-infected ponds or experimental Arcier, J. M., Herman, F., Lightner, D. V., Redman, R. M., Mari,
challenge tests were fixed with RNA-friendly fixative (Hasson et al., J. & Bonami, J. R. (1999). A viral disease associated with mortalities
1997) for 24 h and then transferred into 70 % ethanol for storage. The in hatchery-reared postlarvae of the giant freshwater prawn
fixed tissues were embedded in paraffin blocks and sectioned (6 mm Macrobrachium rosenbergii. Dis Aquat Organ 38, 177–181.
thick) in accordance with standard methods (Lightner, 1996). The Bell, T. A. & Lightner, D. V. (1988). A Handbook of Normal Penaeid
sections were then subjected to in situ hybridization assays according Shrimp Histology. Baton Rouge, LA: World Aquaculture Society.
to the protocol supported by the PCR Fluorescein Labelling Mix with
Bonami, J. R., Shi, Z., Qian, D. & Sri Widada, J. (2005). White tail
minor modifications adopted by Alcivar-Warren et al. (2006) and
disease of the giant freshwater prawn, Macrobrachium rosenbergii:
Panphut et al. (2011). The PCR amplification product with
separation of the associated virions and characterization of MrNV as a
Fluorescein-12-dUTP and nucleic acid from healthy shrimp were
new type of nodavirus. J Fish Dis 28, 23–31.
used in negative control hybridization. After incubation, the speci-
mens were washed and viewed under a confocal microscope. The Briddon, R. W., Ghabrial, S., Lin, N.-S., Palukaitis, P., Scholthof, K.-B. G.
cytoplasm was stained by Evans blue. & Vetten, H.-J. (2012). Satellites and other virus-dependent nucleic
acids. In Virus Taxonomy: Ninth Report of the International Committee
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cephalothoraxes of shrimp using RNAiso Plus (TaKaRa). The extracted M. J. Adams, E. B. Carstens & E. J. Lefkowitz. London: Academic Press.
RNA was denatured initially at 65 uC for 5 min and then cooled on ice FAO (2013). Report of the FAO/MARD Technical Workshop on Early
for 2 min. The denatured RNA was then reverse transcribed using Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis
SuperScript III Reverse Transcriptase (Invitrogen) at 50 uC for 60 min Syndrome (AHPNS) of Cultured Shrimp (under TCP/VIE/3304).
and then denatured at 70 uC for 15 min. The reaction mixture contained Hanoi, Viet Nam, 25–27 June 2013. FAO Fisheries and Aquaculture
2 ml cDNA, 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, Report 1053. Rome: FAO.
1.5 mM dNTP, 0.4 mM primers (CMNV-7F1: 59-AAATACGGCGA-
Flegel, T. W. (2012). Historic emergence, impact and current status of
2.5 U TaKaRa EX Taq DNA polymerase (TaKaRa). The PCR was shrimp pathogens in Asia. J Invertebr Pathol 110, 166–173.
performed at 94 uC for 4 min, followed by 35 cycles of 94 uC for 30 s, Flegel, T. W. & Lo, C.-F. (2013). Announcement regarding free release
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GCGTAAACAGCGAAGG-39) were included in the reactions. The (2003). Experimental infection of Atlantic halibut Hippoglossus
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20 s, 50 uC for 20 s and 72 uC for 20 s, and a final extension at 72 uC for
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Hang, J., Forshey, B. M., Kochel, T. J., Li, T., Solórzano, V. F., Halsey,
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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 shrimp samples for virological detection using cDNA genomic
State University) for his generous help in the grammatical correction of probes. J Virol Methods 66, 227–236.
the manuscript. The authors also thank Dr Leigh Owens (James Cook Huang, J. (2012). Experience in EMS/AHPNS from China. In:
University), Dr Timothy W. Flegel (Mahidol University) and Dr Report of the Asia Pacific Emergency Regional Consultation on the
Donald V. Lightner (University of Arizona) for their advice on Emerging Shrimp Disease: Early Mortality Syndrome (EMS)/Acute
histopathology. This work was supported by the projects under the Hepatopancreatic Necrosis Syndrome (AHPNS), pp. 21–23. Edited by
Special Fund for Agro-scientific Research in the Public Interest (grant NACA Bangkok: Network of Aquaculture Centres in Asia-Pacific.
201103034), China Agriculture Research System (CARS-47), the
Lightner, D. V. (1996). A Handbook of Shrimp Pathology and
Special Scientific Research Funds for Central Non-profit Institutes,
Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. Baton
Chinese Academy of Fishery Sciences (2013A0601 and 2014A06XK01),
the Construction Programme for ‘Taishan Scholarship’ of Shandong Rouge, LA: World Aquaculture Society.
Province of China, and the Programme for Chinese Outstanding Lightner, D. V., Redman, R. M., Pantoja, C. R., Noble, B. L. & Tran, L. H.
Talents in Agricultural Scientific Research. (2012). Early mortality syndrome affects shrimp in Asia. Glob.
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