Aquaculture Reports 25 (2022) 101238

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Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Cases report of covert mortality nodavirus infection in indoor farming

Penaeus vannamei
Liang Yao a, b, Chong Wang b, Wei Wang a, b, Yingxia Li b, Shuang Liu b, Jie Kong b,
Qingli Zhang a, b, *
a
National Demonstration Center for Experimental Fisheries Science Education, National Pathogen Collection Center for Aquatic Animals, International Research Center
for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China
b
Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of
Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research
Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China

A R T I C L E I N F O A B S T R A C T

Keywords: In late 2020, unusual patterns of shrimp mortality were reported in semi-intensive shrimp culture systems in
Disease case Dongying city and Weifang city, China. In order to elucidate the cause of continual death that occurred in local
Penaeus vannamei shrimp (Penaeus vannamei) farms. Samples of shrimp and their frozen bait were collected and analyzed from 4
Covert mortality nodavirus (CMNV)
diseased farms in Dec 2020. Diseased shrimp showed abnormal behavior and symptoms including gathering at
Artemia
Pathogen carrier
the pond bottom, whitish muscle, hepatopancreatic atrophy with color fading, etc. The molecular detecting
results showed that 100 % of P. vannamei samples were tested to be positive of covert mortality nodavirus
(CMNV). The histopathological lesion of tissues taken from moribund shrimp was similar to typical histopa­
thology features of CMNV-infection. In situ hybridization (ISH) results indicated that massive CMNV-positive
hybridization signal was observed in diseased shrimp tissues. Meanwhile, all of samples of frozen bait (i.e.,
Artemia sp. and Acetes sp.) were determined to be CMNV positive with much high viral load. The healthy shrimp
individuals could be infected by the purified CMNV from Artemia sp.in the artificial challenged test. This study
demonstrated that the frozen bait Artemia infected with CMNV was confirmed to be the pathogen carrier in the
CMNV infection case, and represented a new potential pathogenic introduction threat to indoor shrimp farming.
It was highly recommended to avoid using pathogen status unknown frozen bait in the shrimp aquaculture.

1. Introduction farming industry.

Aquaculture, a vital economic activity, contributes significantly to
global nutrition and food security, whose production peaked at 82.1
million tons and sale value was estimated at USD 250 billion in 2018
(FAO, 2020). China is the country with the largest aquaculture producer
in the world, accounting for around 58 % of total global aquaculture
production, far exceeding the total output of the second- and
third-ranked countries combined, of which Pacific white shrimp
(Penaeus vannamei) occupies an economically important position in
aquaculture (FAO, 2020). However, several emerging pathogens,
including covert mortality nodavirus (CMNV) (Zhang et al., 2014),
Vibrio causing acute hepatopancreatic necrosis disease (VAHPND) (Lai
et al., 2015), and shrimp hemocyte iridescent virus (SHIV) (Qiu et al.,
2017), etc. have posed many great challenges on the global shrimp
In the second half of 2020, unusual mortality events of cultured
P. vannamei occurred in local farms in Dongying City and Weifang City,
China, some diseased shrimp showed symptoms of hepatopancreatic
atrophy, midgut empty and shell softening. In this report, we analyzed
and detected the pathogens that could be infected by the diseased
shrimp and its feed organisms, verified through histology and molecular
biology methods, and finally determined the cause of outbreak death of
farming shrimp.
2. Case description
At the end of 2020, continual mortality of cultured P. vannamei
generally occurred in local farms in Dongying and Weifang City, China.
Over 80 % of local shrimp farms have been impacted. In Dec 2020, the

* Correspondence to: NO. 106, Nanjing Road, Shinan District, Qingdao City 266071, China.
E-mail address: zhangql@ysfri.ac.cn (Q. Zhang).

https://doi.org/10.1016/j.aqrep.2022.101238
Received 7 April 2022; Received in revised form 14 June 2022; Accepted 26 June 2022
Available online 4 July 2022
2352-5134/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
L. Yao et al. Aquaculture Reports 25 (2022) 101238

author’s laboratory was asked to perform a local investigation into some
shrimp farms breeding white leg shrimp. Four indoor semi-intensive
aquaculture farms were visited.
It is understood that greenhouse aquaculture is one of the important
local aquaculture modes, and underground brine is an important source
of water for aquaculture due to the northern part of the city is located in
the coastal area. The aquaculture water was aerated with air stone, the
water temperature was 28–30 ◦ C, and the salinity was 18–25 ‰. During
the breeding period, the shrimps were fed with mixed bait and frozen
bait (i.e., Artemia sp. and Acetes sp.). The morbidity of shrimp was
characterized by continual death. The onset time mainly occurred in the
two stages of shrimp larvae population separating and shrimp juvenile
population separating (the separating operation was used to reduce
breeding density in the indoor farming process). The final density was
500–1000 individuals/m2 after the shrimp larvae population separated.
Mortality would be observed to start 3–7 days post-transfer. At the
beginning of the disease (after shrimp population separated for reducing
the density in ponds), the number of shrimp deaths was small, but the
number of shrimp death reached 100–150 individuals/pond after 7
days. Shrimp death continued, with the high number of dead shrimps
exceeded 150 kg/pond 3 days after the onset of illness in some adult
shrimp farms.
The diseased individual (about 5–10 cm in body length) of the
P. vannamei showed obvious clinical symptoms, including hep­
atopancreatic atrophy with color fading, empty stomach and guts, shell
softening (Fig. 1b1). Mild muscle whitening and necrosis occurred in
most P. vannamei individuals in the VCMD case, and a few diseased in­
dividuals that being at the acute stage showed obvious large proportion
whiteness of abdominal segment muscle (Fig. 1b2). Meanwhile, the
diseased shrimp was weak in vitality and usually sunk to the bottom of
the pond without moving. What’s more, shrimp grew slowly on some
farms.
2.1. Sample collection
A total of 28 samples of P. vannamei (about 3–8 g in body wight) and
its frozen bait (Artemia sp. and Acetes sp.) obtained from 4 local farms in
Dongying City, Weifang City, China, in December 2020. The cephalo­
thoraxes and muscle samples from shrimp individual were divided into
four parts and then preserved immediately in 4 % paraformaldehyde
(PFA), RNAstore solution (Tiangen Biotech (Beijing) Co., Ltd.), 2.5 %
glutaraldehyde, and 95 % ethanol solution for further analysis,
respectively.
2.2. Total RNA and DNA extraction
Total RNA was extracted from the samples preserved in RNAstore
solution by using the RNAiso Plus (Takara, Dalian, China). The DNA was
extracted by using TIANamp Marine Animal DNA Kit (Tiangen, Beijing,
China) according to the manufacturer’s instructions. The purity and
concentration of all RNA and DNA samples were determined using a
NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA,
USA). Nucleic acid concentration for all samples was diluted to ~200
ng/μL with absorbance ratios between 1.8 and 2.1 at 260 and 280.
2.3. Detection of shrimp common pathogens
According to the symptoms of disease shrimps, seven shrimp com­
mon pathogens (i.e., CMNV, VpAHPND, SHIV, EHP, IHHNV, IMNV and
WSSV) that could be infected by shrimp were detected.
For CMNV, real-time TaqMan probe-based reverse transcription
quantitative PCR (RT-qPCR) was performed using a specific protocol (Li
et al., 2018). For detection of VpAHPND, the rapid diagnostic kit was used.
And SHIV was quantified in all samples by TaqMan probe qRT-PCR
according to the previously reported method (Qiu et al., 2018). All
samples were processed by an independent laboratory using the TaqMan
probe real-time qPCR, for the detection of EHP (Y.M. Liu et al., 2018).
The IHHNV, IMNV and WSSV were detected using the method

Fig. 1. Investigation results of outbreak death of cultured Penaeus vannamei in Dongying and Weifang City, China. a, Map of the sampling area in Dongying City and
Weifang City, China. The red dots indicate the sampling locations. b, Representative image of diseased P. vannamei from faming ponds. Most of the diseased
P. vannamei individuals showed the syndromes with hepatopancreatic atrophy with color fading, empty stomach and guts, and shell softening, mild muscle whitening
and necrosis (red arrows), as showed the up individual (b1). A few diseased P. vannamei individuals showed the syndromes of acute infection (below one) with typical
muscle whitening and necrosis of the abdominal segment, as showed the blow individual (b2). c. the detection result of CMNV by RT-nPCR in collected samples. M,
2 kb maker. “+”, positive control. “-”, negative control.

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recommended by the OIE (OIE, 2021).
2.4. Determine the evolutionary relationship of CMNV by RT-nPCR and
phylogenetic tree analysis
All samples were amplified and prepared for sequencing using a two-
step, reverse transcription nested polymerase chain reaction (RT-nPCR)
protocol with two pairs of primers. More specifically, the amplification
products (619 bp) of the 1st-step PCR were obtained using a Prime­
Script™ One Step RT-PCR Kit Ver.2 (TaKaRa, Dalian, China). Then, the
secondary PCR was carried out by using a TaKaRa Ex Taq® Hot Start
Version (TaKaRa, Dalian, China) with the templates of the 1st-step RT-
PCR products to obtain 413 bp target products. The procedures and
primers used were identical to those described as reported previously
(Wang et al., 2021b). Following amplifications, products were separated
in an agarose gel electrophoresis and bands were sequence verified at
Sangon Biotech (Shanghai, China) Co., Ltd.
The sequence was identified through BLAST searches, and the
deduced amino acid sequences of CMNV target RdRp gene fragments
from positive samples and RdRp amino acid sequences from other
nodavirus were selected for phylogenetic analysis by using MEGA X
software (Kumar et al., 2018).
2.5. Histology
The samples of the cephalothoraxes and muscle segment were fixed
with 4 % PFA for 24 h at 4 ◦ C and then transferred to a graded ethanol
series for dehydration, followed by treatment with 100 % xylene and
infiltrating in paraffin. The sections (3 μm) were obtained and stained
with conventional H&E staining according to the previous procedures
(Lightner, 1996). Subsequently, the sections were scanned through the
PANNORAMIC Pathology Diagnostic Scanners (3DHISTECH Ltd, Buda­
pest, Hungary) to obtain good quality images.
2.6. In situ hybridization (ISH) for CMNV
In situ hybridization (ISH) was performed on serial tissue sections. In
briefly, the sections were dewaxed in xylene, followed by rehydration
with successively dilute solutions of ethanol. Then ISH using CMNV as a
probe was performed on three sections according to the protocols
described previously (Li et al., 2020; Zhang et al., 2017). After color
reaction, counterstaining of the sections was carried out by using the
Nuclear Fast Red solution (Sigma-Aldrich), followed by dehydration in
alcohol and mounting with water-soluble sealant (Boster, Beijing,
China). Finally, the sections were scanned to obtain extra-quality images
by PANNORAMIC Pathology Diagnostic Scanners (3DHISTECH Ltd,
Budapest, Hungary).
2.7. Transmission electron microscopy (TEM) analysis
Ultrathin sections of the hepatopancreas from diseased shrimp were
analyzed using TEM. The tissue of hepatopancreas (~1 mm3) was
sampled as rapidly as possible and immediately transferred to a 1.5 mL
EP tube containing fixative, 2.5 % glutaraldehyde in 0.1 M PBS (pH =
7.2), and held overnight at 4 ◦ C in fixative. Subsequently, the sample
was secondarily fixed with osmium tetroxide, dehydrated with graded
ethanol, and then embedded in Spurr’s resins. Ultrathin sections (50 nm
in thickness) were cut with a diamond knife and collected on collodion-
coated grids by staff in the Equipment Center of the Medical College of
Qingdao University. The sections were stained with uranyl acetate and
lead citrate and then observed with a JEOL JEM-1200 electron micro­
scope operating at 80–100 kV.
2.8. Challenge test of the purified CMNV from Artemia
P. vannamei shrimp (about 5–7 cm in body length, SPF juvenile) were
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Aquaculture Reports 25 (2022) 101238
collected from a breeding base of BLUMP Seed Industry Technology Co.,
Ltd. in Weifang City, China. For the challenge test, the shrimp were
divided into viral injection group and control group. Each group
included two replicates and each replicate included 35 individuals. The
CMNV was purified from the CMNV positive Artemia sp. according to
previous report (Zhang et al., 2014). Purified CMNV (about 105 viral
copies per mg healthy shrimp) or equal volume TN buffer were injected
into the lateral area of the fourth abdominal segment of the shrimp, and
used as CMNV infection group and control group, respectively. The
shrimp were farmed indoor with commercial feeds for 7 days. Finally,
shrimp were collected and used for RT-qPCR analysis.
3. Results
3.1. Results of detection of suspicious pathogens
CMNV was detected in all 26 samples by TaqMan qPCR. Among
them, the viral load in 82 % (23/28) sample exceeds 103 copies/μg total
tissue RNA. Except for one of the samples, the viral loads of the
remaining samples were all higher than 102 copies/μg total tissue RNA.
In addition, 75 % (9/12) shrimp individuals were detected to be EHP-
positive, and the load in the samples was between 101–4 copies/μg
total tissue DNA. What’s more, no VpAHPND, IHHNV, IMNV, WSSV and
SHIV were detected in all samples (Table 1).
3.2. Phylogenetic analysis of CMNV RdRp gene sequence
After conventional PCR amplification, all secondary PCR products
were detected by running agarose gel, and single bands of the 413 bp
targeted gene amplicons were detected in the all the samples, as well as
in positive control (Fig. 1c). The sequences of the PCR products were
subjected to BLAST analysis. BLAST analyses indicated that all se­
quences of CMNV RdRp gene from the collected samples showed as high
as 98–100 % nucleotide identity with the original CMNV isolate (Gen­
Bank number: KM112247.1) from P. vannamei. The phylogenetic anal­
ysis showed that all the CMNV target fragments from three different
farm’s isolates were clustered tightly into a branch of known CMNV
isolate, which demonstrated higher similarity with genus Alphanodavirus
rather than Betanodavirus (Fig. 2).
3.3. Histopathology analysis of disease shrimps
Histological examination confirmed that histopathological changes
occurred in multiple tissues and organs of diseased shrimp infected with
CMNV. Hepatopancreatic tubules underwent necrosis with atrophy and
sloughing of tubular epithelium cells. Meanwhile, haemocytic infiltra­
tion, karyopyknosis and eosinophilic inclusion bodies were observed
between the atrophic hepatopancreas tubules (Fig. 3a and b). In addi­
tion, extensive karyopyknosis and severe muscular lysis and myonec­
rosis of muscle fibers that the cell boundaries disappeared were observed
in the whitish muscle lesions (Fig. 3e and f). What’s more, inspection of
Fig. 3j indicated massive vacuolation in the cytoplasm of the abdominal
nerve. Furthermore, necrosis and exfoliation of intestine epithelial cells
were also observed in the diseased shrimp (Fig. 3n).
3.4. Verification of CMNV infection in shrimp by ISH
For further confirmation of CMNV infection in diseased shrimp, ISH
was performed by using CMNV-specific RNA probes. The results showed
that blue-violet hybridization signals of CMNV probes were evident in
the hepatopancreas, striated muscle, abdominal nerve and intestinal
epithelium of diseased shrimp (Fig. 3). Massive purple signals of CMNV
probes were observed in the tubular epithelium of hepatopancreas,
especially the inclusion (Fig. 3c and d). Notably, the probes reacted
intensely with the karyopyknosis in the necrotic abdominal muscle and
vacuolated nerve cells (Fig. 3g and h; k and l). Meanwhile, purple
L. Yao et al. Aquaculture Reports 25 (2022) 101238

Table 1
Detection of shrimp common pathogens.
Case number Species Result of detection of common pathogens (copies/μg)

CMNV VpAHPND EHP SHIV WSSV IHHNV IMNV

A1 P. vannamei 2.0 × 103 _

2.0 × 101 _ _ _ _

A2 P. vannamei 2.9 × 103 _

2.1 × 104 _ _ _ _

A3 P. vannamei 5.5 × 103 _

1.6 × 104 _ _ _ _

A4 P. vannamei 6.0 × 103 _

4.8 × 103 _ _ _ _

B1 P. vannamei 2.4 × 103 _

2.0 × 103 _ _ _ _

B2 P. vannamei 8.5 × 102 _ _ _ _ _ _

B3 P. vannamei 8.5 × 102 _ _ _ _ _ _

B4 P. vannamei 1.8 × 103 _ _ _ _ _ _

B5 Artemia sp. 1.1 £ 107 _ _ _ _ _ _

B6 Artemia sp. 4.2 £ 106 _

4.3 × 102 _ _ _ _

B7 Artemia sp. 4.2 × 105 _

2.8 × 103 _ _ _ _

B8 Artemia sp. 2.4 £ 106 _

1.1 × 103 _ _ _ _

B9 P. vannamei 9.0 × 102 / / / / / /

B10 P. vannamei 6.5 × 103 / / / / / /
B11 P. vannamei 8.5 × 103 / / / / / /
B12 Artemia eggs 1.6 × 102 / / / / / /
C1 P. vannamei 5.5 × 103 _
1.1 × 101 _ _ _ _

C2 P. vannamei 7.5 × 104 _

4.2 × 103 _ _ _ _

C3 P. vannamei 2.4 × 103 _

1.4 × 104 _ _ _ _

C4 P. vannamei 1.9 × 103 _

6.5 × 103 _ _ _ _

C5 Artemia sp. 2.1 £ 108 _

9.0 × 103 _ _ _ _

C6 Acetes sp. 2.3 × 104 _

3.4 × 104 _ _ _ _

D1 P. vannamei 2.2 × 105 / / / / / /

D2 P. vannamei 2.7 × 105 / / / / / /
D3 P. vannamei 2.1 × 104 / / / / / /
D4 P. vannamei 2.6 × 105 / / / / / /
D5 Artemia sp. 2.5 £ 106 / / / / / /
D6 Artemia eggs 2.5 × 101 / / / / / /

Notes: "numbers" showed pathogen load; "_" and "/" showed the negative result and not done, respectively.

hybridization signals were also detected in the intestinal epithelial cells
(Fig. 3o and p).
3.5. TEM observation of ultrathin sections
TEM observation revealed that massive spherical unenveloped
CMNV-like particles (Fig. 4a and b) with a diameter of about 30 nm
could be observed in the ultrathin sections of hepatopancreas tissue of
P. vannamei from typical VCMD infection under the TEM.
3.6. Pathogenicity of CMNV isolated from the Artemia in artificial
challenged test
Artificial challenged test was conducted for further confirmation of
the pathogenicity of CMNV isolated from the frozen bait Artemia sp. The
challenge test results showed that cumulative mortality of P. vannamei in
the viral injection group was 31.5 % by Day 7 post-injection. While,
there was no mortality of P. vannamei in the control group injected with
TN buffer (Fig. 5). Further, RT-qPCR analysis indicated that shrimp in­
dividuals from the infected group were positive of CMNV with variable
viral dose, whereas shrimp from the control group were negative of
CMNV (data note shown).
4. Discussion
Outbreaks of disease that cause significant morbidity and/or mor­
talities due to high-density farming and environmental changes in an
aquaculture operation are always a major concern (Ramirez-Paredes
et al., 2021; Yao et al., 2022). This case report specifically confirmed via
PCR, histopathology, ISH, and TEM outbreaks of disease causing by
CMNV in local semi-intensive farms in Dongying City and Weifang City,
China.
Detection results of suspicious pathogens showed that all 28 samples
from 4 farms were detected to be CMNV-positive and the viral load of 82
% of samples exceeded 103 copies/μg total tissue RNA. Among them, the
frozen bait samples from all of three farms were detected high viral
loads, especially the sample of C5-Artemia sp. as high as 2.1 × 108
copies/μg total tissue RNA (Table 1). Meanwhile, it is worth noting that
CMNV-positive was detected in all frozen baits from more other local
shrimp farms by investigators (data not fully shown). Further, the se­
quences of PCR amplicons both from the disease shrimp and the Artemia
in the phylogenetic tree were highly identical to that of the original
CMNV isolate. What’s more, the challenge test results showed that
CMNV purified from Artemia can infect healthy P. vannamei and cause a
31.5 % mortality of the infected shrimp within 7 days. Considering that
the shrimp post-larva used in the farms were Specific Pathogen Free, and
aquaculture water used in diseased farms was underground brine which
was free of known pathogens, the CMNV from the frozen bait, Artemia
sp. and Acetes sp., was highly suspect to be the origin causative agent of
disease on the investigated farms. These results indicated that most
likely CMNV was derived from frozen baits, and then played a signifi­
cant role in the outbreak of CMNV-infection and high mortality of in
indoor farming shrimp that were investigated.
In previous reports, at farm ponds level, the cumulative mortality of
diseased P. vanname with CMNV-infected could reached up to 80 %
(Zhang et al., 2014). Whereas, the cumulative mortality (31.5 %) of
CMNV infected shrimp in the challenge test in indoor farming in present
study was significantly lower than intensive pond farming. We deduced
that the stable and good farming environment might be conducive to
reducing the mortality of shrimp infected with CMNV. And this result is
also consistent with a recent report in which the result indicated that the
lethal capacity of CMNV was related with the farming environment, and
the stable farming environment was conducive to reducing the mortality
of shrimp caused by CMNV infection (Liu et al., 2022).
Although EHP was detected in the samples, which may be related to
the slow growth of shrimps in local farms (Sritunyalucksana et al., 2014;
Tourtip et al., 2009), there is no report that EHP can cause obvious
shrimp death. Whether the prevalence of EHP might aggravate mortality
in shrimp infected with CMNV is unclear. Additionally, in this case,
VpAHPND, IHHNV, IMNV, WSSV and SHIV were not the causal agents

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Fig. 2. Phylogenetic tree of different CMNV isolates and other nodaviruses based on RNA dependent RNA polymerase (RdRp) gene generated by the neighbor-joining
method using the MEGA X. Bar = 0.5.
causing mass mortality of shrimp because none of these five pathogens
were detected in all samples.
Shrimp sampled from local farms during the outbreak period showed
severe clinical and pathological symptoms, typically related to CMNV
infections. The moribund shrimp with whitish plaques on the abdominal
muscle at the viral infection acute stage was commonly found in the
bottom of the pond instead of swimming to the surface or edges. This
phenomenon was consistent with that previously observed in
P. vannamei infected with CMNV (Zhang et al., 2014). Although several
RNA viruses have been found to cause typical muscle whitening and
necrosis of farmed shrimp, it is somewhat different from CMNV infec­
tion. For instance, shrimp infected with Infectious Myonecrosis Virus
(IMNV) will display evident signs of extensive white necrotic areas in
skeletal muscles, especially in the sixth abdominal segment and tail fan,
which can become necrotic and reddened in some shrimp (Lightner
et al., 2004; Poulos et al., 2006). Likewise, Penaeus vannamei nodavirus
(PvNV) infected P. vannamei causing muscle necrosis, but the mortality
rate was lower than CMNV infection (Tang et al., 2007; Zhang et al.,
2014). What’s more, even though P. vannamei is another species that is
susceptible to Macrobrachium rosenbergii nodavirus (MrNV), this situa­
tion usually only occurs under low temperature together with low
salinity of aquaculture water leading to significant mortality (Senapin
et al., 2012).
The principal target tissues or organs of CMNV are not completely
consistent with those of IMNV, MrNV, and PvNV. In this case, the results
of histopathology together with ISH showed that atrophic and necrotic
tubule epithelium of hepatopancreas with massive purple CMNV-
positive hybridization signal was observed (Fig. 2a–d), which provides
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Aquaculture Reports 25 (2022) 101238
reliable evidence that hepatopancreas is the one of the important target
organs of CMNV. Meanwhile, CMNV-like particles also were observed in
hepatopancreas tissue under the TEM (Fig. 4). But, until now, there was
no report demonstrated that hepatopancreatic atrophy and necrosis
caused by MrNV, PvNV and IMNV infection. The evidences above
strongly supported that CMNV was the causative agent of shrimp
epidemic in Dongying and Weifang City.
CMNV, a novel member of Alphanodavirus, has been found to have a
wide susceptible host range, including invertebrates and poikilothermic
vertebrates. Initially discovered in P. vannamei (Zhang et al., 2014), the
virus has been reported to naturally infect Mugilogobius abei (Zhang
et al., 2018), Danio rerio (Wang et al., 2021a), Larimichthys polyactis (Xu
et al., 2021), and Apostichopus japonicas (Wang et al., 2021c), etc.
causing damage to multiple tissues and organs. Additionally, previous
studies had shown that a variety of cultured crustaceans and in­
vertebrates from shrimp ponds affected by viral covert mortality disease
(VCMD) can be infected by CMNV (S. Liu et al., 2018; Zhang et al.,
2017). In this case, through artificial infection experiments, it was
confirmed that healthy P. vannamei could be infected with CMNV iso­
lated from Artemia.
Although VCMD is a disease that can cause high mortality in farmed
shrimp at acute infection stage, and its pathogens also have a wide range
of hosts, it is not listed as a notifiable or significant disease of shrimp by
OIE at present (OIE, 2021). VCMD outbreaks have been related to high
temperature together with stressful events such as sudden temperature
or salinity changes or even high levels of NO-2 - N caused by poor farming
environment, and sudden weather changes, even the operation of
dividing growing shrimp population to different ponds for reducing
L. Yao et al. Aquaculture Reports 25 (2022) 101238

Fig. 3. Micrographs of haematoxylin and eosin (H&E) staining and in situ hybridization (ISH) of the main tissue and organs of Penaeus vannamei infected with CMNV.
(a, b) Micrographs of H&E staining of the atrophied and necrotic tubular epithelium of hepatopancreas. (b) Karyopyknosis (blue arrows) and the inclusions (red
arrows) were observed in the hepatopancreatic epithelium. (c, d) Micrographs of ISH of the hepatopancreatic epithelium. (d) CMNV-positive hybridization signals
(colored purple) were detected in the tubular epithelium of hepatopancreas. (e, f) Micrographs of H&E staining of the whitish abdominal muscle lesion. (f) Note that
muscle-lytic necrosis with karyopyknosis (red arrows). (g, h) Micrographs of ISH of the necrosis muscle lesion. (h) Note intense hybridization signals (colored deep-
purple) were detected in the necrosis muscle tissue. (i, j) Micrographs of H&E staining of the abdominal nerve. (j) Severe cavitation was observed in the abdominal
nerve. (k, l) Micrographs of ISH of the abdominal nerve. (l) Intense deep-purple hybridization signals were detected in the vacuolated abdominal nerve. (m, n)
Micrographs of H&E staining of the intestine. (n) Loose epithelium and karyopyknosis (blue and red arrows) in the infected intestine. (o, p) Micrographs of ISH of the
intestine. (p) Purple hybridization signals were detected in the intestinal epithelium. b, d, f, h, j, l, n, and p are magnified panel from the yellow-framed areas of a, c,
e, g, i, k, m, and o, respectively. Bar scales, (a, c, e, g, i, k, m, and o) = 100 µm, (b, d, f, h, j, l, n, and p) = 20 µm.

density (Zhang, 2020; Zhang et al., 2014). Meanwhile, CMNV has been
shown to be endemic in many countries around the world (Zhang, 2019)
and caused significant production losses at a national or regional level.
This case reported another significant typical VCMD in shrimp culture of
China since 2014. Those results also demonstrated that CMNV is a
serious threat to the sustainability of penaeid shrimp and other aquatic
organisms’ aquaculture.
5. Conclusion
This case report specifically confirms via PCR, histopathology, ISH,
and TEM another typical disease outbreaks causing by CMNV in local
shrimp farms of Dongying City and Weifang city, China since 2014. This
study demonstrated that the frozen bait Artemia infected with CMNV
was the pathogen carrier in the CMNV infection case, and represented a
new potential pathogenic introduction threat to the indoor shrimp
farming. It was highly recommended to avoid using pathogen status
unknown frozen bait in the shrimp aquaculture.
Funding
This research was funded by the National Natural Science Founda­
tion of China (32073016; 31672695), the earmarked fund for CARS-48,
the Major Applied Technology Innovation Project of Agriculture in
Shandong Province (NO. SD2019YY001), the Central Public-interest
Scientific Institution Basal Research Fund, CAFS (NO.2020TD39), and
the Central Public-interest Scientific Institution Basal Research Fund,
YSFRI, CAFS (NO.202213-2).

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Fig. 4. Transmission electron micrographs of ultrathin section of the hepatopancreas of diseased Penaeus vannamei naturally infected with CMNV. a, micrograph of
TEM of hepatopancreas tissue. Note that aggregated distribution of virus-like particles can be observed in the black frame. b, magnified micrograph of the area in the
black frame in a. Note that spherical unenveloped CMNV-like particles can be observed. Scale bars: a = 500 nm, b = 100 nm.
Fig. 5. Pathogenicity analysis of CMNV isolated from the Artemia in artificial
challenged test.
CRediT authorship contribution statement
Liang Yao: Investigation, Visualization, Writing – original draft.
Chong Wang: Investigation, Validation. Wei Wang: Investigation, Data
curation. Yingxia Li: Data curation, Validation. Shuang Liu: Investi­
gation. Jie Kong: Resources. Qingli Zhang: Project administration,
Funding acquisition, Conceptualization, Writing – review & editing.
Acknowledgments
We are thankful to Dr. Tingting Xu for assistance in mapping.
Author contributions
Liang Yao contributed to the sample collection, histopathology
analysis, ISH analysis, and the writing of the manuscript. Chong Wang
contributed to the histopathology analysis and ISH analysis. Wei Wang
and Yingxia Li contributed to the detection of suspicious pathogens,
Shuang Liu contributed to the sample collection, and Jie Kong supplied
the SPF shrimp. Qingli Zhang revised the manuscript and guided the
entire project. All authors interpreted the data and contributed the
manuscript.
Declaration of competing interest
The authors have declared no conflict of interest. All co-authors have
seen and agree with the contents of the manuscript and there is no
financial interest to report. We certify that the submission is original
work and is not under review at any other publication.
7
Aquaculture Reports 25 (2022) 101238
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