Presentation 19: Covert Mortality Nodavirus (CMNV): the pathogen, pathogenesis, transmission, distribution, impacts, co-infection with EMS/AHPND (Dr Huang Jie, China)

FAO TCP/INT/3502 “Reducing and managing the risk of
Acute Hepatopancreatic Necrosis Disease (AHPND) of Cultured Shrimp”
Covert mortality nodavirus (CMNV):
the pathogen, epidemiology, and co-
infection with EMS/AHPND
Jie Huang, Qing-Li Zhang, Nan Bai, Xiao-Yuan Wan, Hai-Liang Wang,
Guo-Si Xie, Bing Yang, Xiu-Hua Wang, Chen Li, Xiao-Ling Song
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery
Sciences, Qingdao 266071, China; National Laboratory for Marine
Science and Technology, Qingdao 266071, China
huangjie@ysfri.ac.cn
2015-06-24
International Technical Seminar/Workshop “EMS/AHPND: Government, Scientist and Farmer Responses”
1

Production of farmed shrimp in China
0
20
40
60
80
100
120
140
160
180
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
Area(kha)
Production(milliontons)
L. vannamei (fresh)
L. vannamei (marine)
Other shrimp
Area of other shrimp
Area of L. vannamei (marine)
Whitespot
Reddishbody
Covertmortality
Earlymortality
Whitefeces
Slowgrowth
2015-06-24
International Technical Seminar/Workshop “EMS/AHPND:
Government, Scientist and Farmer Responses”
2

Differences among WSD, CMD, & EMS
2015-06-24
3
WSD CMD EMS
Earliest observation 1992 2002 2010
Disease mode Rapid mortality Cumulative mortality Sudden mortality
Peak disease period 30-60d post-stocking 60-80d post-stocking 10-30d post-stocking
Worse condition
Stress due to change of
envir.
High temp., NO-
2 or
NH3
Over feeding
Oxygen requirement More requirement No significant change No significant change
Color of HP Normal to faded Faded, yellow brown Pale to whitish
Color of skin Reddish Normal Whitish
Whitish muscle Sometimes Typical Sometimes
Slow growth No relationship Likely related High mortality

Viral Covert Mortality Disease
2015-06-24
4
• Pathogen
– Covert mortality nodavirus (CMNV)
• Clinical signs
– Atrophic or faded HP
– Whitish muscle in abdomen
– Some empty stomach and guts
– Soft shell, slow growth
• Mortality
– Died at the bottom of pond
– Cumulative mortality during 30—80 days post stocking
– Moribund and dead shrimp can be found every day
– Disease go worse after 60—80 days post-stocking accompanied
by the increase of NO2
– -N under high temperature above 28

Viral Covert Mortality Disease
2015-06-24
5

Histopathology of Viral Covert Mortality Disease
• Hepatopancreas
– Separated HP tubules with
hemocytic inflammation
– Nuclear karyomegaly and
eosinophilic intranuclear
inclusions in the tubular
epithelium of HP
– Early development small
eosinophilic intranuclear
inclusions in nuclei of HP
tubule epithelium
2015-06-24
6

• Muscle
– Muscle fragmentation tending towards
coagulative, hyaline degeneration, muscular lysis,
and myonecrosis
– Multifocal myonecrosis in the striated muscle
accompanied by the hemocytic infiltration and
karyopyknosis of hemocytes
2015-06-24
7

• LO
– Disorganization and accumulation of the cells in
lymphoid organ cords formed lymphoid spheroids
– Intranuclear inclusions, nuclear pyknosis and
karyomegaly in the lymphoid spheroids
2015-06-24
8

Transmission Electromicroscopy
• HP
– Presence of spherical
unenveloped virus-
like particles with
diameter about 25
nm
– The virus-like
particles gather in a
cytoplasm region
surrounded with a
double-layer
membrane
2015-06-24
9

Transmission Electron microscopy
• Muscle
– Presence of spherical
unenveloped virus-
like particles in
average diameter 29
nm and empty
capsid-like particles
in average diameter
49 nm.
– The virus-like
particles gather in a
cytoplasm region
surrounded with
membrane in cross-
sectioned cells of
muscle fibre and
neuro cord.
2015-06-24
10

Transmission Electron Microscopy
• Negative staining of virus
purification
– Unenveloped virus-like particles
with a wild diameter range like
MrNV and PvNV
– The virus inoculation might
contain two kinds of spherical
particles
– The size of the larger virus-like
particles was (32.1±5.5) nm in
diameter, while that of the
smaller spherical particles was
(19.0±1.9) nm in diameter
which may be a Nodavirus-
associated satellite virus.
2015-06-24
11

Phylogenetic tree of CMNV in Nodaviridae
• Identity of AA sequences of RNA-dependent RNA polymerase
– The amino acid sequence of a 1185 bp fragment (CMNV-7) shares 54%,
53% and 39% identity with the amino acid sequences of RNA-
dependent RNA polymerase from FHV, BBV and MrNV, respectively.
NoV: Nodamura virus
BoV: Boolarra virus
BBV: Black beetle virus
FHV: Flock house virus
DmANV: Drosophila
melanogaster
American nodavirus
PaV: Pariacoto virus
(host: armyworm)
2015-06-24
12

Challenge tests of CMNV
• Challenge tests with filtered or purified
CMNV inoculation showed different
results in mortality.
– Some results of the challenge tests
showed that cumulative mortalities of
shrimp by injection or per os infection
were up to 80—100% during two weeks
post-challenge, but some did not result in
significant mortality.
• The mortality difference of challenge tests
seems relating with the temperature
during the experiment.
– Water temperature higher than 28°C may
result in a significant mortality.
– Faked HP and whitish muscle can be
observed in the challenge tests under a
lower temperature.
• Histopathology, TEM, and RT-PCR
supported the infection in challenged
groups.
2015-06-24
13

Fluorescence in situ hybridization
(FISH)
• Fluorescent signals were
evident in the HP, LO, and
muscle of infected shrimp.
– The probe reacted most
intensely in the tubular
epithelium cells of HP.
– A few inclusion-like bodies
show very strong
fluorescent signal in muscle.
– The probe also reacted
intensely with the inclusions
in LO.
2015-06-24
14

Molecular detection methods for
CMNV• Nested RT-PCR
– Outer primers:
• CMNV-7F1: 5’-AAA TAC GGC GAT GAC G-3’
• CMNV-7R1: 5’-ACG AAG TGC CCA CAG AC-3’
– Inner primers:
• CMNV-7F2: 5’-CAC AAC CGA GTC AAA CC-3’
• CMNV-7R2: 5’-GCG TAA ACA GCG AAG G-3’
– Key parameters:
• RT: RNA denatured at 65°C and chilled on ice, then RT at 50°C.
• Optimized conditions: 4 mM MgCl2, annealing at 72°C
• 1st step PCR: amplicon 619 bp
• 2nd step PCR: amplicon 165 bp
2015-06-24
15

Molecular detection methods for
CMNV
• qRT-LAMP
– Good linearity in a range of106 dilution of tissue RNA (R2 = 0.9953).
• Development of a high sensitive and rapid detection kit
– Developed based on a high sensitive & rapid detection kit platform
– All protocol, including sample preparation, can be finished in 1.5 hour.
– False reactions and contamination risk controlled.
– Sensitivity up to 102 copies.
2015-06-24
16

Surveillance of CMNV using the
molecular detection methods
• It seems that CMNV can be vertical transmitted and was already
wildly spread to shrimp farms in different countries, although it a
newly found virus.
– About 18% positives in >300 diseased farm samples collected in 2014
from 10 coastal provinces
– Positives were not only detectable in samples of L. vannamei, but also
in samples of F. chinensis, M.p.. japonicus, M.b. rosenbergii, and
swimming crab Portunus trituberculatus
– Positives in samples of different life stages, including nauplii, postlarva,
growth-out juveniles, and broodstock, were detected
– Samples from some other countries in Asia and Americas can also be
detected positive.
2015-06-24
17

Confusing names of diseases in China
• EMS/AHPND was thought to
represent severe cases of CMD
as its clinical signs were similar
to that of CMD to some
degree.
• CMS represents symptoms of
several diseases currently due
to the difficulty in distinguish
of the gross signs of CMD and
AHPND for farmers.
• HPNS referred to a group of
symptoms in the HP resulted
from infection with different
pathogenic bacteria combined
with impacts of environmental
stresses, such as NH3-N and
NO2
–-N (Dr. J-G He in Sun Yat-
Sen University).
Covert mortality syndrome
(CMS)
HPNS
AHPND
VCMS
NCMD
2015-06-24
18

Surveillance in 2009
2015-06-24
19
• Through March to November, 2009
• Two cycles of national wide sampling
– All 11 coastal provinces, the mainland of China
– 70 sampling sites, over ~25000 km driving
• 400 samples collected
– 5 species of shrimp
• Main diseases diagnosed
– WSD/Reddish body
– CMD
– No EMS-like case found

Isolation of a earlies V. parahaemolyticus strain
2015-06-24
20
• Samples of suspected
EMS/AHPNS from
Guangxi Province (E) in
2010
• Juvenile L. vannamei in
the second crop
• Yellow to pale and
atrophy HP
• Mortality around 90% Location of the reported cases of suspected
EMS/AHPNS in 2010

Virulence and antibiotic resistance of V.
parahaemolyticus strain from AHPND in 2010
2015-06-24
21
• LD50: 3.6×104CFU/shrimp by injection
Antibiotics
Conc.
(μg/disc)
Inh. zone
(mm)
Sens. Antibiotics Conc. (μg/disc)
Inh. zone
(mm)
Sens.
Cefalexin 30 - R Lomefloxacin 18 - R
Cefazolin 30 11 I Norfloxacin 10 15 I
Cefradine 30 - R Ofloxacin 5 11 R
Ceftazidime 30 13 R Metronidazole 5 11 R
Cefatrizine - 17 I Pipemidic acid 30 9 R
Amikacin 30 13 R Rifampicin 5 16 R
Gentamicin 10 10 R Novobiocin 30 13 I
Neomycin 30 12 R Kanamycin 30 15 I
Streptomycin 10 12 I Minocycline 30 - R
Erythrocin 15 12 R Doxycycline 30 - R
Clarithromycin 15 11 R Florfenicol 30 24 S
Azithromycin 15 8 R SMZco 3.75/1.25 16 R
Nalidixic acid 30 18 R
Zhang et al., 2012

Strains No. Pathogenicity Adhesion Checked with the primers
based on VPP19LT50 by shrimp LC50 by GART
20130726001S03^ 9.96±0.81 a 2.73 2.36±0.45 h +
20130726001S01^ 9.60±0.53 a 3.05 2.42±0.34 h +
20130629001AS04^ 9.60±0.53 a 3.29 2.12±0.04 gh +
20130721002GUTS03^ 9.95±0.31 a 3.38 2.37±0.03 h +
20130629001S04^ 9.60±0.53 a 3.61 2.00±0.21 fgh +
20130722001S01 10.69±0.04 a 6.42 2.34±0.22 h +
20100612001 10.67±0.53 a 6.71 1.40±0.33 def +
20130629004WS02 12.89±1.02 a 6.82 2.03±0.42 fgh +
20130629002S01 11.73±0.53 a 6.83 2.16±0.32 gh +
20130629004GS02* 25.38±1.10 b 6.92 0.49±0.08 a +
20131216001HHPS01* 25.90±2.78 b 7.27 0.52±0.10 ab +
20130629004WS05* 23.14±1.91 b 7.31 0.53±0.07 ab +
20131216001HHPES02* 25.45±6.05 b 7.32 0.62±0.13 abc +
20130722003HPES03 25.17±1.44 b 7.34 1.60±0.19 efg +
20130721001S04 27.80±2.68 b 7.49 1.16±0.02 bcde +
20130629005S05 29.17±4.10 b 7.68 0.99±0.07 abcde +
20130629005S07 43.86±2.91 c 7.88 1.18±0.10 cde +
20130722KXHPES03 47.28±1.48 c 7.94 1.16±0.01 bcde +
20130629005S03 47.28±1.48 c >8 0.74±0.11 abc +
20130628001S02 46.14±2.45 c >8 0.94±0.15 abcd +
20130628001AS01 47.98±2.27 c >8 0.89±0.11 abcd +
20130629005AS01 49.91±3.07 c >8 0.88±0.20 abcd +
20130722DXES02 N.V.
N.V.
N.V. 0.58±0.14 abc -
20130629002AS02 N.V. 0.59±0.13 abc -
Pathogenicity tests for V.p. strains
2015-06-24
22

Detection of 7 pathogens in diseased
farm samples
2015-06-24
23
0%
10%
20%
30%
40%
50%
60%
EHP WSSV Vahpn IHHNV CMNV YHV TSV
4 pathogens infection
Single pathogen infection
Free of infection

Coinfection cases of CMNV, VPAHPND, EHP, or YHV
• Coinfection CMNV and VPAHPND
– Cases seems not regularly happen in the
diseased farm samples.
– Gross signs during the early stage post-stocking
are mainly caused by VPAHPND in the coinfection
cases, while gross signs during the late growth-
out stage are likely mixed or unclear.
• Coinfection of CMNV and EHP
– Several cases were noticed, it seems having
additive impacts to the growth rate of shrimp.
• Coinfection of YHV and VPAHPND
– One case was noticed in the later growth-out
stage. Histopathology of both pathogens
appeared.
• Coinfection with multiple pathogens caused
significant complexity to disease profiles in
shrimp farms.
2015-06-24
24

Relationship between EHP loading and growth of shrimp
2015-06-24
25
• qPCR results showed the negative correlation between EHP loading and
growth of shrimp.
– The EHP load above 103 copies/(ng HpDNA) represents a high risk level.
– Some unpredictable cases with low EHP loading may due to coinfection?
0
1
2
3
4
5
6
2.0 2.5 3.0 3.5 4.0 4.5 5.0
ExponentialrelativecopiesofEHP
Body length (cm)
Mean Sample Tendence
1
2
3
4
5
6
7
4 5 6 7 8
ExponentialrelativecopiesofEHP
Body length (cm)
Mean Sample Tendance
R = -0.0876 R = -0.7128 R = -0.5009 R = -0.4413

Discouragement due to complexity of
coinfection with pathogens
• The farmers tend to choose the better stocks of
shrimp based on the source and prefer
environmental management technologies, rather
than consideration of the risk of pathogens, due
to frequent coinfections with multiple pathogens
in shrimp farms and the cost or difficulty of
detection of multiple pathogens.
• Ignorance of the existence of pathogens in the
shrimp health management may mislead key
strategies for disease control.
2015-06-24
26

Biosecurity concept needs to be introduced and developed for
shrimp aquaculture
27
• Biosecurity
– means a set of management and physical measures designed to reduce the risk of
introduction, establishment and spread of pathogenic agents to, from and within an aquatic
animal population.
• Biosecurity plan
– Disease surveillance plan: to understand the status of diseases
– Biological risk assessment: to investigate the sources and priorities of the risks.
– Biological risk management plan: to establish the control measures for the risk
– Traceability system: to audit the implementation of biosecurity plan
• Biosecurity ladder: based on the performed biosecurity conditions
– Grade I: partial prevention and control under disease diagnosis, without disease surveillance
and risk assessment
– Grade II: integral prevention and control with improvement of management and physical
measures, with insufficient disease surveillance and risk assessment
– Grade III: Integral prevention and control under the direction of risk analysis, with imperfect
traceability system
– Grade IV: Disease free status achieved under complete biosecurity, with auditability and
sustainability
2015-06-24

Acknowledgements
2015-06-24
28
• The authors appreciate FAO's invitation and support for attending of the FAO International Technical Seminar/Workshop
‘EMS/AHPND: Government, Scientist and Farmer Responses’,
– Especially Dr. Melba B. Reantaso in FAO and Dr. Victoria Alday-Sanz in the National Aquaculture Group in the arrangements for
the workshop.
• This presentation is mainly a combination of the following published and unpublished contributions,
– Zhang Q-L, Liu Q, Liu S, Yang H-L, Liu S, Zhu L-L, Yang B, Jin J-T, Ding L-X, Wang X-H, Liang Y, Wang Q-T, Huang J. 2014. A new
nodavirus is associated with covert mortality disease of shrimp. J Gen Virol, 2014, 95: 2700—2709.
– Zhang B-C, Liu F, Bian H-H, Liu J, Pan L-Q, Huang J. 2012. Isolation, identification, and pathogenicity analysis of a Vibrio
parahaemolyticus strain from Litopenaeus vannamei. (Chinese J.) Progress in Fishery Sciences, 33(2): 56—62.
– Liu Q, Huang J, Yang H-L, Yang B, Liu S, Wang H-L, Wang Q-T, Liu F, Zhang Q-L. 2014. Detection of a new genotype of Yellow-head
virus in farmed shrimp suspicious of EMS/AHPNS infection. Oceanologia et Limnologia Sinica, 45(4): 703-709.
– Liu Z, Zhang Q-L, Wan X-Y, Huang J. Development of real-time PCR assay for detection of microsporidian Enterocytozoon
hepatopenaei and detection with shrimp samples under different growth rates. (Chinese J.) Progress in Fishery Sciences,
(Accepted)
– Zhang Q-L, Liu S, Yang H-L, Zhu L-L, Wan X-Y, Huang J. Reverse transcription loop-mediated isothermal amplification for rapid and
quantitative assay of covert mortality nodavirus.(Submitted)
– Wan X-Y, Yang B, Zhang Q-L, Huang J, et al. Detection and Surveillance of the diseases in farm shrimp in China. (unpublished).
– Wang H-L, Wang Y-J, Yang H-L, Wang N, Huang J. Time-saving and specific methods with high sensitivity detecting acute
hepatopancreatic necrosis disease (AHPND). (unpublished).
– Bai N, Wang H-L, Zhu L-L, Huang J. Applying gnotobiotic Artemia saline to evaluate virulence of 24 stains of Vibrio
parahaemolyticus from shrimps with acute hepatopancreatic necrosis disease. (unpublished).
– Zhang Q-L et al., Existence and prevalence of a emerging shrimp virus, covert mortality, in China. (unpublished)
• This study 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 foundation under the Construction Program for
‘Taishan Scholarship’ of Shandong Province of China, and the Programme for Chinese Outstanding Talents in Agricultural
Scientific Research.

Presentation 19: Covert Mortality Nodavirus (CMNV): the pathogen, pathogenesis, transmission, distribution, impacts, co-infection with EMS/AHPND (Dr Huang Jie, China)

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Presentation 19: Covert Mortality Nodavirus (CMNV): the pathogen, pathogenesis, transmission, distribution, impacts, co-infection with EMS/AHPND (Dr Huang Jie, China)

Similar to Presentation 19: Covert Mortality Nodavirus (CMNV): the pathogen, pathogenesis, transmission, distribution, impacts, co-infection with EMS/AHPND (Dr Huang Jie, China) (20)

More from ExternalEvents

More from ExternalEvents (20)

Recently uploaded

Recently uploaded (20)

Presentation 19: Covert Mortality Nodavirus (CMNV): the pathogen, pathogenesis, transmission, distribution, impacts, co-infection with EMS/AHPND (Dr Huang Jie, China)