Reviews in Aquaculture, 1–44 doi: 10.1111/raq.

12352

Causative agent, diagnosis and management of white spot

disease in shrimp: A review
Bipul K. Dey , Girsha H. Dugassa, Sheban M. Hinzano and Peter Bossier
Department of Animal Sciences and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

Correspondence
Bipul K. Dey, Department of Animal Sciences
and Aquatic Ecology, Faculty of Bioscience
Engineering, Ghent University, Coupure
Links 653, 9000 Ghent, Belgium. Email:
bipulkumar.jstu@gmail.com
Received 23 January 2019; accepted 2 May
2019.
Abstract
White spot syndrome virus (WSSV) is the pathogen behind white spot disease
(WSD) in shrimp and many other crustaceans. It is a highly contagious virus cap-
able of causing total mortality in 3–10 days of outbreak in normal culture condi-
tions. Since the first report of occurrence in China and Taiwan between 1991 and
1992, WSD outbreak caused tremendous losses at farm level throughout the
world. Most of the published reviews on WSSV emphasize advanced genetic stud-
ies and biosecurity measures in terms of disease management. Recently, some new
technologies such as greenhouse, polyculture, biofloc and minimal water
exchange have been proposed for WSD management, which is the trigger for this
review. However, further research is needed on those new technologies enhancing
their efficient application.
Key words: disease, management, shrimp, technologies, WSSV.

et al. 2012; World Bank 2014; Verbruggen et al. 2016; Wu

Introduction
White spot syndrome virus (WSSV) is a pathogen behind
white spot disease (WSD) in shrimp and many other crus-
taceans (Pradeep et al. 2008; Flegel 2012; Joseph et al.
2015). WSSV is highly contagious capable of causing total
mortality in 3-10 days in normal culture conditions (Chou
et al. 1995; Pradeep et al. 2008; Leu et al. 2009; Joseph
et al. 2015). WSSV infection in shrimp can be easily recog-
nized by its characteristic white spots on the carapace
(Chou et al. 1995; Pradeep et al. 2012). However, this clini-
cal sign is displayed long after infection at a point where
the animal is at the verge of death. Diagnosis techniques
have thus evolved in the past from morphological-based
identification techniques using electron microscopy (EM)
to highly sensitive immunological and molecular tech-
niques that can detect the virus even in asymptomatic carri-
ers (Joseph et al. 2015). However, white spots may not
develop in all infections and may not present in all species
(Rajan et al. 2000).
Since the first report of occurrence in China and Taiwan
between 1991 and 1992, the WSSV has been associated with
huge economic losses in shrimp production industry in
many countries (Lightner 1996 2003; Lundin 1996; Chan-
ratchakool & Phillips 2002; FAO, 2005; Zhao et al. 2007;
Pradeep et al. 2008; Leu et al. 2009; Walker & Winton
2010; Tendencia et al. 2011; Debnath et al. 2012; Moss
et al. 2017; Chandrakala & Mekala 2018; Oidtmann et al.
2018). Globally, the total economic losses caused by the dis-
ease to the shrimp industry have been estimated to be
around USD 8-15 billion since its emergence (Lightner
et al. 2012). The economic losses had been increasing by
USD 1 billion yearly (Flegel et al. 2008; Stentiford et al.
2012). Several authors have indicated that the annual eco-
nomic losses due to WSSV have conventionally equated to
approximately one tenth of the global shrimp production
(Stentiford et al. 2012). Regionally the estimated loss of
revenue in Asia was in the range of USD 4-6 billion whereas
in the Americas, a loss of USD 1 billion was reported dur-
ing the same period (Walker & Winton 2010; Tendencia
et al. 2011). Thus, WSD making shrimp industries such as
farms, feed manufacture industries, processing plants and
hatcheries unsecured, and eventually increasing the unem-
ployment problems worldwide (FAO, 2005). Although
WSD continuing its notorious occurrences in the shrimp
farms worldwide since 1991 (Chanratchakool & Phillips
2002; FAO, 2005; Zhao et al. 2007; Pradeep et al. 2008;
Walker & Winton 2010; Tendencia et al. 2011; Debnath
et al. 2012; Moss et al. 2012; World Bank 2014), but
remarkably, the disease is not under control even to date at
the farm level (Verbruggen et al. 2016; Wu et al. 2017).
Therefore, management of WSD in shrimp farms is a buz-
zword nowadays. Considering the importance of

© 2019 Wiley Publishing Asia Pty Ltd 1

B. K. Dey et al.
controlling WSD, the World Organization for Animal
Health (OIE), an intergovernmental organization responsi-
ble for improving animal health worldwide, has already
included this disease in their prioritized list.
Much success has been achieved in WSSV research
notwithstanding the challenges brought by its unique geno-
types and its infection mechanism that is completely differ-
ent from those of already known viruses (van Hulten et al.
2001a; Witteveldt et al. 2004b; Li et al. 2006). Classification
and complete genome sequencing of the WSSV have been
reported by Murphy et al. (2012) and van Hulten et al.
(2000a) respectively. Identification of the virion structural
proteins, the genes behind their expression and their role in
host infection have also been documented (Wan et al.
2008; Chang et al. 2010; S
anchez-Paz 2010). In other stud-
ies, the transmission of the virus from one host to another
and the role of water physico-chemical parameters in infec-
tion has been investigated (Supamattaya et al. 1998; Corsin
et al. 2001; Vidal et al. 2001; Rahman et al. 2006; Reyes
et al. 2007). Finally, a correlation between proximity of a
shrimp farm to the sea and WSSV infection have also been
reported by Mohan et al. (2008).
To date, like most of the other viral diseases (Menasveta
2002), WSD cannot be treated, and thus, excluding the
virus from shrimp rearing facilities is recommend (Menas-
veta 2002; Pan et al. 2017). In this regard, several biosecu-
rity measures have been reported in literatures. Moreover,
the effects of incoming water screening and disinfection
have widely been reported. Other biosecurity measures that
have been exploited in preventing introduction of WSSV
include domestication of specific pathogen-free (SPF)
broodstock, low water exchange systems and complete dry-
out of culture units after every culture cycle (Tendencia
et al. 2011; Wyban 2015). The use of crab fencing, bird
scares, foot baths and limited access to shrimp facilities are
common practices in excluding vectors and pathogen in
most shrimp farms (World Bank 2014). In addition to
biosecurity measures, the use of immunostimulants and
vaccines to improve response among shrimps against
WSSV infection have also been explored (Satoh et al. 2008;
Sajeevan et al. 2009).
Most of the reviews on WSSV have dwelled on
advanced genetic studies and on biosecurity measures
employed in WSD management (e.g. Escobedo-Bonilla
et al. 2008; Stentiford et al. 2009; Xiaoyan et al. 2012;
Ganjoor 2015; Vaseeharan et al. 2016; Bir et al. 2017;
Sivasankar et al. 2017). The shrimp farming practices,
however, are dynamic and in the recent past, the indus-
try has seen some new technologies being implemented
not only to increase yields but also to improve WSD
management. The present review highlights some of the
technologies and their potential in managing the WSD
scourge in shrimp farming.
2 © 2019 Wiley Publishing Asia Pty Ltd
Biology
Taxonomy
Initially, as a non-occluded baculovirus, WSSV was placed
in the subfamily Nudibaculoviridae (Francki et al. 2012)
belonging to the family Baculoviridae (Wang et al. 1995;
Francki et al. 2012). However, it was reclassified into a new
family named Whispoviridae (Van Hulten & Vlak 2001).
Later, WSSV has been accommodated into a new family
called Nimaviridae containing a single genus, Whispovirus,
containing a single species, WSSV (Lo et al. 2012). How-
ever, according to the earlier publications of the Interna-
tional Committee on Taxonomy of Viruses (ICTV),
inclusion of only one species (WSSV) into Nimaviridae
family was acknowledged as unusual because some other
viruses found in several species of crabs have been
described as ‘similar’ to WSSV, at least based upon mor-
phology of the virion (Vlak et al. 2005). Hence, the taxo-
nomic position of those viruses was tentatively defined as
within the Nimaviridae family by the ICTV (Vlak et al.
2005) and, still this position awaiting for more detail evi-
dence of those viruses (Lo et al. 2012).
Morphology
Morphologically, the WSSV is large (Fauquet et al. 2005;
S
anchez-Paz 2010), enveloped, rod-shaped (Wang et al.
1995; Fauquet et al. 2005) to elliptical (Fauquet et al. 2005).
The size of the virion was estimated to be 80–120 9 250–
380 nm (Fauquet et al. 2005), that contains a tail-like
appendage (Durand et al. 1997; Fauquet et al. 2005). The
viral envelope is 6–7 nm thick (Wongteerasupaya et al.
1995; Durand et al. 1997; Nadala et al. 1998), even some-
times ~12 nm (Amano et al. 2011). There are many tad-
pole-shaped spikes on the outer surface of envelop (Figs. 1
and 2) that facilitate them to attach host cells. The length of
the spike is 5–6 nm and the spike head is 4–5 nm in diameter
(Amano et al. 2011). Viral DNA is situated inside the nucle-
ocapsid (Durand et al. 1997; Wang et al. 1999; van Hulten
et al. 2001a) and nucleocapsid is covered by the envelope
(Durand et al. 1997; Nadala & Loh 1998). Nucleocapsid is a
rod-like structure (Huang et al. 2001) and 54–85 9 180–
440 nm in size depending on the isolates (Kasornchandra
et al. 1998; Hameed et al. 1998; Rajendran et al. 1999;
Amano et al. 2011); and it has a 5–6 nm thick membrane
(Amano et al. 2011; Huang et al. 2001). Nucleocapsid is
shaped by fifteen conspicuous helices that are arranged
along its long axis (Figs. 3a and 4) with 7 nm space between
them (Huang et al. 2001). Huang et al. (2001) found a ter-
minal single ‘ring’ structure in some of the degraded nucleo-
capsid (Fig. 3b). In accordance of Amano et al. (2011), the
15 ring structures are not united. The structural units of the
rings have been noticed as equilateral triangle and rhombus
Reviews in Aquaculture, 1–44
 White spot syndrome virus in shrimp

body units, whereas each rhombus is comprised of two uni-

ted triangle body units. In such a way, one ring is composed
of 36 triangle bodies and 18 rhombs bodies (Fig. 4) (Amano
et al. 2011). A thin flexible structure has been found in
between the rings which confers a loose connection between
rings allowing the enhancement of length of nucleocapsid
outside the envelop (Amano et al. 2011). Thus, when
released from the envelope, the nucleocapsid increases in
length indicating that it is tightly packed within the virion.
The length of the naked nucleocapsid (350 nm) has been
found as approximately 40% longer than the viral particles
(Huang et al. 2001). The nucleocapsid core contains
unknown substance(s), and get oval shaped whenever the
core is empty and thus the width of empty nucleocapsid
increases (Amano et al. 2011).
Figure 1 Electron microscopic analysis of a broken WSSV virion.
Spikes can be seen (indicated by arrows) clearly on the broken mem- Genome
brane. (Source: Amano et al. 2011)
White spot syndrome virus is made up of double-stranded
(Wang et al. 1995) circular and highly coiled DNA (Fig. 5)
Spike which has been reported to be about 300–305 kbp long
Envelop extension depending on the isolates (Yang et al. 2001; van Hulten
et al. 2001b; Lightner et al. 2012; Parrilla-Taylor et al.
2018) with 180 open reading frames (ORFs) and nine
regions of repeated sequences in tandem (van Hulten et al.
2000a). However, the length of DNA could be 281–312 kbp
Virion length: Nucleocapsid that has been reported by Oakey and Smith (2018). Most of
250–380 nm the proteins encoded by the genome have been reported to
lack homologues in sequence databases except those that
encode for enzymes such as protein kinases and ribonu-
cleotide reductases and constitute only 5% of the total
Double layered ORFs (van Hulten et al. 2001a; Witteveldt et al. 2004b; Li
envelop et al. 2006). Currently, the tools used in WSSV genomic
and epidemiology studies include minisatelites (ORF94,
Virion width:
80–120 nm ORF75 and ORF125) and WSSV-based microsatellites.
Many research on functional analysis of the genetic prod-
Figure 2 Structure of WSSV has been drawn based on the informa- ucts of WSSV genome, structural proteins in particular,
tion previously mentioned in morphology section. have carried out (Li et al. 2006). Genomic sequences and
proteomic analyses of WSSV strains isolated from different
(a) (b)

Figure 3 Scanning image of nucleocapsid core of WSSV. (a) The nucleocapsid comprising 15 conspicuous vertical helices. (b) ‘Ring’ structure (indi-
cated by arrows) is visible at the termini of some of the naked nucleocapsid.(Source: Huang et al. 2001)

Reviews in Aquaculture, 1–44

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B. K. Dey et al.

Nucleocapsid Cross section Side view

1
(a)
(b)
3
70 nm
5

180–440 nm 7
9
(c)
11
80 nm
13
15

Figure 4 Structure of a WSSV nucleocapsid has been drawn based on the information previously addressed in morphology section. (a) Electron
microscopic analysis of WSSV nucleocapsid comprising 15 conspicuous vertical helices. (b) A single helices (cross section) of nucleocapsid having a
diameter of 70 nm (side view). (c) An empty nucleocapsid (cross section) having an 80 nm diameter (side view). (Source: Amano et al. 2011). The
cross-hatched appearance of the nucleocapsid is unique diagnostic feature of WSSV (Verbruggen et al. 2016).

VP35 is also absent in WSSV-MX08 (Rodriguez-Anaya

VP28 ORF14/15VR
ORF23/24VR
WSSV-TH
292,967 bp
VNTR ORF75
VNTR ORF125 VNTR ORF94
Figure 5 Genome structure of WSSV from Thailand (WSSV-TH) show-
ing some of the ORFs and the dominant envelope protein coding gene
VP28. (Source: Shekar et al. 2012)
parts of the globe have reported some differences among
them. For instance comparative genomic studies of isolates
from Thailand (WSSV-TH), China (WSSV-CN) and Tai-
wan (WSSV-TW) reported considerable differences in their
genome sizes but with 99.32% overall similarity (Shekar
et al. 2012). Recently, the genome of a Mexican strain
named WSSV-MX08 was examined; that was 99.50–
99.64% similar with the genomes of other strains. The gen-
ome of this Mexican strain was most distant to that of Kor-
ean strain (WSSV-KR) (Rodriguez-Anaya et al. 2016).
However, there was a lack of ORFs 122 and 123 both in
WSSV-MX08 (Rodriguez-Anaya et al. 2016) and WSSV-
KR (Chai et al. 2013). In fact, the G+C content of WSSV-
TH was found to be 41% which matches with that of the
other WSSV genomes (Chai et al. 2013). Furthermore,
et al. 2016), as detected in both the WSSV-TH (Marks
et al. 2004) and WSSV-KR (Chai et al. 2013), suggesting
that VP35 is not essential for replication (Rodriguez-Anaya
et al. 2016). Marks et al. (2004) reported that one of the
biggest difference among the isolates is a genetic variation
in ORFs 14 and 15. Rodriguez-Anaya et al. (2016) also
observed 575 bp deletion in this region in WSSV-MX08
(Rodriguez-Anaya et al. 2016). In addition, out of the three
Saudi Arabian WSSV strains reported, two differ signifi-
cantly from those reported in Asian and American because
of a 1522 bp deletion in the ORF94. Moreover, some differ-
ences exist between the Saudi Arabian strains themselves in
the number of repeated base pair sequences in the ORF125
(Tang et al. 2012). Recently, the genome sequence of an
Australian strain (WSSV-AU) showed 91–97% similarities
comparing with that of other strains. In WSSV-AU several
deletions in structural proteins encoding regions of genome
have been reported (Oakey & Smith 2018). Furthermore, in
an Ecuadorian strain (WSSV-EC), two major fragments
containing ORFs VP22 and VP25 were appeared to be
deleted. Interestingly, the authors observed that WSSV-EC
strain causes the lowest shrimp mortality despite its wider
spreading comparing with those of strains possessing VP22
and VP25. At this point the authors stated that the less vir-
ulence could be due to the deletion of ORFs VP22 and
VP25 (Restrepo et al. 2018).
Virulence
Virulence may indicate the degree of pathogenicity within a
group or species of pathogens. Different studies have shown

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4 © 2019 Wiley Publishing Asia Pty Ltd

that slight differences in virulence have been observed
among different strains of WSSV (Pradeep et al. 2012). Fur-
thermore, many similarities including morphology and pro-
teomes have been found among the various isolates of the
virus from different geographical locations (S
anchez-Paz
2010) although direct comparisons were not made (Wang
et al. 1999; Lan et al. 2002). Wang et al. (1999) compared
the virulence of six different geographical origins, namely:
China, India, Thailand, Texas and South Carolina as well as
from infected crayfish kept at the USA National Zoo park of
WSSV isolates in Penaeus vannamei (postlarvae stage) and
Farfantepenaeus duorarum (juveniles) (Wang et al. 1999).
The authors demonstrated that all the six isolates were infec-
tious and highly virulent to P. vannamei. However, the cray-
fish WSSV isolate was detected as the least virulent among
the six isolates, whereas the Texas isolate was the most viru-
lent strain among them (Wang et al. 1999). Thus, the study
revealed slight differences in virulence among the six geo-
graphical isolates of WSSV. Interestingly, in that experiment
the juveniles of F. duorarum have shown moderate resistance
to WSSV infection (Wang et al. 1999). Similarly, in some
other recent studies, different strains showed different viru-
lence (Li et al. 2017; Ramos-Paredes et al. 2017). However,
the important remark is that one should consider the labora-
tory standards when comparing results between laboratories.
In fact, in an experiment, the mortalities in Macrobrachium
nipponense due to WSSV infection via oral administration,
muscle injection and immersion have been found to be
100%, 75% and 0% respectively. However, the fastest infec-
tion was observed in terms of muscle injection (Yin et al.
2017). Thus, interestingly this research revealed the inconsis-
tency between infection speed and mortality.
The studies by Marks et al. (2005) compared the viru-
lence between two strains, namely: WSSV-TH-96-II (Thai-
land WSSV isolate with the largest genome reported to
date) and WSSV-TH (WSSV isolate containing the smallest
genome). The authors suggested that the isolate with the
smallest genome is more virulent and fit compared with the
WSSV-TH-96-II isolate containing the largest genome
(Marks et al. 2005). Moreover, other comparative study
was conducted by Stalinraj et al. (2009) to compare the vir-
ulence between six isolates of WSSV from farms experienc-
ing outbreaks (virulent WSSV: vWSSV), and three isolates
of WSSV which were taken from farms that had infected
shrimps without the occurrence of outbreaks (non-virulent
WSSV: nvWSSV). The study indicated both vWSSV and
nvWSSV isolates have led to 100% mortality within 5 days
on challenge in shrimps (Stalinraj et al. 2009). Rahman
et al. (2008) collected three WSSV isolates from naturally
infected P. monodon, two of them from Thailand (WSSV
Thai-1, WSSV Thai-2) and one from Vietnam (WSSV
Viet); and the authors detected WSSV Thai-1 to be most
virulent where WSSV Viet was the least virulent.
Reviews in Aquaculture, 1–44
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White spot syndrome virus in shrimp
The studies by Pradeep et al. (2012) have compared the
virulence and fitness between three different Indian geno-
types of WSSV by infecting shrimps and crabs. The three
different strains of the virus were isolated from disease out-
break ponds from West and East coast of India. The results
of this study have shown that there was no difference in vir-
ulence among the three genotypes used for the infection of
shrimps and crabs. Furthermore, all the three strains
resulted in mortality in both shrimps and crabs within the
same period. The study has revealed that there were no sig-
nificant differences in viral loads as shown using real-time
PCR (RT-PCR) between the two species. This study has
indicated that when three strains were mixed and used,
only the strains which have a smaller genome size (ORF94,
3RU and ORF94, 6RU) resulted in disease in both shrimps
and crabs. The authors have also conducted a study on sub-
sequent passage of the three strains to determine which
strain(s) can cause disease. Finally, the study revealed that
the strain with ORF94, 3RU caused disease. These findings
can suggest that although all the strains of WSSV used in
the experiment are equally virulent; there is a difference in
fitness among the three mixed strains (Pradeep et al. 2012).
This finding is supported by the study of Hoa et al. (2011)
demonstrating that WSSV mixed genotype infections result
in lower outbreak rate compared with that of single geno-
type infections (Hoa et al. 2011).
Structural proteins
Structural proteins have been the main subject of research
in the past and this could be largely attributed to their cru-
cial role in WSSV infection (Wu et al. 2005; Li et al. 2006).
One of the techniques employed in WSSV protein identifi-
cation is a combination of sodium dodecyl sulphate-poly-
acrylamide gel electrophoresis (SDS-PAGE) with mass
spectrometry (Xie et al. 2006). The structural proteins play
vital roles in cell targeting, virus entry, assembly and bud-
ding (Chazal & Gerlier 2003; Rajc
ani 2003; Mettenleiter
2004; Mettenleiter et al. 2006; Campadelli-Fiume et al.
2007). So far, 58 structural proteins have been identified
out of which more than 30 are recognized as envelope pro-
teins (see S
anchez-Paz 2010). Among the envelop proteins,
VP19, VP24, VP26 and VP28 are major proteins (Momtaz
et al. 2018) and, VP28 and VP26 are the most abundant,
comprising approximately 60% of the envelope (Tang et al.
2007). VP28 plays an important role in the early stages of
virus infection (van Hulten et al. 2000b, 2001b). Chang
et al. (2010) formed a 3D model in which a structural pro-
tein, VP24, has been demonstrated as a core protein
directly associating with VP26, VP28, VP38A, VP51A and
WSV010, and thus forming a membrane-associated protein
complex. In that model, VP19 and VP37 were shown to be
attached to the complex associating with VP51A and VP28
5
B. K. Dey et al.
respectively. This envelope complex was reported to anchor
to the nucleocapsid through the VP26–VP51C interaction
(Chang et al. 2010). Some envelope proteins are expressed
within the virion, whereas others are exposed at the surface
(Fig. 6). Those on the surface are generally involved in
binding to receptors, fusion and interaction with the host
immune system (Chang et al. 2010). On the other hand,
those expressed within the virion aid in the transmission of
the WSSV nucleocapsid to the nucleus of the host cells
(Wan et al. 2008). In enveloped viruses like WSSV, interac-
tions between structural proteins are usual, however, such
kind of interactions have been reported precisely only in
nine WSSV virion proteins (VP19, VP24, VP26, VP28,
VP37 or VP281, VP38A or VP38, VP51C or VP51, VP51A
and WSV010) (Zhang et al. 2004; Xie & Yang 2006; Chen
et al. 2007; Chang et al. 2008 2010; Jie et al. 2008; Wan
et al. 2008; Liu et al. 2009; Zhou et al. 2009), even some of
them (VP19, VP24 and VP51A) have affinity for self-inter-
action (Chang et al. 2010). Identification and description
of envelope proteins was driven by the need to understand
their profiles which would hopefully provide an opportu-
nity for management of the virus through production of
neutralizing antibodies or use the proteins as targets sites
for vaccines production (Witteveldt et al. 2004b). However,
in the recent past, the orientation of studies on envelope
Outside the virion
Envelope
Inside the virion
Figure 6 Three-dimensional presentation of WSSV envelope proteins
showing the putative relative localizations of the WSSV virion protein
complex composed of VP19, VP24, VP26, VP28, VP37, VP38A, VP51A,
VP51C and WSV010. Each protein is rendered using a unique colour.
The cylinders represent transmembrane helices.(Source: Chang et al.
2010)
6 © 2019 Wiley Publishing Asia Pty Ltd
proteins was towards understanding the interactions of
envelope proteins with each other and to the potential host
surface proteins. These knowledge would be effective in
elucidating the mechanisms of WSSV infection (Chang
et al. 2010).
Replication
The life cycle of viruses is well described and studied by dif-
ferent authors. Accordingly, the life cycle can be generally
divided into different phases namely: entry into the host
cell (via fusion or endocytosis), uncoating of the genome,
replication, assembly and maturation of virions and releas-
ing of virus particles via either budding or cytolysis (Esco-
bedo-Bonilla et al. 2008). Attachment of WSSV to the
surface of the host cell membrane initiates the replication
cycle of this virus. The attachment is followed by the entry
of the virus into the cell by endocytosis. The WSSV envelop
fuses with endosome, and this fusion triggers the release of
nucleocapsid into the cytoplasm. The released nucleocapsid
comes to close contact with nucleus and inserts the viral
genome into nucleus via nuclear pore. Meanwhile, degrada-
tion of envelope and capsid proteins occurs in the cyto-
plasm (Li et al. 2015b). After the virus reach inside the host
nucleus, the virus particle has to express its own gene for
replication. Since all viruses including WSSV do not con-
tain their own transcriptional machinery, they have to
depend initially on the host cell to get this machinery in
order to start new replication phases (S
anchez-Paz 2010).
Inside the nucleus, the binding of the host transcription
factors to viral promoters brings about the activation of the
transcription process (S
anchez-Paz 2010; Verbruggen et al.
2016) produces the mRNAs of immediate-early genes (Li
et al. 2015b) such as wsv477 (Yang et al. 2001), the genes
that are essential for encoding transcription factors and
other regulators which can allow the transcription of the
viral genes (S
anchez-Paz 2010; Verbruggen et al. 2016) that
are necessary for succeeding stages of the viral life cycle and
are expressed prior to the replication of DNA (Jensen et al.
1996; Li et al. 2009a; S
anchez-Paz 2010; Verbruggen et al.
2016). Immediate-early genes subsequently move to the
cytoplasm via nuclear pores. Subsequently, cytoplasmic-
free ribosomes transform the immediate-early genes into
proteins, when major capsid protein of the WSSV, VP664,
is expressed. All these mechanisms were detected to happen
within 1 hour of postinoculation in laboratory condition.
The immediate-early proteins activate early and late pro-
teins (Li et al. 2015b). Late genes are those that are
expressed after the onset of DNA replication (Jensen et al.
1996). The transcription of the mRNA for envelop proteins
starts after 3 hours postinoculation. VP28, major envelope
protein of the WSSV, is expressed in the cytoplasmic rough
endoplasmic reticulum and subsequently transported to
Reviews in Aquaculture, 1–44

the inner nuclear membrane, which then proliferates within
the nucleus. The virus genome multiplies in the nucleus
and the virus capsids become assembled around new viral
genomes (started from 6 hours postinoculation). After-
wards, nucleocapsids are formed and subsequent budding
occurs at the inner nuclear membrane with VP28. The
newly formed virus particles temporarily stay in the lumen
of the expanded inner nuclear membrane, and finally the
new WSSV particles are released from the cell through cell
lysis (Li et al. 2015b).
During the phases of the life cycle, there is a wide range
of molecular interactions which occur between the WSSV
proteins and its host cells. These molecular interactions
play an important role in determining host susceptibility
and pathogenicity. They can also provide great opportuni-
ties for treatment interventions and control measures. The
host cell membrane acts as primary obstacle for the entry of
viruses into the host cell. However, viruses are very active,
and they have evolved several mechanisms to tackle this cell
barrier. These mechanisms may include lipid fusion and
membrane perforation (mainly for non-enveloped virus)
and endocytosis (for enveloped viruses like WSSV) which
help the viruses to entry into the host cell. In endocytosis,
the viruses bind with the host cell surface proteins, carbo-
hydrates and lipids (Mercer et al. 2010). This interaction
results in triggering of the various endocytic pathways such
as clathrin-mediated endocytosis, caveolar endocytosis and
macropinocytosis (Sodeik 2000; Mercer et al. 2010; Kalia &
Jameel 2011). The interactions between host cell surface
proteins and the virus lead to the activation of the endocy-
totic process which facilitates the entry of WSSV into the
host cell (Mercer et al. 2010).
Different authors have indicated that VP28, an envelope
protein, is one of the major structural proteins of the virus
which plays a key role in host–virus protein interactions
(van Hulten et al. 2001b; Yi et al. 2004; Wan et al. 2008;
Sanjuktha et al. 2012). Several authors have also indicated
some interactions are beneficial for the virus, whereas
others may have negative impacts. WSSV mainly targets for
the integrin receptors on the host cell surfaces for binding
purpose. The structural proteins of the virion envelope
such as VP26, VP31, VP37, VP90 and VP136 interact with
integrin receptors to promote the binding of the virus to
the extracellular matrix (or cell-to-cell adhesion). The inte-
grins are heterodimeric surface receptors which recognize
specific peptides like RGD (Arginine-Glycine-Aspartate)
motifs present in structural proteins of the virus particles
(Ruoslahti 1996; Zhang et al. 2014).
Host range and susceptibility
White spot syndrome virus can infect a wide range of
potential shrimp species including penaeid shrimps. The
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White spot syndrome virus in shrimp
host range of WSSV includes many other species of deca-
pod and non-decapod (e.g. crabs, lobsters, prawns, cray-
fishes, copepods and arthropods) that has been reviewed by
several authors (see Oidtmann & Stentiford 2011; Pradeep
et al. 2012). More than 100 species of arthropods either
from culture facilities, the wild or experimental infection
have been reported to be the host or carriers of WSSV
(Hameed et al. 1998 2000 2001 2003; Musthaq et al. 2006).
This wide range of hosts plays an important role for the
transmission and occurrence of the disease outbreak in
farmed shrimps. Currently, more than 98 species of deca-
pod and non-decapod have been identified as potential
host for WSSV (Stentiford et al. 2009). These authors have
indicated that there are also cases in which many organisms
living in the virus infected pond without exhibiting clinical
signs (Stentiford et al. 2009). These organisms can serve as
vector for circulating WSSV in the pond. The other studies
by Walker and Mohan (2009) have reported that high levels
mortality was observed in all cultured shrimps infected
with WSSV although it is not fatal to other hosts species
(Walker & Mohan 2009).
Disease susceptibility varies across the Crustacean, and
this is an interest to many researchers as it may provide them
an approach to disease resistance (Nakano et al. 1994; Chen
et al. 2000; Yoganandhan et al. 2003; Bateman et al. 2012).
For example the studies by Bateman et al. (2012) demon-
strated that the shore crab (Carcinus maenas) can be infected
by the WSSV. However, this species appears to be refractory
to development of the disease leading to little pathology and
low mortality rates compared with farmed shrimps (Bate-
man et al. 2012). The virulence of WSSV can be affected as
the virus passes between two host species. For example
according to the studies of Waikhom et al. (2006), when
WSSV was passed via Macrobrachium rosenbergii, a reduc-
tion in its virulence was observed that resulted in low mor-
tality rates in P. monodon compared with non-transmitted
virus. This change in virulence is not yet well understood.
However, it may be related to variations in tandem repeat
regions in the WSSV genome (Waikhom et al. 2006). In
general, several authors have suggested that WSSV can infect
a wide range of potential hosts ranging from penaeid
shrimps to other crustaceans. However, the susceptibility of
the potential hosts to WSSV may vary from one species to
another species (Reddy et al. 2013).
Transmission
White spot syndrome virus can be transmitted via horizon-
tal and vertical routes (Lo et al. 1997; Soto & Lotz 2001;
Prior et al. 2003; Soowannayan & Phanthura 2011). The
transmission of the virus between the shrimp and other
Decapod crustacean may occur via three routes, namely: (i)
oral route by feeding of the infected organisms or
7
B. K. Dey et al.
contaminated food. Moreover, transmission of the virus is
also possible through consumption of infected tissue by
predation and cannibalism, (ii) via water borne in which
the virus invades through gills and other body surfaces by
direct exposure to virus particles in water (Lo et al. 1997;
Chou et al. 1998) and (iii) through the vertical transmis-
sion in which the virus is passed from an infected brood-
stock to offspring via oocytes (S
anchez-Paz 2010; Pradeep
et al. 2012). Different findings have demonstrated that the
transmission rates are similar in both species of P. van-
namei and P. monodon. However, the authors have sug-
gested there may be difference in the relative contributions
of direct and indirect transmission rates (Tuyen et al.
2014). The virus particles have been reported in oocytes,
however, the virus particles are absent in mature eggs. This
may indicate that the oocytes which contain the virus can-
not develop into mature eggs (Pradeep et al. 2012). The
transmission of WSSV between different geographical loca-
tions can be facilitated by the transport of live and frozen
uncooked shrimp (Nunan et al. 1998), and import of
broodstock (Stentiford et al. 2012) from one region to
other locations. Several studies have indicated that some
environmental factors like temperature, salinity drop and
pH are known as main stress factors which can influence
the transmission and the occurrence of WSSV infection
outbreaks (Peinado-Guevara & L
opez-Meyer 2006; Ten-
dencia et al. 2010). A study had been conducted to know
the effect of climate factors on WSD occurrence in an
intensive shrimp culture area in Thailand, where the analy-
sis of collected data revealed a high incident rate ratio of
WSD when average atmospheric temperature ranged
between 24.5 and 27.2°C (Piamsomboon et al. 2016).
Pathogenesis
White spot syndrome virus (WSSV) affects all the tissues of
ectodermal and mesodermal origins (Lightner 1996). Sev-
eral studies have shown that WSSV infections can be
detected in different tissues including haemolymph, gills,
stomach, body cuticular epithelium, haematopoietic tis-
sues, lymphoid organ, antennal glands, foregut, testes and
ovaries of naturally and experimentally infected shrimp
(Rajan et al. 2000; Yoganandhan et al. 2003; Escobedo-
Bonilla et al. 2007). Besides, several authors have also indi-
cated that the gills, stomach, body cuticular epithelium,
haematopoietic tissues, lymphoid organ and antennal
glands are the major target tissues for replication of WSSV
(Tan et al. 2001; Durand & Lightner 2002; Escobedo-
Bonilla et al. 2007). The studies by Escobedo-Bonilla et al.
(2007) have also explained that the foregut, gills, antennal
gland and integument are essential for maintenance of
shrimp homeostasis. The WSSV infection of these organs
leads to the death of the animals due to dysfunction of the
8 © 2019 Wiley Publishing Asia Pty Ltd
tissues (Escobedo-Bonilla et al. 2007). The epithelial cells,
haematopoietic tissues and tubules of antennal gland are
degenerated during a terminal stage of WSSV infection
(Chang et al. 1996; Lo et al. 1997). An experimental study
revealed that after 6 h of infection WSSV occurred to meta-
bolic changes through neutralizing the host’s oxidative
stress defences (Chen et al. 2016a). Different studies have
explained that previous results obtained with experimental
inoculations of WSSV into culture water of shrimps are
highly variable in the percentage of infection. Some studies
showed a high percentage of infected shrimp after exposure
to virus particles in water (Supamattaya et al. 1998),
whereas others studies indicated that shrimps were not
infected, even when exposed to high virus doses as deter-
mined by intramuscular titrations (Prior et al. 2003). Fur-
thermore, the results obtained with experimental WSSV
infection in shrimps via ingestion of infected tissues or via
contaminated feed are in contradictory findings. Some
researchers found that the oral route as powerful tool to
induce infection in shrimp (Lightner et al. 1998; Vidal
et al. 2001; Escobedo-Bonilla et al. 2006). However, others
authors had got difficulties to reproduce these results
(P
erez et al. 2005; Gitterle et al. 2006; Laramore 2007).
Furthermore, Thuong et al. (2016) have indicated that
WSSV contaminated feed is poorly infectious per oral
route. However, it is highly infectious when injected to the
shrimp (Table 1). The authors have also demonstrated that
peritrophic membrane has no significant role as an internal
barrier of shrimp against WSSV infection (Thuong et al.
2016). These controversial observations may be associated
with differences in virulence of the WSSV strain, route of
administration, virus dose, experimental animals and other
experimental conditions (Thuong et al. 2016).
The infectivity titres of a WSSV stock solution has been
determined primarily by oral route challenge for the experi-
mental inoculation in shrimps (Escobedo-Bonilla et al.
2005). Furthermore, studies by Escobedo-Bonilla et al.
(2006) have developed a standardized oral inoculation pro-
tocol which can deliver an exact amount of the virus titre
to all inoculated shrimps (Escobedo-Bonilla et al. 2006).
Using this standardized inoculation technique, the primary
replication sites of the virus were determined with
immunohistochemistry (IHC) detection method. Accord-
ingly, the primary replication sites are the epithelial cells in
the foregut, cells in the gills, and only with a high dose (10
000 SID50) also cells in the antennal gland (Escobedo-
Bonilla et al. 2007).
Several authors have indicated that the mechanism of
WSSV spread from the primary replication sites to other
target organs is highly controversial. Some studies indicated
that the virus infects haemocyte in crayfish. Subsequently,
the virus can spread from the primary replication sites to
other distant target organs by the help of these infected cells
Reviews in Aquaculture, 1–44

Table 1 WSSV inoculation techniques in
shrimp and their effectiveness to cause infec- Species
tion to the host
Litopenaeus
vannamei
[1], Thuong et al. (2016); [2], Escobedo-Bonilla et al. (2006); [3], Escobedo-Bonilla et al. (2005);
SID50, Shrimp Infectious Dose 50, for instance the infective dose of WSSV that infects 50% of
exposed shrimps.
(Wang et al. 2002; Di Leonardo et al. 2005). However,
other studies have revealed that the circulating haemocyte
in freshwater prawns and shrimps are refractory to WSSV
infection (Van de Braak et al. 2002; Escobedo-Bonilla et al.
2007). Thus, these studies can indicate that the virus might
travel from the primary replication site to other target
organs via haemolymph circulation in a cell-free form
(Escobedo-Bonilla et al. 2007). Based on these studies, it is
possible to suggest that the mechanism of WSSV spread
may depend on the type of host species.
Several environmental factors, for example temperature,
salinity and pH, are known to be associated with the out-
breaks and intensity of WSD in shrimp (Peinado-Guevara
& L
opez-Meyer 2006; Tendencia et al. 2010). For example
in a laboratory experiment, the temperatures below 32°C
(16, 25, 27, 28 and 30°C) occurred at a constant high level
mortality in P. monodon, whereas a lower rate of mortality
was observed at 32 and 36°C. However, all shrimps that
survived at higher temperatures (32 and 37°C) were
claimed to be second step positive for WSSV. But in the
same experiment, salinity variables (0.5, 5, 10, 15, 30 and
45 g L
1) did not show any significant impact on the mor-
tality of the experimented animals (Stalin et al. 2012). In
another experiment, at the salinity of 30, 40 and 50 g L
1,
the minimum and maximum count of mortality were
found to be 3.5 and 8.5, 0.5 and 4.5 and 1.5 and 7.5 respec-
tively. Finally, the authors concluded that the lesser or
greater level of salinity than the normal level could lead to
severe mortality in Litopenaeus vannamei under WSD con-
dition (Kakoolaki et al. 2011). Furthermore, a study con-
ducted by Gunalan et al. (2010) revealed that lower
(a)
Figure 7 WSSV infected P. monodon indicating white spot symptoms. (a) White spots on carapace. (b) White spots on last abdominal segment.
(Source: Pradeep et al. 2012)
Reviews in Aquaculture, 1–44
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White spot syndrome virus in shrimp
Inoculation route Detection of virus titre/mortality by different research groups
[1] [2] [3]
Intramuscular 108.77 SID50 g
1 Early onset of mortality 106.9 SID50 mL
1
Oral 101.23 SID50 g
1 Late onset of mortality 105.4 SID50 mL
1
Feeding 100.73 SID50 g
1 – –
temperature (22–23°C) and high pH (9.5–9.7) influence
WSSV infection in cultured shrimps, P. monodon.
Clinical signs and pathology
Several authors have reported that WSSV may take some
time to express itself, but after expression onset, the
infected animals die within 3–8 days leading to high mor-
tality (Corbel et al. 2001). The infected shrimps usually
prefer to gather near the pond edge, and show clinical signs
in 1 or 2 days before occurrence of any mortality. The clin-
ical signs and gross pathology of animals suffering from the
disease may include lethargy, reduced food consumption,
reddish discoloration of body and appendages, reduced
preening activities, a loosening of the cuticle, and a discol-
oration of the hepatopancreas, low response to stimulus,
low appetite, swelling of branchiostegites due to accumula-
tion of fluid (Hameed et al. 2003), and thinning and
delayed clotting of haemolymph (Wang et al. 2000) and
gathering near the embankment (Rajan et al. 2000; Pradeep
et al. 2012). The expression of white to reddish-brown dis-
coloration may appear over the head and carapace of the
infected animals, and the presence of calcified white spots
on the exoskeleton. The presence of circular white spots or
patches can be detected in the cuticle of cephalothorax and
tail part of the infected shrimps (Lo et al. 1996; Rout et al.
2005) (Fig. 7). The presence of calcified spots appearing on
the exoskeleton of infected animal may be used as tentative
diagnostic tool for the disease at field condition. However,
this clinical sign is usually observed in some but not all host
species (Chou et al. 1995). For example in an experiment,
(b)
9
B. K. Dey et al.

at the phage of acute WSSV infection in Penaeus indicus
and P. monodon, white spots were not recognized, but the
only signs observed are lethargy and lack of appetite. Fur-
thermore, in P. indicus, body turned to reddish colour and
it was required to remove the carapace to confirm WSSV
infection (Rajan et al. 2000).
White spot disease causes rapid mortality in farmed
shrimp, and the cumulative mortality is generally found
between 90% and 100% (Wang et al. 1999; Wang & Zhu
2017). The exact mechanism of white spot formation and
deposition are not yet well known. However, some studies
demonstrated that the WSSV infection may induce the dys-
function of the respiratory and integument systems leading
to the accumulation of calcium salts within the cuticle
which give rise to white spots accumulation (Wang et al.
1999). Histologically, the WSSV infection is characterized
by having eosinophilic inclusion bodies in early stage of
infected cells (Fig. 8a). During advance stage of infection,
the hypertrophied nuclei of infected cells contain the inclu-
sion bodies which stain more basophilic (Lo et al. 1996)
(Fig. 8b). Furthermore, the infected nuclei become pro-
gressively more basophilic and enlarged (Otta et al. 1999;
Wang et al. 2000; Hameed et al. 2003). The cross-hatched
appearance of the nucleocapsid is unique diagnostic feature
to WSSV (Fig. 8c,d). Karyorrhexis and cellular disintegra-
tion of the infected cells may occur in the late stages of
infection resulting in the formation of necrotic areas
characterized by vacuolization (Karunasagar et al. 1997;
Kasornchandra et al. 1998) (Fig. 8c,d).
Diagnostic methods
Electron microscopy
In general, electron microscopy is still used as the first tech-
nique for virus detection (Goldsmith & Miller 2009). With a
few exceptions (the mimiviruses, poxviruses and some iri-
doviruses) the resolution of optical microscope cannot
detect the viruses, whereas the resolution of transmission
electron microscope (TEM) is 1000 times higher than optical
microscope allowing the direct detection of small viruses.
However, with the help of TEM, the virus can be identified
only up to family or genus level, but it is a quick method
(Vale et al. 2010). Although, by using TEM simply, previ-
ously the detection limit of viruses was 106 viruses per mL,
but by applying filtering method the limit has decreased to
102 viruses per mL (Beniac et al. 2014). There are some
issues such as the capital expenses, inapplicable for all
viruses, difficulties in differentiation between similar viruses
and low throughput are considered as the disadvantages of
using EM (Biel & Gelderblom 1999). There are also some
advantages of using EM, for example everything in the sam-
ple can be observed (Beniac et al. 2014), standard for the
identification of new viruses (Biel & Gelderblom 1999),
identification of dual infections whereas more than one

(a) (b)

(c) (d)

Figure 8 Observations on histological staining of white spot syndrome virus (WSSV) infected cells obtained from L. vannamei under electron micro-
scope. (a) During the early stage of infection the cells display enlarged nuclei with marginalized chromatin and a homogenous eosinophilic central
region. These then develop an intranuclear eosinophilic inclusion bodies (*); a clear halo along nuclear membrane can also be observed (white arrow).
Scale bar = 25 lm. (b) The eosinophilic inclusion generally expands and fills the nucleus (*). This inclusion becomes more basophilic and denser in col-
our in staining as the infection progresses (white arrow). Nuclei are then ruptured so that the content is integrated with the cytoplasm (black arrow).
Scale bar = 10 lm. H&E stain. (c) WSSV virions appear to be ovoid in shape containing an electron-dense nucleocapsid (white arrow) within a trilami-
nar envelope (black arrow). Scale bar = 0.2 lm. (Inset) Negative staining showing the presence of cross-hatched or striated material that is structured
as a series of stacked rings of subunits and is a key diagnostic feature of WSSV. Scale bar = 20 nm. (d) Presumptive pre-enveloped nucleocapsid
material within the nucleus, sporadically visible in the manufacture of the WSSV particles, that is cross-hatched or striated in appearance and linear
prior to its incorporation in the formation of mature WSSV particles. Scale bar = 100 nm.(Source: Verbruggen et al. 2016)

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10 © 2019 Wiley Publishing Asia Pty Ltd

viruses are involved (Kennedy 2005; Goldsmith & Miller
2009), and for rapid morphological identification. As such,
EM can be used as a frontline method and be coordinated
with other methods (Hazelton & Gelderblom 2003).
Immunological techniques
It has been demonstrated that the specific monoclonal anti-
bodies (MAbs) can be used to detect WSSV in shrimp by
western blotting, dot blotting and immunohistochemistry
avoiding cross-reactions to other viral or shrimp proteins
(Makesh et al. 2006; Chaivisuthangkura et al. 2010;
Vaniksampanna et al. 2017), whereas specific MAbs can
bind with target protein of WSSV. For example MAb
against VP28 only can bind with VP28 of WSSV but not
bind with any other viral or shrimp proteins (Makesh et al.
2006). Higher diagnostic sensitivity (100%) makes these
immunological techniques preferable comparing with sin-
gle-step polymerase chain reaction (PCR). However, dot
blotting, a well-known antibody-based protein detection
method, is claimed to demand for sufficiently high viral
loads for detection (Sithigorngul et al. 2011). Interestingly,
two MAb cocktail were found to be 100 times more sensi-
tive compared with one-step PCR and almost equivalent to
two-step PCR in detecting WSSV based on its VP28
envelop protein. Furthermore, the detection limit of WSSV,
using a MAb cocktail, was two- to four-fold amplified in
comparison with a single MAb (Vaniksampanna et al.
2017; Inchara et al. 2018). In a study of Chaivisuthangkura
et al. (2010), the combination of VP19 and VP28 (virion
proteins) specific MAbs have been recommended to
enhance the sensitivity of WSSV detection in shrimp in sev-
eral types of antibody-based assays (Chaivisuthangkura
et al. 2010). An immunosensor has been developed for
quantitative detection of WSSV in shrimp pond water. That
immunosensor was found to be highly sensitive for the
analysis of WSSV in the linear range 1.6 9 101–
1.6 9 106 copies lL
1. Furthermore, the immunosensor
were reusable up to approximately 37 times; and simple to
operate (Waiyapoka et al. 2015). In another study, lateral
flow immunoassay (LFIA) has been demonstrated as fast
and effective method to detect WSSV in ~20 min using
gold nanoparticles that conjugate to a polyclonal antibody
against VP28. Interestingly, the LFIA also showed no cross-
reactivity with some other shrimp viruses namely infectious
hypodermal and haematopoietic necrosis virus (IHHNV),
hepatopancreatic parvovirus (HPV) and monodon bac-
ulovirus (MBV) (Kulabhusan et al. 2017). To date, double
antibody enzyme-linked immunosorbent assay (ELISA) has
been adopted to capture polyclonal antibodies that could
recognize multiple epitopes, and to detect specific mono-
clonal antibodies to quantify the antigen at protein level
(Mecham 2006; Niu et al. 2013; Chang et al. 2016). It has
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White spot syndrome virus in shrimp
been recognized as an accurate and sensitive method for
detecting viral pathogens, and further, it could be applied
in WSSV detection for shrimps (Tang et al. 2018). Muru-
gan and Sankaran (2018) proposed a new ELISA technique
where recombinant bacterial lipid modification in proteins
in Escherichia coli system has been discussed. In that study,
a lipid-modified protein ICP11, the most abundantly
expressed white spot syndrome viral protein, was tested as
a new target in the new ELISA method. The authors found
this method as a sensitive measure of antigen in the
infected shrimp tissues where the detection limit for ICP11
protein was 250 pg and the linear range of the assay was
15–240 ng. The advantages of this assay are minimum assay
requirement, naked eye detection and cost-effective (Muru-
gan & Sankaran 2018), however, the authors did not con-
clude the duration of the detection whether it is quick or a
lengthy process. Antibody-based indirect immunofluores-
cence is another technique that is routinely used in virolog-
ical studies, however, although this technique is specific but
less sensitive. In an experiment, the sensitivity and speci-
ficity of indirect immunofluorescence were found to be
29% and 96%, respectively, comparing with PCR (Burges-
ser et al. 1999). As such, indirect immunofluorescence
technique requires comparatively high density of virus par-
ticles in the host tissue, therefore, it is not possible to detect
virus at the early stage of infection (Mott et al. 1997).
Finally, despite of many advantages (e.g. highly sensitive,
specific and accurate diagnosis) of immunological WSSV
detection techniques described above (Poulos et al. 2001;
Makesh et al. 2006), generally these techniques are costly,
time-consuming, require specialized equipment and skilled
personnel that making them not usable under field condi-
tions (Poulos et al. 2001). Recently, a lateral flow
immunoassay (LFIA) employing gold nanoparticles conju-
gated to a polyclonal antibody against dominant envelope
protein of WSSV VP28 has proposed. This method had
been reported to simultaneously detect WSSV, IHHNV (in-
fectious hypodermal and haematopoietic necrosis virus),
MBV, HPV (human papillomavirus), etc. through an easy
specific upgradation of the technique for specific virus.
Moreover, the technique is comparatively less costly (~USD
3 per sample), and the farmer could perform the test with-
out any help of outside professional that can substantially
reduce financial burden on the farmers. Therefore, LFIA
would directly benefit the farmers, hatchery operators and
aquaculture industry (Kulabhusan et al. 2017).
Molecular techniques
After publishing the whole genome sequence of WSSV (van
Hulten et al. 2001a; Yang et al. 2001, 2001b) the PCR as a
molecular technique has been used aiming the most sensi-
tive detection of WSSV. Consequently, nested or two-step
11
B. K. Dey et al.
PCR, that is now recommended by OIE (http://www.oie.
int/fileadmin/Home/eng/Health_standards/aahm/2009/2.2.
05_WSD.pdf), has been recognized to be more sensitive
than the single-step PCR (Lo et al. 1998); both of them are
non-quantitative diagnosis method (Durand & Lightner
2002), but they are similar in terms of sensitivity (Nunan &
Lightner 2011). In these PCR techniques, primers are used
(Durand & Lightner 2002). Later, a real-time PCR (RT-
PCR) has been described (Durand & Lightner 2002) that is
capable of virus quantification in the infected shrimp
depending on nucleic acid quantification, and therefore it
is also called quantitative PCR (qPCR). This method uses
various fluorescent dyes (e.g. CyberGreen) or DNA probes
(e.g. Taqman Probe) along with primer. Either techniques
quantify DNA depending on the intensity of fluorescence,
whereas probe-based technique is extremely sensitive (Dur-
and & Lightner 2002). In contrast, dyes bind to any dsDNA
rather than specific ones, the biggest disadvantage of using
dyes in qPCR. However, nowadays two different types of
dyes namely SyberGreen and EvaGreen are commonly used
in many laboratories throughout the world, whereas Eva-
Green dye is claimed to be the first and only PCR dye to
date that is environmentally safe, however, this dye is cur-
rently the only qPCR dye to be used in droplet digital PCR
(ddPCR) (https://biotium.com/faqs/what-is-the-difference-
between-evagreen-and-sybr-green/). The fluorescence sig-
nal changes proportionally to the amount of replicated
DNA with time and thus the DNA is quantified in ‘real
time’, that is why it is called real-time PCR (http://www.en
zolifesciences.com/science-center/technotes/2017/march/
what-are-the-differences-between-pcr-rt-pcr-qpcr-and-rt-
qpcr?/). However, the real-time PCR is still an expensive
and sophisticated technique, and in such context, nested
PCR represents the best alternative (Peinado-Guevara &
L
opez-Meyer 2006). There is also another method to quan-
tify DNA that is fluorescent quantitative PCR (FQ-PCR),
whereas the result is assesses based on the accumulation of
DNA templates containing fluorescent PCR products (Bus-
tin 2000). This method is cheaper than RT-PCR (Ririe
et al. 1997), but as in dye-based qPCR, any kind of double-
strand DNA can generate fluorescence indicating that it is
less specific than TaqMan RT-PCR (Ririe et al. 1997),
whereas TaqMan probes are used to increase the specificity
of RT-PCR (Holland et al. 1991). Another molecular tech-
nique is loop-mediated isothermal amplification (LAMP)
method that was invented by Kono et al. (2004) as a novel
technique. In that study, non-quantitative and probe-less
LAMP method has been found as highly specific and 10-
fold sensitive diagnostic system for WSSV when compared
with nested PCR. Moreover, this is a rapid method where
the amplification of the target nucleic acids occurred under
isothermal conditions, therefore needing no sophisticated
machine for thermal cycling, making this method
12
convenient (Kono et al. 2004). Recently, Pan et al. (2017)
developed a new technique called pre-amplification PCR
method, where some short-strand sequences were used as
adapters that can ligate to the target restriction fragment
VP28, VP39 and VP108 of WSSV by a linker. Thereafter,
the ligated product is served as the template for pre-ampli-
fication PCR using a universal primer. The target DNA is
greatly enriched by the pre-amplification PCR and then the
pre-amplified product is served as the template for the
specific PCR to complete the final detection. This non-
quantitative diagnosis technique is more efficient than
nested PCR, and is suitable for the super early diagnosis of
WSSV (Pan et al. 2017). However, the method of DNA
extraction, template concentration and the size of the
amplicon considerably impact on the sensitivity of the PCR
technique. Moreover, a positive PCR test, which detects a
DNA fragment, is a poor indicator of viral viability (Cha &
Thilly 1993). In addition, in most of the cases, these tests
require long-distance transportation of samples from field
to lab. And these mode of testing generally require high
cost, much time, highly trained laboratory personnel, and
dedicated laboratory space. To eradicate such problems,
recently PCR-based WSSV test are proposed via a portable
‘GD PCR machine’. This is a non-toxic, single use and bin-
ary format test, designed for non-specialized shrimp farm-
ers. In fact, during the field trial, GD testing had been
recognized as a valuable element. This technique has the
potential to be used as a semi-quantitative approach, how-
ever, this must be further investigated and optimized. This
test could be performed rapidly, and has the potential to be
integrated with mobile app or computer connections to
make the process easier, and thus it can timely deliver out-
break report to the competent authorities (Minardi et al.
2019).
Management aspects of WSSV
Exclusion of WSSV vectors and carrier organisms
Most of the WSSV hosts are carriers and can potentially
transmit the virus to shrimp farms when consumed or
through co-existence with healthy shrimps (Haryadi et al.
2015). In the earthen pond, where farm water is exchanged
periodically with water of natural sources such as rivers and
canals, effective exclusion of vectors and carriers is
appeared to be difficult. However, reduction in vectors and
careers as much as possible by following some cautions
may reduce the risk of WSD. Some practices such as sludge
removal, ploughing, liming and complete system dry-out
between culture cycles can eradicate the existing WSSV vec-
tors and/or carriers such as crabs, polychaete worms and
other benthic species (Corsin et al. 2001; Velasco et al.
2002; Mohan et al. 2008). However, in most instances,
sludge is placed on the dike after removal from the pond
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bottom. This practice might allow harmful micro-organ-
isms and benthic animals present in the sludge to be
washed back into the pond. Therefore, bottom sludge is
recommended to place far from the pond after excavation
(Alapide et al. 2011). During watering the pond, filtration
of inlet water through 300 lm mesh screen is affordable to
prevent the entry of vector and/or carrier animals and their
eggs. Post-watering application of phosphorus through fer-
tilization were reported to reduce risk of WSD outbreak in
the farm (Corsin et al. 2001; Velasco et al. 2002; Mohan
et al. 2008). In most farms, by-pass canals supplying water
to the ponds remain filled with water long after filling the
pond with water. This retention of water in the canal has
been observed to offer room for proliferation of an array of
WSSV hosts which in turn pass the virus into shrimp ponds
during the subsequent filling of the pond. Therefore, drai-
nage gate should be constructed in each by-pass canal that
can drain out the remaining water into it after pond filling,
and allow the canal thorough drying before the next refill-
ing activity (World Bank 2014). Apart from carriers, aqua-
tic birds have been identified as crucial vectors responsible
for spreading WSSV from one pond to another in the same
farm. Although faeces containing WSSV is not infective,
regurgitation of a WSSV-infected shrimp by birds, which is
common in most aquatic birds, can potentially enhance the
spread of WSSV (S
anchez-Paz 2010; World Bank 2014). It
is for this reason that technologies preventing accessibility
of birds to shrimp rearing units were initiated. The use of
birds scare lines (Fig. 9a) has been effectively used in
Malaysia to control the spread of WSSV. It involves cover-
ing a pond with a monofilament net supported by wooden
columns anchored at the pond bottom (World Bank 2014).
Crab fencing (Fig. 9b) is also one of such farm-based
strategies that prevents crabs from accessing shrimp ponds.
The practice involves erection of plastic barriers (50 cm
high) around the pond; and this barrier prevents crabs
from accessing shrimp culture units (World Bank 2014).
Furthermore, shrimp feed could be the cause of WSSV
introduction in the new environment. In fact, in an experi-
ment, SPF L. vannamei fed with Dendronereis spp., a
(a)
Figure 9 Physical barriers for denying crabs and birds access to shrimp ponds. (a) Birds’ scares (arrow). (b) Crab fencing (arrow).(Source: World Bank
2014)
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White spot syndrome virus in shrimp
ubiquitous Polychaetes that is a natural food of shrimp,
were found to be WSSV infected in ponds (Haryadi et al.
2015). As such, feeding SPF broodstock of shrimp is a great
concern as they are fed mostly with dead aquatic animals
especially Polychaetes (Desrina et al. 2012); and the WSSV
can pass from brood to the progeny (S
anchez-Paz 2010;
Pradeep et al. 2012). Therefore, shrimp feed should be free
of viable WSSV particles.
Greenhouse shrimp production
Changes in climatic conditions have been demonstrated to
contribute to shrimp diseases outbreak through alterations
in the distribution, prevalence and virulence of different
types of pathogens such as bacteria, viruses, fungi and para-
sites. On the other hand, changes in various climatic fea-
tures, for example temperature, may change disease
susceptibility of the host (Harvell et al. 1999; Tendencia
et al. 2010). In fact, low atmospheric temperature which
affects water temperature is an important WSSV risk factor
(Tendencia et al. 2010). Lower temperatures enhance viral
replication and decrease the immune response of shrimp
(Vidal et al. 2001; Reyes et al. 2007). As such, application
of greenhouse technique in shrimp production may reduce
the occurrence of WSD as greenhouse technology is usually
affordable to naturally maintain higher temperature inside
compared with that of surrounding environment. Accord-
ing to Vidal et al. (2001), WSD does not develop in WSSV
infected L. vannamei at temperatures above 32°C but when
the temperature is reduced to 26°C, the disease developed
very fast. This results were found to be consistent to that of
Rahman et al. (2006), where the authors stated that the
higher temperature (32–33°C) retards the replication of
WSSV in infected shrimp. The phenomenon has also been
reported for other penaeid species (Guan et al. 2003; Guna-
lan et al. 2010; Kakoolaki et al. 2011; Stalin et al. 2012).
Following the results of these studies, some shrimp farmers
in China, South and Central America resorted to farming
of shrimps in greenhouse enclosed ponds or raceways dur-
ing winter (Sonnenholzner & Calderon 2004; Peng et al.
(b)
13
B. K. Dey et al.
2014). In the same way, this technology may be beneficial
to shrimp culture in the temperate regions of the planet.
On the other hand, higher temperatures may facilitate the
survival and growth of rare animals in some cases. Prelimi-
nary studies have reported an overall survival of 85% and
1.3 g week
1 growth rate (McAbee et al. 2003). Addition-
ally, in aquaculture system, maintenance of proper biosecu-
rity is important in terms of disease prevention. Being an
enclosed system, greenhouse can protect WSSV vectors like
birds; and also prevent the addition of rainwater that may
cause reduction in pH, temperature and salinity. Several
authors stated that, as greenhouse system allows for strin-
gent biosecurity implementation and less water exchange
with the external environment, it can minimize diseases
introduction in areas where endemism is experienced
(McAbee et al. 2003; Kumara & Hettiarachchi 2018). In
Mexico, a three-phase nursery system has been imple-
mented where after a period in the hatchery, the larvae are
held in greenhouse raceway ponds until they are above 45
days old before they are transferred into grow out ponds
(Abad 2015). The technology has been successful in Malay-
sia with mixed results of success and failures being reported
in Thailand and Vietnam (Abad 2015). The materials used
for construction of greenhouses (Fig. 10) differ depending
on the level of investment and range from expensive metals
and concrete structures to cheap bamboo columns and
plastic polymers (Sonnenholzner & Calderon 2004; Peng
et al. 2014), suggesting that the construction of greenhouse
is not always much costly. Primarily, the technology was
aimed at enhancing shrimp supply during winter but has
been critical in controlling WSSV by maintaining higher
water temperature in rearing units (Peng et al. 2014). Son-
nenholzner and Calderon (2004) noted that a temperature
difference between 3 and 5°C could be obtained in green-
houses compared with outdoor systems. In the same study,
it was also reported that the shrimps stocked in
Figure 10 Raceway ponds enclosed in a green house. The raceways
are covered with waterproof plastic sheet (arrow).(Source: McAbee
et al. 2003; World Bank 2014)
14
greenhouses did not suffer mortalities due to WSSV infec-
tion as it was the case in outdoor shrimp farms. The nurs-
ery system as a WSSV management tool enables shrimp
farmers to hold postlarvae in warm conditions in green-
houses to avoid the potential risk of WSD during cold sea-
son before stocking in outdoor grow out ponds is done
when conditions improve (World Bank 2014).
Shrimp polyculture
As defined by Mart
ınez-Porchas et al. (2010) polyculture is
the addition of one or more species on top of the main cul-
tured species. It can be direct, where both species share the
same unit; cage-cum-pond where the added species is put
in a cage or sequential where the added species are placed
in different interconnected units (Yi & Fitzsimmons 2004).
As a farming practice, polyculture provides a cheaper way
of producing an extra crop with the already existing
resources besides mitigating the problems associated with
the shrimp industry (Muangkeow et al. 2007; Troell et al.
2009). In South Korea, after the outbreak of WSD in 2004,
polyculture of shellfish, seaweeds and finfish was embraced
to cushion shrimp farmers against future losses because of
massive shrimp mortalities (Jang et al. 2007). The red algae
(Kappaphycus spp.) is commonly grown in polyculture with
shrimp and has been reported to possess antiviral and
antimicrobial properties (Trono 1999). However, its appli-
cation in WSD management has not been reported. Shrimp
polyculture seems to have huge potential, however, the fact
is that the polyculture is not a common practice; and this
culture system is rarely studied by the researchers. In a
report, the authors stated that polyculture of shrimp-fish
and shrimp-crab reduce the susceptibility of shrimp to dis-
eases. Several authors mentioned some fish, especially
omnivorous fish and crustacean species such as mullet, tila-
pia, Fugu spp., perch, sea bream and crab that are useful in
shrimp polyculture (see Shengli & Qinying 1996). In an
experiment, potential of polyculture in management of
WSD has been demonstrated where shrimps were stocked
with an omnivorous fish river puffer. By stocking L. van-
namei and Fenneropenaeus chinensis separately in monocul-
tures and polycultures with river puffer (Takifugu obscurus)
in shrimp ponds, higher survivals (Fig. 11) were observed
in the polycultures compared with monocultures. However,
the authors also reported that the application of the strat-
egy in Chinese shrimp (P. chinensis) did not provide simi-
lar benefits because Chinese shrimp are more susceptible to
WSSV compared with L. vannamei and F. chinensis (Jang
et al. 2007). Hence, the impact of polyculture in reducing
the WSD risk is appeared to be species specific. The expla-
nation of better performance in shrimp polyculture with
omnivorous fish species was given by the authors is that the
omnivorous fish would consume dead and moribund
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40
30
Survival (%)
20
10
0 WSSV-microbes synergism in infection in shrimp. Several
Lv Lv+P Fc Fc+P
Type of culture
Figure 11 Survival of L. vannamei and F. chinensis after WSSV out-
break in two different culture patterns. Lv = L. vannamei in monocul-
ture; Lv + P = L. vannamei in polyculture with T. obscurus; Fc =
F. chinensis in monoculture; Fc + P = F. chinensis in polyculture with
T. obscurus. (Source: Jang et al. 2007)
shrimp hence reducing the viral load and chances of shrimp
getting infection. Additionally, such types of polyculture
systems help in balancing the mini-ecology of shrimp
ponds (Shengli & Qinying 1996).
Moreover, efficiency of nitrogen utilization has been
recorded as higher in polyculture systems compared with
that in monoculture systems (Zhen-xiong et al. 2001). Thus,
polyculture systems ensure better management in water
quality. For instance in an experiment, integrated culture of
shrimp, shellfish and seaweed found to be beneficial, where
hairy cockle (Scapharca inaequivalvis) and seaweed
(Gracilaria spp.) contributed in reduction in total ammonia
nitrogen (TAN), total nitrogen (TN) and total phosphorous
(TP) concentrations from shrimp effluents by 61, 72 and
71% respectively (Bunting 2006). In another polyculture
experiment, tilapia has been reported to improve water qual-
ity by feeding on excess organic matter, and thus, to increase
shrimp production (Akiyama & Anggawati 1998). Wang
et al. (1998) reported the similar results in Chinese shrimp–
hybrid tilapia polyculture system. Furthermore, antibacterial,
antifungal and cytotoxic activity of mucus of many fish
(Magarinos et al. 1995; Hellio et al. 2002) [e.g. Siganus fus-
cescens, S. guttatus (Mart
ınez-Porchas et al. 2010), Ore-
ochromis urolepis, milkfish (Tendencia et al. 2004; Tendencia
et al. 2006)] may contribute in reducing bacterial load from
the culture water. In fact, in an experiment, genetically
improved farmed tilapia (GIFT) decreased luminous bacte-
rial counts and as a consequence increased the survival of
shrimp (P. monodon) Tendencia et al. (2006). But, to date,
concrete report is not available on the impact of polyculture
in controlling Acute Hepatopancreatic Necrosis Disease
(AHPND) or Early Mortality Syndrome (EMS), a critical
bacterial disease in shrimp farms. Some aquatic weeds such
as red seaweeds (Gracilaria salicornia and Gracilaria fisheri),
green seaweed (Caulerpa macrophysa) and brown seaweed
(Sargassum polycystum) were found to be capable of effec-
tively assimilating TAN, NO2 and NO3 (>40%) (Kaewsura-
likhit 1994; Khidprasert 1995). All these benefits of
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White spot syndrome virus in shrimp
polyculture do not prevent WSD directly, but perhaps do
partially at least by reducing stress in shrimps by ensuring
better water quality. In addition, antibacterial, antifungal
and cytotoxic activity of mucus of subordinate species in
shrimp polyculture may reduce shrimp’s susceptibility to
bacterial diseases and thus may also reduce the impact of
experiments revealed better performance in growth of
shrimp in mixed culture. For example Ghosh et al. (2016)
conducted an experiment on L. vannamei-Mugil cephalus
mixed culture in floating cages and found significantly
higher production of L. vannamei comparing with mono-
culture, where the species complemented each other and did
not interfere in their growth. Even, mixed culture of L. van-
namei at high density with M. cephalus offered optimal uti-
lization of the available space inside the cages. Likewise,
Yuan et al. (2010) recorded the best shrimp production,
FCR and shrimp survival rate, and net profit in L. van-
namei-Oreochromis spp. polyculture in tanks. The authors
recommended to manipulate further the feed and feeding of
the animals.
Despite advantages, there are several disadvantages of
shrimp polyculture to be mentioned. Antagonism between the
species may be claimed as one of the main problems of poly-
culture, where carnivorous fish can consume shrimps or
shrimps can attack other organisms (Mart
ınez-Porchas et al.
2010). But antagonism can be controlled using appropriate
polyculture, simple engineering (e.g. biomass, space, dissolved
oxygen) and proper sequencing of the ponds as described by
Chien and Tsai (1985). Focusing on shrimp polyculture,
Mart
ınez-C
ordova (1999) suggested not to stock shrimp at
intensive densities in the farm to reduce the possibility of
increased oxygen consumer biomass. Although this problem
could be solved through intensive aeration, however, the feasi-
bility and sustainability of this practice could be questionable.
In the economic aspect, polyculture may require compara-
tively higher initial investment because of infrastructure, aera-
tion, feed and human effort, but indeed, investment varies
with type of polyculture, the intensification and the design of
the system (Chien & Tsai 1985; Gonzales-Corre 1988).
Disinfection and batch production
Stocking ponds should be disinfected before starting a new
production cycle. In some small intensive production units,
the ponds are chlorinated between production cycles. Fur-
thermore, liming of larger ponds has been practiced in
many areas for disinfection purpose. Contamination from
nearby farms is a main problem facing shrimp growers
nowadays with disinfection of small ponds in areas with
many smaller producers exist in each area (Cock et al.
2015; Kumara & Hettiarachchi 2018). Lucas and Southgate
(2012) suggested that batch production system can reduce
15
B. K. Dey et al.
the risk of WSD in aquaculture systems. The authors have
also indicated that the batch production can give a chance
for shrimp growers to clear and disinfect all the ponds in
each area at the same time, and then to stock all shrimps as
a single batch. This production system may help larger
organizations, even with small ponds, to move towards
batch production (Lucas & Southgate 2012). However, this
is not normally feasible for the smaller producers unless
they are socially organized in such a manner that they can
manage many small units coherently (Olesen et al. 2015).
In Thailand, WSSV is prevented from getting into the
ponds by combining batch production and stocking with
SPF stocks in the hot dry season where higher temperature
and general growth conditions are not conducive for the
occurrence of the disease (Briggs et al. 2005). Correspond-
ingly in Malaysia, several authors reported that WSD is more
severe in the wet season compared with the dry season
(Oseko et al. 2006). These studies can indicate the best pre-
vention method of WSSV is simply not to stock during the
winter and monsoon season when the conditions are favour-
able for the occurrence of WSD outbreaks. Therefore, WSSV
can be prevented by combining a batch production system
with production cycles synchronized with environmental
conditions which not favourable for the disease outbreaks
(Briggs et al. 2005; Oseko et al. 2006; Olesen et al. 2015).
A wide variety of disinfectants are used in aquaculture.
Among which, most commonly used disinfectants are
formaldehyde, potassium permanganate, chlorine and chlo-
rine-containing compounds (e.g. calcium hypochlorite,
sodium chloride, benzalkonium chloride) and iodine (see
Rico et al. 2012). Overall, aquaculture disinfectants, how-
ever, are moderately to highly toxic to planktonic and
macroinvertebrates. For example chlorine disinfectants
(e.g. calcium or sodium hypochlorite) react with organic
matter, giving rise to significant concentrations of organic
chlorine compounds such as halogenated hydrocarbons,
which are highly toxic to aquatic life and are persistent
environmental contaminants (Emmanuel et al. 2004). In
addition, application of disinfectants to the farm water or
soil kills beneficial microbes along with harmful ones,
therefore, it can harm the microbial maturity of water. On
the other hand, disinfectants those negatively impact plank-
tons may harm green water technique in shrimp culture.
However, it is perhaps a matter of time to get back the
microbial maturity of culture water in the disinfected farm.
Immune stimulation
It is well known that the invertebrates only relay on their
innate immune defences as they do not have adaptive
immune system (see He et al. 2015; Li et al. 2019). How-
ever, evidence shows that invertebrate-haemocytes play a
crucial role both in their cellular and humoral immunity
16
(Lavine & Strand 2002; Vazquez et al. 2009). The cellular
immune responses include apoptosis, encapsulation,
phagocytosis and nodule formation, whereas the humoral
responses mediated by haemocytes that include the prophe-
noloxidase (proPO) system, the clotting cascade and secre-
tion of antimicrobial peptides (Ganz 2003; Li & Xiang
2013). Several research reported that WSD can be pre-
vented by enhancing immune system of the shrimps (Itami
et al. 1998; Thitamadee et al. 2014; Chen et al. 2016c; Sinu-
rat et al. 2016). Many studies showed that prebiotics and
probiotics are capable of controlling diseases in aquaculture
through their many beneficial activities associated to the
environment and the physiology of animals. Many prebi-
otics such as inulin, fructooligosaccharides, short-chain
fructooligosaccharides, mannanoligosaccharides, galac-
tooligosaccharides, xylooligosaccharides and arabinoxy-
looligosaccharides, isomaltooligosaccharides have
experimentally used in aquaculture, and to date several
commercial prebiotics are available (see Ringø et al. 2010).
Similarly, many beneficial Gram-positive and Gram-nega-
tive bacterial species, are probiotics, have been recom-
mended to use in aquaculture (see Hai 2015). Many of the
prebiotics and probiotics studied till date, can cause
immunostimulation along with other benefits in fish, shell-
fish and shrimp (see Ringø et al. 2010; Cruz et al. 2012)
(Tables 2 and 3). However, fish have extensively studied in
this regard, comparing with shrimp. Moreover, most of the
research on prebiotic and probiotic use in aquaculture deal
with bacterial infections (see Ringø et al. 2010). In contrast,
their use in controlling viral diseases is less studied. How-
ever, a few studies show notable benefits of prebiotic and
probiotic use in aquaculture in controlling viral diseases in
shrimp (Tables 2 and 3). For example in an experiment,
dietary administration of a prebiotic inulin decreased the
prevalence of WSSV in L. vannamei, however, there was no
difference between treatment and control groups in terms
of survival of experimental animal (Luna-Gonz
alez et al.
2012). Supplementation of a combination of a prebiotic
isomaltooligosaccharides and a probiotic Bacillus OJ
enhanced resistance of L. vannamei to WSSV and signifi-
cantly increased the survival recorded after 14 days of
postinfection (Li et al. 2009b). Some other probiotics such
as engineered Lactobacillus plantarum (Thammasorn et al.
2017), Bacillus PC465 (Chai et al. 2016) and Bacillus OJ (Li
et al. 2009b) significantly enhanced several immune param-
eters including transcription of penaeidin 3a, peroxinectin,
C-type lectin 3 and thioredoxin, haemocyanin, prophe-
noloxidase; transcription of crustin (Chai et al. 2016),
phagocytosis and acid phosphatase (Li et al. 2009b); and
developed resistance to YHV (Thammasorn et al. 2017)
and WSSV (Li et al. 2009b; Chai et al. 2016) and increased
survival of L. vannamei in experimental conditions (Li
et al. 2009b; Chai et al. 2016; Thammasorn et al. 2017).
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 White spot syndrome virus in shrimp

Table 2 Effects of prebiotics on the physiology of shrimp and lobster

Prebiotics Experimental species Dose Treatment Changes Survival References

period

Inulin from Agave tequilana Litopenaeus vannamei 2.5 or 5 g kg
1 62 or 73 d proPO↑; WSSV prevalence↓ NE Luna-Gonz
alez
et al. (2012)
Short-chain Litopenaeus vannamei 0.25, 0.5, 0.75, 6w PO↑; RB↑*; WG, FCR# NE Li et al. (2007)
fructooligosaccharides 1, 2, 4 or 8 g kg
1
Short-chain Litopenaeus vannamei 0.4, 0.8, 1.2 8w WG, SGR↑; FCR↓; ↑ Zhou et al.
fructooligosaccharides or 1.6 g kg
1 promote intestinal (2007)
microflora
Mannanoligosaccharides Homarus gammarus 20 ppt – Disease resistance↑ ↑* Daniels et al.
(2006)
Mannanoligosaccharides Penaeus semisulcatus 3 g kg
1 48 d Growth↑; FCR↓ ↑ Genc et al.
(2007)
Mannanoligosaccharides Litopenaeus vannamei Encapsulated 13 d THC, PO, RB, expression of ↑* Hamsah et al.
Artemia serine protein, peroxinectin, (2019)
lipopolysaccharide and b-1,
3-glucan-binding protein,
resistance to Vibrio harveyi
MR5339↑*
Isomaltooligosaccharides Litopenaeus vannamei 2 g kg
1 28 d Ph, PO, RB, AP↑*; intestinal ↑* Li et al.
(+ Bacillus OJ) Vibrio↓*; resistance to (2009a,2009b)
WSSV↑

AP, Acid phosphatase; d, Day; FCR, Feed conversion ratio; NE, No effect; Ph, Phagocytosis; PO, Phenoloxidase; proPO, prophenoloxidase; RB, Respira-
tory burst; SGR, Specific growth rate; w, Week; WG, Weight gain; #, No change; *, Significant change; ↑, Increase; ↓, Decrease.

Interestingly, a study revealed that the injection of meta-
bolic products of an actinomycetes isolate (Streptomyces
ghanaensis CHASH-2) is capable of inactivating WSSV in
shrimp, whereas the inactivation of WSSV was confirmed
by several techniques namely PCR, RT-PCR, Western blot
and ELISA. The results also showed a reduction in mortality
in experimental animals (Rajkumar et al. 2018).
Furthermore, immunostimulation can be achieved by
feeding with compounds containing pathogen-associated
molecular patterns (PAMPs) which are known to activate
the host innate immune system. The studies by Itami et al.
(1998) indicated that feeding P. japonicus with peptidogly-
cans over a period has increased the phagocytic activity of
granular cells of the host haemocyte leading to a significant
decrease in mortality upon WSSV exposure (Itami et al.
1998). Likewise, the other studies by Thitamadee et al.
(2014) revealed that injection of b-glucan prior to WSSV
infection has resulted in activation of the prophenoloxidase
system. The activation of this system has led to a reduction
in mortality (25–50% as compared to 100% in controls).
However, these studies revealed that repeated dosages of b-
glucan resulted in high mortality rates in the host. This
may be associated with excess generation of reactive oxygen
species (Thitamadee et al. 2014).
Since the side-effects of synthesized chemical (e.g. antibi-
otics) use to prevent diseases in aquaculture have been clar-
ified, herbal immunostimulants, as a better alternative, are
being studied in the field of disease control in aquaculture.
In an experiment, the addition of 4% of dried Undaria pin-
natifida (brown seaweed) in feed lowered the mortality in
L. vannamei challenged with WSSV, where the survival was
found to be much better (48%) compared with that of con-
trol (74%); however, the authors did not mention any
immunological parameters of the host (Schleder et al.
2018) and, a similar finding was reported by Niu et al.
(2018). In another experiment, post-vaccination supple-
mentation of extracted herbal immunostimulants from
Acalypha indica, Cynodon dactylon, Picrorrhiza kurrooa,
Withania somnifera and Zingiber officinalis showed a dose-
dependent significant survival (<60%) of P. monodon. In
the same experiment, supplementation of herbal immunos-
timulants provided with better performance in growth of
the experimental animal (Yogeeswaran et al. 2012). Dietary
administration of equally mixed methanolic extracts of five
different herbal medicinal plants namely Cyanodon dacty-
lon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa
and Eclipta alba at a rate of 800 mg kg
1 feed enhanced
haematological, biochemical and immunological parame-
ters; and significantly enhanced the survival (74%) in
WSSV infected shrimp, P. monodon (Citarasu et al. 2006).
Chang et al. (2018) reported the immunestimulating
capacity of Astragalus polysaccharides, whereas the supple-
mentation of this polysaccharides at a rate of 0.2 g kg
1
feed enhanced several immune parameters in shrimp suf-
fering WSSV challenge, however, the survival did not sig-
nificantly change (Chang et al. 2018). Therefore, herbal

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© 2019 Wiley Publishing Asia Pty Ltd 17
B. K. Dey et al.

Table 3 Effects of probiotics on the physiology of shrimp

Probiotics Experimental species Dose Treatment Physiological effects Survival Survival References
period checked

Engineered Shrimp 1011 CFU g
1 5d Resistance to YHV or AHPND↑ ↑* 8 dpi Thammasorn
Lactobacillus feed et al. (2017)
plantarum
Bacillus PC465 Litopenaeus 107 or 109 30 d WG↑*; transcription of ↑* 22 dpi Chai et al.
vannamei CFU g
1 feed penaeidin 3a, peroxinectin, (2016)
C-type lectin 3, thioredoxin,
haemocyanin, proPO↑*;
transcription of crustin↓;
resistance to WSSV
Bacillus OJ Litopenaeus 1010 CFU 28 d Ph↑; AP↑*; resistance to WSSV↑ ↑* 14 dpi Li et al.
vannamei g
1 feed (2009b)
Lactobacillus spp. Litopenaeus 5, 10 or 15% 21 d THC, Ph, PO, IgG, IgA, IgM↑*; – – Sandeepa
vannamei in feed resistance to WSSV↑ and Ammani
(2017)
Bacillus licheniformis Litopenaeus 1 9 106, 2 9 12 d Dose dependent changes: NE 20 dpi S
anchez-Ortiz
Mat32 + B. subtilis vannamei 106, 4 9 106 SGR↑*; up-regulation of SOD, et al. (2016)
MAt43 + B. subtilis or 6 9 106 proPO, LvToll and Hsp70 gene↑*;
GAtB1 CFU g
1 TGase gene↓#; WSSV and
feed (1:1:1) IHHNV prevalence↓*;
Bacillus S11 Penaeus – 100 d Resistance to Vibrio harveyi D331↑ ↑* 10 dpi Rengpipat
monodon et al. (1998)
Saccharomyces Penaeus 1% in feed 7w Resistance to Vibrio harveyi BP05# NE – Scholz et al.
cerevisiae, vannamei (1999)
Phaffia rhodozyma
Bacillus P64 or Litopenaeus 3% in feed 25 d Global immunity index↑* – – Gullian et al.
V. alginolyticus vannamei (2004)
Pseudoalteromonas Litopenaeus Encapsulated 13 d THC, PO, RB, expression of ↑* 5 dpi Hamsah et al.
piscicida or P. vannamei Artemia serine protein, peroxinectin, (2019)
piscicida + MOS lipopolysaccharide and b-1,
3-glucan-binding protein,
resistance to Vibrio harveyi
MR5339↑*

AHPND, Acute hepatopancreatic necrosis disease; AP, Acid phosphatase; d, Day; dpi, Day post infection; IHHNV, Infectious hypodermal and
haematopoietic necrosis; NE, No effect; Ph, Phagocytosis; PO, Phenoloxidase; proPO, prophenoloxidase; RB, Respiratory burst; SGR, Specific growth
rate; SOD, Superoxide dismutase; THC, Total haemocyte count; w, Week; WG, Weight gain; YHV, Yellow head virus; #, No change; *, Significant
change; ↑, Increase; ↓, Decrease.

treatment could open the door towards the control of WSD
in shrimp. However, multiple research have already been
conducted in controlling a wide variety of bacterial infec-
tions in fish using herbal products. But the effect of many
herbal immunostimulants in controlling WSD in shrimp is
yet to be unravelled.
Vaccination against WSSV
Invertebrates lack an adaptive immune system, and they
depend on innate immune defences (reviewed in He et al.
2015; Li et al. 2019). Thus, it is not possible to develop vac-
cine in the conventional way as it is done in mammals and
other vertebrates. However, some studies have provided
evidence that certain forms of pathogen-specific ‘immune
priming’ are possible in invertebrates (Musthaq & Kwang
2015; Momtaz et al. 2018). To date, many reports on vacci-
nation of shrimp against WSSV are available, and most of
which focused on WSSV protein subunit vaccines. Among
nearly 40 identified envelop proteins of WSSV, a few have
been extensively studied in terms of their potential in devel-
oping recombinant WSSV subunit vaccines. The WSSV
subunit vaccines might be categorized as monovalent and
multivalent vaccines (Feng et al. 2017). Furthermore, there
are also reports on inactivated whole WSSV vaccines and
DNA vaccines.
Targeting monovalent WSSV vaccine development, sev-
eral envelop proteins such as VP28, VP19, VP26, VP24,
VP292 and VP466 were focused, whereas, VP28, as a first
protein candidate, has had been extensively studied since it
is known to play an important role in early stage of virus
infection (van Hulten et al. 2000b 2001b). In an

Reviews in Aquaculture, 1–44

18 © 2019 Wiley Publishing Asia Pty Ltd

experiment, vaccination with VP28 enhanced the immunity
against WSSV in L. vannamei by significantly increasing
HSP70 and two serine proteases, chymotrypsin and trypsin
(Chen et al. 2016b). Aiming at vaccine production, various
expression systems have been developed nowadays, such as
bacteria, virus, algae, yeast and transgenic animals
(Tables 4 and 5). Among these systems, E. coli is the most
commonly used expression system that produces most
effective VP28 vaccine to date. However, all the expression
systems have some limitations described in Table 6. Along
with VP28, some other monovalent WSSV vaccine also
confer protection to shrimp (Table 4). For example in an
experiment, vaccine targeting VP26 envelop protein
showed 100% protection to shrimp against WSSV infection
(Satoh et al. 2008). But, unlike VP28, lack of effective deliv-
ery systems and practical pilot tests restrict such vaccines
from their commercial use in shrimp farming industry
(Feng et al. 2017). The characteristics of each subunit vac-
cine can be compared with respects to gene expression, the
form of the protein subunits used, the mode of administra-
tion and the level of protection. Focusing on different
administration methods, the chronology of effectiveness is
injection > immersion > oral vaccination (Jha et al. 2006;
Syed & Kwang 2011), but indeed, vaccination dose and
number-dependent survival also observed in many experi-
ments (Table 4). However, orally vaccinated shrimp also
show higher survival rates when challenge test also follows
oral pathway (Satoh et al. 2008). Although the injection
method of vaccines provides best protection, this adminis-
tration mode is not suitable for commercial scale opera-
tions, and therefore, improving the immune effect of
vaccines administered by the oral mode could be the best
practical choice for the shrimp farming industry. In fact,
this simple method saves time and effort, avoids any oper-
ating pressure, and easily applicable to small and large sized
animal (Kang et al. 2018). However, to date there is a lim-
ited success in vaccination through oral administration. An
antiviral vaccine report in 2014 revealed that only two of
more than 17 commercially available vaccines were orally
usable (Dhar et al. 2014), indicating a weak market of com-
mercial oral vaccine (Mutoloki et al. 2015). Furthermore,
commercialized oral vaccines are claimed to have low yields
because of their rapid degradation by gastric acid and other
digestive fluids before reaching the immune site (Mutoloki
et al. 2015). The development of high-quality oral vaccines
is complex and difficult because the immune mechanism
for oral vaccines is still to be clarified (Kang et al. 2018).
Hence, much studies is needed in the field of development
of amenable vaccination of shrimp against WSSV.
Aiming the enhancement of protective effects of vaccines,
polyvalent WSSV vaccines have been developed through
fusion expression or combine use of different monovalent
subunit vaccines. In a study, 100% protection rate of
Reviews in Aquaculture, 1–44
© 2019 Wiley Publishing Asia Pty Ltd
White spot syndrome virus in shrimp
shrimp was achieved by administrating CotB-VP28 fusion
protein vaccine (Ning et al. 2011). Therefore, CotC could
draw an attention to the researchers towards the develop-
ment of a novel vaccine usable in broad scale (Valdez et al.
2014). Moreover, several studies revealed better results in
combine use of monovalent vaccines instead of using them
separately (Jha et al. 2006 2007; Lee & Chen 2017)
(Table 5), urging an increased research efforts in this area.
Vaccination with inactivated whole WSSV shows success
in protecting shrimp against WSD (Table 7). In this prac-
tice, WSSV is inactivated by means of heat and binary
ethylenimine (BEI) (Zhu et al. 2009), formalin (Amar &
Faisan 2011), or gamma irradiation (Heidareh et al. 2014).
Among these inactivation practices, formalin-inactivated
WSSV is appeared to confer highest protection (100%) as
shown in the experiment conducted by (Singh et al. 2005)
where the vaccine was orally administrated. A recent study
revealed a 91% survival rate in L. vannamei vaccinated with
electron beam irradiated WSSV (Motamedi-Sedeh et al.
2017). However, despite having potential, this system still
lagging behind because of difficulties in mass production of
the virus (Feng et al. 2017).
DNA vaccines are also studied in several shrimp species;
and so far, many studies have demonstrated notable protec-
tion of DNA vaccines to shrimp against WSSV infection
(Table 8). Most of the DNA vaccine studies focused on
those that encode VP28, and revealed highest protection
rates (Table 8). DNA vaccination in shrimp enhances the
survival by means of significant stimulation of their
prophenoloxidase (proPO) and superoxide dismutase
(SOD) activity (Kumar et al. 2008), haemocyte phagocyto-
sis (Chotigeat et al. 2007), expression of lysozyme (Li et al.
2010) and immune-related proteins (Kono et al. 2009;
Musthaq & Kwang 2014). Furthermore, DNA vaccines are
advantageous due to its inexpensive cost and easy mainte-
nance as it is easy to mix transgenic E. coli (Mu et al.
2012), S. typhimurium (Ning et al. 2009) and baculovirus
(Rattanarojpong et al. 2016), the different delivery systems,
with feed. In addition, a study checked the effect of DNA
vaccination (VP28 and VP19 DNA constructs) on the
growth performance of shrimp, where no significant differ-
ence was observed between treatment and control groups
(Das et al. 2010) and, similar findings were reported by
Ning et al. (2009). Hence, this technique seems suitable for
the mass production of shrimp.
Gene silencing
In many eukaryotes, double-stranded RNA (dsRNA) is an
important regulator of gene expression. Therefore, different
types of gene silencing that are collectively referred to as
RNA silencing or RNA interference (RNAi) are triggered by
dsRNA. In the mechanism of RNAi, processing of dsRNAs
19
 Table 4 Trials of different monovalent vaccines against WSSV in shrimp

20
Protein Expression host Protein form Experimented Administration Vaccination Challenge Challenge Protection Survival References
species mode period mode rate checked
B. K. Dey et al.

VP28 E. coli Purified VP28 P. monodon Oral 7d Imm. 0 dpv 70% 15 dpc Witteveldt et al. (2004b)
VP28 E. coli Purified VP28 P. monodon Oral 7d Imm. 3 dpv 70% 14 dpc Witteveldt et al. (2004b)
VP28 E. coli Purified VP28 P. monodon Oral 7d Imm. 7 dpv 77% 14 dpc Witteveldt et al. (2004b)
VP28 E. coli Purified VP28 P. monodon Oral 7d Imm. 21 dpv 50% 14 dpc Witteveldt et al. (2004b)
VP28 E. coli Purified VP28 M. japonicus Oral (10 µg g
1) 15 d Oral 10 dpv 100% 30 dpc Satoh et al. (2008)
VP28 E. coli Purified VP28 M. japonicus Oral 15 d Imm. 10 dpv 71% 17 dpc Satoh et al. (2008)
VP28 E. coli Purified VP28 M. japonicus Oral 15 d Injc. 10 dpv 61% 29 dpc Satoh et al. (2008)
VP28 E. coli Purified VP28 P. clarkii Oral 25 d Oral 3 dpv 57% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 P. clarkii Oral 25 d Oral 21 dpv 51% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 3 dpv 60% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 21 dpv 60% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 3 dpv 54% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 21 dpv 54% 25 dpc Jha et al. (2006)
VP28 E. coli Purified VP28 L. vannamei Injc. 1st-(4 d)-2nd Injc. 3 dpv 0% 12 dpc Yang et al. (2012)
VP28 B. subtilis Transgenic B. subtilis spores P. clarkii Oral 20 d Imm. 3 dpv 68% 25 dpc Fu et al. (2008)
VP28 B. subtilis Transgenic B. subtilis spores P. clarkii Oral 20 d Imm. 14 dpv 78% 25 dpc Fu et al. (2008)
VP28 B. subtilis Transgenic B. subtilis spores P. clarkii Oral 20 d Imm. 28 dpv ~60% 25 dpc Fu et al. (2008)
VP28 B. subtilis Transgenic B. subtilis vegetative P. clarkii Oral 20 d Imm. 3 dpv 43% 25 dpc Fu et al. 2008;
VP28 B. subtilis Transgenic B. subtilis vegetative P. clarkii Oral 20 d Imm. 14 dpv 52% 25 dpc Fu et al. (2008)
VP28 B. subtilis Transgenic B. subtilis vegetative P. clarkii Oral 20 d Imm. 28 dpv ~40% 25 dpc Fu et al. (2008)
VP28 B. subtilis Transgenic B. subtilis F. chinensis Oral 20 d - 3 dpv 77% 25 dpc Fu et al. (2010)
VP28 B. subtilis Transgenic B. subtilis F. chinensis Oral 20 d – 14 dpv 83% 25 dpc Fu et al. (2010)
VP28 B. subtilis Transgenic B. subtilis F. chinensis Oral 20 d - 28 dpv 72% 25 dpc Fu et al. (2010)
VP28 E. coli Transgenic E. coli F. chinensis Oral 20 d – 3 dpv ~65% 25 dpc Fu et al. (2010)
VP28 E. coli Transgenic E. coli F. chinensis Oral 20 d – 14 dpv ~72% 25 dpc Fu et al. (2010)
VP28 E. coli Transgenic E. coli F. chinensis Oral 20 d – 28 dpv ~68% 25 dpc Fu et al. (2010)
VP28 E. coli Transgenic E. coli E. carincauda Injc. 1st-(7 d)-2nd Injc. 2nd & 3rd ~ 50% 7 dpc Sun et al. (2013)
-(7 d)-3rd vac. day
VP28 M13 Recombinant M13 phage L. vannamei Injc. 48 h Injc. 0 dpv 45% – Sol
ıs-Lucero et al. (2016)
filamentous
phage
VP28 Baculovirus Transgenic baculovirus P. monodon Oral 7d Imm. 3 dpv 82% 15 dpc Syed and Kwang (2011)
VP28 Baculovirus Transgenic baculovirus P. monodon Imm. 1st-(5 d)-2nd Imm. 3 dpv 75% 15 dpc Syed and Kwang (2011)
VP28 P. pastories rVP28 P. pastories (whole) P. clarkii Oral 25 d Oral 3 dpv 74% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (whole) P. clarkii Oral 25 d Oral 21 dpv 40% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (supernatant) P. clarkii Oral 25 d Oral 3 dpv 87% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (supernatant) P. clarkii Oral 25 d Oral 21 dpv 67% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (cells+PBS) P. clarkii Oral 25 d Oral 3 dpv 27% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (cells + PBS) P. clarkii Oral 25 d Oral 21 dpv 20% 25 dpc Jha et al. (2007)
VP28 P. pastories rVP28 P. pastories (sonicated) P. clarkii Oral 25 d Oral 3 dpv 87% 25 dpc Jha et al. (2007)

© 2019 Wiley Publishing Asia Pty Ltd

Reviews in Aquaculture, 1–44
 Table 4 (continued)

Protein Expression host Protein form Experimented Administration Vaccination Challenge Challenge Protection Survival References
species mode period mode rate checked

VP28 P. pastories rVP28 P. pastories (sonicated) P. clarkii Oral 25 d Oral 21 dpv 74% 25 dpc Jha et al. (2007)
VP28 Silkworm pupae HyNPV-VP28-infected pupae P. clarkii Oral 28 d Injc. 0 dpv 52% 20 dpc Wei and Yang (2011)
VP28 Silkworm pupae HyNPV-VP28-infected pupae P. clarkii Oral 28 d Injc. 0 dpv 43% 20 dpc Wei and Yang (2011);
VP28 D. salina Transgenic D. salina Crayfish Oral 10 d Imm. 0 dpv 41% 20 dpc Feng et al. (2014)

Reviews in Aquaculture, 1–44

VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 14 d Injc. 3 dpv 95% 15 dpc Caipang et al. (2008)
VP28 B. brevis Sonicated B. brevis P. japonicus Oral 14 d Injc. 3 dpv 10% 15 dpc Caipang et al. (2008)

© 2019 Wiley Publishing Asia Pty Ltd

VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 14 d Injc. 14 dpv 60% 15 dpc Caipang et al. (2008)
VP28 B. brevis Sonicated B. brevis P. japonicus Oral 14 d Injc. 14 dpv 56% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 14 d Injc. 7 dpv 57% 15 dpc Caipang et al. (2008)
VP28 B. brevis Sonicated B. brevis P. japonicus Oral 14 d Injc. 7 dpv 15% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (10 µg sh.
1) 14 d Injc. 3 dpv 68% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (1 µg sh.
1) 14 d Injc. 3 dpv 67% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 1d Injc. 1 dpv 77% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 3d Injc. 1 dpv 79% 15 dpc Caipang et al. (2008)
VP28 B. brevis Purified VP28 P. japonicus Oral (50 µg sh.
1) 7d Injc. 1 dpv 81% 15 dpc Caipang et al. (2008)
VP28 E. coli Purified VP28C, VP28N L. vannamei Oral (40 µg sh.
1) – – 7 dpv RPS 48%, 13% 14 dpc Qiu et al. (2012)
VP28 E. coli Purified VP28C, VP28N L. vannamei Oral (40 µg sh.
1) – – 14 dpv RPS 23%, 17% 14 dpc Qiu et al. (2012)
VP28 E. coli Purified VP28C, VP28N L. vannamei Injc. (40 µg sh.
1) – – 7 dpv RPS 30%, 8% 14 dpc Qiu et al. (2012)
VP28 E. coli E. coli Top 10 L. vannamei Oral – – 7 dpv RPS 8% 14 dpc Qiu et al. (2012)
VP28 E. coli E. coli Top 10 L. vannamei Oral - – 14 dpv RPS 10% 14 dpc Qiu et al. (2012)
VP28 B. choshinensis rVP28-MLV, rNVP28 M. japonicus Oral (25 µg sh.
1) 3d Injc. 3 dpv 68%, 55% 16 dpc Mavichak et al. (2010)
VP28 B. choshinensis rVP28-MLV, rNVP28 M. japonicus Oral (25 µg sh.
1) 7d Injc. 3 dpv 79%, 78% 16 dpc Mavichak et al. (2010)
VP28 E. coli Purified VP28 L. vannamei Injc. (10 µg g
1) Together Injc. – 8% 7 d pc Lee and Chen (2017)
VP28 E. coli Purified rVP28 C. clarki Oral (100 µg an.
1) 7d Injc. 1 dpv 40% 14 dpc Zhang et al. (2012)
VP28 E. coli Purified VP28 P. monodon Injc. 1st-(4 d)-2nd Injc. 7 dpv 77% 20 dpc Rout et al. (2007)
VP28 E. coli Purified VP28 P. monodon Injc. 1st-(4 d)-2nd Injc. 14 dpv 70% 20 dpc Rout et al. (2007)
VP28 E. coli Purified VP28 P. monodon Injc. 1st-(4 d)-2nd Injc. 25 dpv 17% 20 dpc Rout et al. (2007)
VP28 E. coli Purified VP28 P. monodon Injc. 1st-(4 d)-2nd Injc. 50 dpv 4% 20 dpc Rout et al. (2007)
VP28 - Purified VP28 P. monodon Injc. (5 µg sh.
1) Once Injc. 0 dpv 60% 14 dpc Aksono and Suprapto
(2018)
VP19 P. pastories rVP19 P. pastories (whole) P. clarkii Oral 25 d Oral 3 dpv 18% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (whole) P. clarkii Oral 25 d Oral 21 dpv 14% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (supernatant) P. clarkii Oral 25 d Oral 3 dpv 27% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (supernatant) P. clarkii Oral 25 d Oral 21 dpv 14% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (cells + PBS) P. clarkii Oral 25 d Oral 3 dpv 21% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (cells + PBS) P. clarkii Oral 25 d Oral 21 dpv 7% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (sonicated) P. clarkii Oral 25 d Oral 3 dpv 34% 25 dpc Jha et al. (2007)
VP19 P. pastories rVP19 P. pastories (sonicated) P. clarkii Oral 25 d Oral 21 dpv 23% 25 dpc Jha et al. (2007)
VP19 E. coli Purified VP19 P. monodon Oral 7d Imm. 0 dpv 7% 15 dpc Witteveldt et al.
(2004b)
White spot syndrome virus in shrimp

21
 22
B. K. Dey et al.

Table 4 (continued)

Protein Expression host Protein form Experimented Administration Vaccination Challenge Challenge Protection Survival References
species mode period mode rate checked

VP19 E. coli Purified VP28 P. clarkii Oral 25 d Oral 3 dpv 23% 25 dpc Jha et al. (2006)
VP19 E. coli Purified VP28 P. clarkii Oral 25 d Oral 21 dpv 8% 25 dpc Jha et al. (2006)
VP19 E. coli Purified VP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 3 dpv 47% 25 dpc Jha et al. (2006)
VP19 E. coli Purified VP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 21 dpv 53% 25 dpc Jha et al. (2006)
VP19 E. coli Purified VP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 3 dpv 17% 25 dpc Jha et al. (2006)
VP19 E. coli Purified VP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 21 dpv 14% 25 dpc Jha et al. (2006)
VP19 Sf21 insect cells Purified rVP19 P. chinensis Injc. Once Injc. 7 dpv 50% 15 dpc Ha et al. (2008)
VP19 Sf21 insect cells Purified rVP19 P. chinensis Oral Once Injc. 10 dpv 51% 15 dpc Ha et al. (2008)
VP19 - Purified VP19 P. monodon Injc. (5 µg sh.
1) Once Injc. 0 dpv 20% 14 dpc Aksono and Suprapto
(2018)
VP24 E. coli Purified rVP24 P. monodon Oral (25 µg g
1) 10 d Oral 1 dpv 64 10 dpc Thomas et al. (2014)
VP26 E. coli Purified rVP26 M. japonicus Oral (10 µg g
1) 15 d Oral 10 dpv 100% 30 dpc Satoh et al. (2008)
VP26 E. coli Purified rVP26 M. japonicus Oral 15 d Imm. 10 dpv 79% 17 dpc Satoh et al. (2008)
VP26 E. coli Purified rVP26 M. japonicus Oral 15 d Injc. 10 dpv 69% 29 dpc Satoh et al. (2008)
VP36B E. coli Purified VP36B L. vannamei Injc. 1st-(4 d)-2nd Injc. 3 dpv 0% 12 dpc Yang et al. (2012)
VP39B E. coli Purified VP39B L. vannamei Injc. (10 µg g
1) Together Injc. – 6% 7 dpc Lee and Chen (2017)
VP51B E. coli Purified VP51B L. vannamei Injc. (10 µg g
1) Together Injc. – 39% 7 dpc Lee and Chen (2017)
VP292 E. coli Purified rVP292 P. monodon Injc. (0.1 mg g
1) Once Injc. 30 dpv 37% 10 dpc Vaseeharan et al. (2006)
VP292 E. coli Purified rVP292 P. monodon Injc. (0.1 mg g
1) 1st-(19 d)-2nd Injc. 10 dpv 60% 10 dpc Vaseeharan et al. (2006)
VP466 Sf21 insect cells Purified rVP466 P. chinensis Injc. Once Injc. 7 dpv 48% 15 dpc Ha et al. (2008)
VP466 Sf21 insect cells Purified rVP466 P. chinensis Oral Once Injc. 10 dpv 11% 15 dpc Ha et al. (2008)

an., Animal; B. brevis, Brevibacillus brevis; B. choshinensis, Brevibacillus choshinensis; d, Day; D. salina, Dunaliella salina; dpc, Day post challenge; dpv, Day post vaccination; E. carincauda, Exopalamon
carincauda; E. carincauda, Exopalamon carincauda; F. chinensis, Fenneropenaeus chinensis; h, Hour; hpc, Hour post challenge; Imm. , Immersion; Injc., Injection; M. japonicas, Marsupenaeus japoni-
cas; P. chinensis, Penaeus chinensis; P. clarkii, Procambarus clarkii; P. japonicas, Penaeus japonicas; P. pastories, Pichia pastories; RPS, Relative percentage survival; sh., Shrimp.

© 2019 Wiley Publishing Asia Pty Ltd

Reviews in Aquaculture, 1–44
 Table 5 Trials of different polyvalent vaccine against WSSV in shrimp

Protein Expression host Protein form Experimental Administration Vaccination Challenge Challenge Protection Survival References
species mode period mode rate checked

Reviews in Aquaculture, 1–44

VP19 + VP28 P. pastories Mixture of VP19 and VP28 (whole) P. clarkii Oral 25 d Oral 3 dpv 49% 25 dpc Jha et al. (2007)
VP19 + VP28 P. pastories Mixture of VP19 and VP28 (whole) P. clarkii Oral 25 d Oral 21 dpv 27% 25 dpc Jha et al. (2007)

© 2019 Wiley Publishing Asia Pty Ltd

VP19 + VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 3 dpv 67% 25 dpc Jha et al. (2007)
(supernatant)
VP19 + VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 21 dpv 47% 25 dpc Jha et al. (2007)
(supernatant)
VP19 +VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 3 dpv 32% 25 dpc Jha et al. (2007)
(cells + PBS)
VP19 + VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 21 dpv 14% 25 dpc Jha et al. (2007)
(cells + PBS)
VP19 + VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 3 dpv 74% 25 dpc Jha et al. (2007)
(sonicated)
VP19 + VP28 P. pastories Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 21 dpv 67% 25 dpc Jha et al. (2007)
(sonicated)
VP19 + VP28 E. coli Mixture of VP19 and VP28 P. monodon Oral 7d Imm. 0 dpv 50% 15 dpc Witteveldt et al.
(2004b)
VP19 + VP28 E. coli Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 3 dpv 47% 25 dpc Jha et al. (2006)
VP19 + VP28 E. coli Mixture of VP19 and VP28 P. clarkii Oral 25 d Oral 21 dpv 43% 25 dpc Jha et al. (2006)
VP19 + VP28 E. coli Mixture of rVP19 and rVP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 3 dpv 63% 25 dpc Jha et al. (2006)
VP19 + VP28 E. coli Mixture of rVP19 and rVP28 P. clarkii Injc. (2 µg g
1) 7 h-(4 d)-7 h Oral 21 dpv 53% 25 dpc Jha et al. (2006)
VP19 + VP28 E. coli Mixture of rVP19 and rVP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 3 dpv 47% 25 dpc Jha et al. (2006)
VP19 + VP28 E. coli Mixture of rVP19 and rVP28 P. clarkii Imm. 7 h-(4 d)-7 h Oral 21 dpv 43% 25 dpc Jha et al. (2006)
VP51B + VP39B E. coli Mixture of rVP51B and rVP39B L. vannamei Injc. (20 µg[1:1] g
1) Together Injc. – 62% 7 dpc Lee and Chen (2017)
VP51B + VP28 E. coli Mixture of rVP51B and rVP28 L. vannamei Injc. (20 µg[1:1] g
1) Together Injc. – 43% 7 dpc Lee and Chen (2017)
VP39B + VP28 E. coli Mixture of rVP39B and rVP28 L. vannamei Injc. (20 µg[1:1] g
1) Together Injc. – 23% 7 dpc Lee and Chen (2017)
VP51B + E. coli Mixture of rVP51B, rVP39B L. vannamei Injc. (24 Together Injc. – 50% 7 dpc Lee and Chen (2017)
VP39B + VP28 and rVP28 µg[1:1:1] g
1)
rTAT-VP28 E. coli Purified rTAT-VP28 C. clarkii Oral (100 µg an.
1) 14 d Injc. 1 dpv 57% 14 dpc Zhang et al. (2012)
CotB-VP28 B. subtilis spores Transgenic spores L. vannamei Oral 7d Injc. 7 dpv 65% 14 dpc Nguyen et al. (2014)
CotB-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 0 dpv ~40% 15 dpc Ning et al. (2011)
CotB-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 7 dpv 50% 15 dpc Ning et al. (2011)
CotB-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 21 dpv 30% 15 dpc Ning et al. (2011)
CotC-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 0 dpv ~45% 15 dpc Ning et al. (2011)
CotC-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 7 dpv 53% 15 dpc Ning et al. (2011)
CotC-VP28 B. subtilis spores Transgenic spores C. clarkii Oral 7d Injc. 21 dpv 32% 15 dpc Ning et al. (2011)

an., Animal; C. clarkia, Cambarus clarkii; d, Day; dpc, Day post challenge; dpv, Day post vaccination; h, Hour; Imm., Immersion; Injc., Injection; P. clarkii, Procambarus clarkii; P. pastories, Pichia pasto-
ries.
White spot syndrome virus in shrimp

23
B. K. Dey et al.

Table 6 Main advantages and disadvantages experienced in VP28 targeting vaccine production systems

Expression systems Advantages Disadvantages References

Bacteria High protection to animal Formation of insoluble inclusion bodies; Satoh et al. (2008), Yang et al. (2012)
complicated purification
Virus High protection to animal Strict culture requirements; contamination Musthaq et al. (2009), Syed and Kwang
risks to the environment (2011), Feng et al. (2017)
Yeast Rapid cell division; high High mannose glycosylation of Jha et al. (2007), Feng et al. (2017)
yield for vaccine recombinant WSSV proteins
Transgenic High protection to animal Too long production period; very difficult preparation Wei and Yang (2011), Feng et al. (2017)
silkworm pupae
Dunaliella salina Moderate protection Low yield or inconsistent Wei and Yang (2011), Feng et al. (2014)

into short RNA duplexes of characteristic size and structure
is a key step (Meister & Tuschl 2004). Viral envelop pro-
teins are known to play significant role in infection as these
are necessary to bind with host cell surface (Zhang et al.
2004; Wu et al. 2005). In this context, inhibition of WSSV
gene expression or gene silencing, envelop protein encod-
ing genes, for instance through RNAi could be the new
approach to control viral disease in shrimp (Gitlin et al.
2002; Giladi et al. 2003; Lu et al. 2005; Xu et al. 2007; Atta-
sart et al. 2009; Mej
ıa-Ru
ız et al. 2011; Sanjuktha et al.
2012; Leu et al. 2013; Sangsuriya et al. 2014; Li et al. 2015a;
Yuan et al. 2016; Ufaz et al. 2018). To date, many WSSV
genes have already been used as target genes for RNAi neu-
tralization (Table 9). For example in an in vivo experiment
on P. japonicas a short (21 bp) interfering RNA targeting a
major envelop protein gene VP28 of WSSV has been used
to silence the VP28-specific gene (Xu et al. 2007; Ufaz et al.
2018). In an experiment, significant reduction in apoptotic
ratio of the haemocytes due to the knocked-down expres-
sion of apoptosis signal-regulating kinase 1 gene (LvASK1)
by RNAi has been observed in WSSV-infected L. vannamei.
In the same experiment, the cumulative mortality was
decreased in WSSV-infected L. vannamei due to the down-
regulation of LvASK1 (Yuan et al. 2016). Although this
silencing process is very sensitive, sequence variation as
small as 1 nucleotide can affect the silencing. However, this
technique has been found to reduce the viral DNA produc-
tion in infected shrimp and thus increased the survival (Xu
et al. 2007). Recently, silencing of a novel WSSV gene
(ORF89) has been found to reduce the viral replication sig-
nificantly and increased the survival of shrimp. Therefore,
the author represented WSSV ORF89 as a novel RNAi tar-
get against WSSV infection (Escobedo-Bonilla et al. 2015).
However, in some instances, long dsRNA were required
engaging antiviral mechanisms and gene silencing, whereas
short RNAs failed to induce similar responses (Robalino
et al. 2005; Liu et al. 2016). Hence, appropriately modify-
ing the structure and size of dsRNA molecules could be an
effective strategy for obtaining a high protection rate.
MicroRNAs (miRNAs), a class of small non-coding RNAs,
are known to regulate gene expression at post-transcriptional
level. Increasing evidence has shown that the expression of
miRNAs is affected during virus or bacterial infection and
upon stress in shrimp (Gonz
alez-Duarte & Garc
ıa-Carre~ no
2018). However, different miRNAs expressed differentially
in response to the pathogen. In a study, 31 miRNAs were
differentially expressed in response to the virus, 25 of them
were up-regulated and 6 of them were down-regulated
(Huang et al. 2012a). Another study revealed that miR-7 in
shrimp targets the 3’UTR of mRNA from viral ‘early gene’
wsv477 (involved in early DNA replication and virus prolif-
eration), and thus, plays crucial roles in the virus–host inter-
actions. Perhaps this knowledge can open a door for
controlling WSSV infection by regulating miR-7 (Huang &
Zhang 2012). The overexpression of another miRNA named
miRNA-965 showed a reduction in WSSV copies and infec-
tion-induced mortality by targeting the wsv240 gene, a gene
required for the WSSV infective process in shrimp (Shu
et al. 2016). Other two miRNAs named miR-71 and miR-
13b from M. japonicus were reported to simultaneously reg-
ulate autophagy, known to be involved in viral disease, and
WSSV infection (He et al. 2017). As the evidence shows that
miRNAs may play a role in the shrimp immune system
(Ruan et al. 2011) against virus infection (Kaewkascholkul
et al. 2016), therefore, most of the recent research on miR-
NAs’ role in shrimp has been focused in immune defence
mechanisms (Table 10). Recently, a novel miRNA called
miR-1959, identified from L. vannamei, could enhance the
activity of the promoter of WSSV immediate-early gene ie1.
Hence, inhibition of miR-1959 decrease the mortality of
WSSV-infected shrimp where the genome copies and expres-
sion of WSSV ie1 were decreased; and VP28 genes was
down-regulated (Xu et al. 2016). p38 mitogen-activated pro-
tein kinases (MAPKs) are found in the organisms ranging
from yeast to mammals; and take part in various cellular
responses to several stimuli. The activation of p38MAPKs
can be induced by the MAPK kinase 6 (MKK6). Interest-
ingly, silencing of MKK6 in L. vannamei lowered the virus
loads; and suppressed viral gene VP28 expression when chal-
lenged with WSSV (Li et al. 2016).
Since the mechanisms of RNAi in terms of viral disease
control were unravelled, several studies have had been

Reviews in Aquaculture, 1–44

24 © 2019 Wiley Publishing Asia Pty Ltd
 White spot syndrome virus in shrimp

Table 7 Trials of whole virus inactivated vaccines against WSSV infection

Inactivation method Experimented Administration Vaccination Challenge Challenge Protection Survival References
species mode period mode rate checked

Formalin-inactivated F. indicus Oral 7d Oral 1 dpv 0% 10 dpc Singh et al. (2005)

Formalin-inactivated F. indicus Oral 7d Oral 5 dpv 100% 10 dpc Singh et al. (2005)
Formalin-inactivated F. indicus Oral 7d Oral 10 dpv 100% 10 dpc Singh et al. (2005)
Formalin-inactivated F. indicus Oral 7d Oral 15 dpv 0% 10 dpc Singh et al. (2005)
Formalin-inactivated P. monodon Injc. (30 µg sh.
1) Once Injc. 3 dpv 83% 15 dpc Amar & Faisan (2011)
Formalin-inactivated P. monodon Injc. (30 µg sh.
1) Once Injc. 15 dpv 67% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Injc. (30 µg sh.
1) Once Injc. 30 dpv 33% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Imm. 2h Imm. 3 dpv 86% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Imm. 2h Imm. 15 dpv ~75% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Imm. 2h Imm. 30 dpv ~70% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Imm. 2h Imm. 45 dpv 66% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Oral (50 µg sh.
1) 14 d Imm. 30 dpv ~75% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Oral (50 µg sh.
1) 14 d Imm. 45 dpv 64% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Oral (50 µg sh.
1) 14 d Imm. 60 dpv 8% 15 dpc Amar and Faisan (2011)
Formalin-inactivated P. monodon Oral 7d Oral 1 dpv 100% 8 dpc Sudheer et al. (2015)
Formalin-inactivated P. monodon Oral 7d Oral 7 dpv 50% 8 dpc Sudheer et al. (2015)
Formalin-inactivated; P. monodon Oral 14 d Imm. – 73% 15 dpc Amar and Faisan
MSM (2011)
Formalin-inactivated; P. monodon Injc. Once Injc. 60 dpv 30, 50, 20 dpc Yogeeswaran et al.
herbal feeding 60% (2012)
Formalin-inactivated; P. monodon Oral 14 d Imm. – 73% 15 dpc Amar and Faisan
wheat grass (2011)
Heat-inactivated P. clarkii Injc. Once Injc. 7 dpv 10% 18 dpc Zhu et al. (2009)
Heat-inactivated P. clarkii Injc. Once Injc. 21 dpv 3% 18 dpc Zhu et al. (2009)
2 mM Binary P. clarkii Injc. Once Injc. 7 dpv 77% 18 dpc Zhu et al. (2009)
ethylenimine-inactivated
2 mM Binary P. clarkii Injc. Once Injc. 21 dpv 60% 18 dpc Zhu et al. (2009)
ethylenimine-inactivated
3 mM Binary P. clarkii Injc. Once Injc. 7 dpv 63% 18 dpc Zhu et al. (2009)
ethylenimine-inactivated
3 mM Binary P. clarkii Injc. Once Injc. 21 dpv 30% 18 dpc Zhu et al. (2009)
ethylenimine-inactivated
Electron beam L. vannamei Imm. Once Injc. – 75% 10 dpc Motamedi-Sedeh
irradiated et al. (2017)
Electron beam L. vannamei Injc. Once Injc. – 73% 10 dpc Motamedi-Sedeh
irradiated et al. (2017)
EBI-WSSV + Prebiotic L. vannamei Imm. Once Injc. – 91% 10 dpc Motamedi-Sedeh
Immunogen et al. (2017)
EBI-WSSV + Prebiotic L. vannamei Injc. Once Injc. – 82% 10 dpc Motamedi-Sedeh
Immunogen et al. (2017)

an., Animal; d, Day; dpc, Day post challenge; dpv, Day post vaccination; EBI, Electron beam irradiated; F. indicus, Fenneropenaeus indicus; h, Hour;
Imm., Immersion; Injc., Injection; MSM, Methyl sulfonyl methane; P. clarkia, Procambarus clarkia; sh., Shrimp.

conducted targeting the enhancement in survival of shrimp
(Table 9). In most of the studies, particular dsRNA was
injected into the shrimp, where up to 90% survival was
achieved. However, this technique protected shrimp maxi-
mum 25 days postinfection. In addition, injection of
recombinant ferritin protein reduced the viral (WSSV) load
in L. vannamei by inhibiting virus replication. The authors
presumed that the reduced iron availability by ferritin inhi-
bit the activity of ribonucleotide reductase and thus inter-
rupt the replication of virus genome (Ye et al. 2015). The
injection of recombinant downstream dynamin (Dnm)
proteins: Dnm1, Dnm2, or Dnm3 might inhibit WSSV
replication in the hosts (Huang et al. 2016). As the
injection practice is not commercially amenable comparing
with oral administration, therefore, extensive research is
necessary to speed up the process of vaccine maturation
and application in broad scale at the farm level. In this
regard, improvement in oral administration of dsRNAs
could possibly bring a revolution in protecting shrimp
against WSSV infection.
Quarantine measures
Since the transmission of WSSV between different geo-
graphical locations had been reported to occur by the trans-
port of live and frozen uncooked shrimp (Nunan et al.

Reviews in Aquaculture, 1–44

© 2019 Wiley Publishing Asia Pty Ltd 25
 Table 8 Comparison of different DNA vaccines against WSSV infection

26
Details of vaccine Expression vector Experimental Administration Challenge Vaccination Challenge Protection Survival References
species mode mode period rate checked
B. K. Dey et al.

VP28 DNA constructs pVAX1 P. monodon Injc. Injc. 1st-(4 d)-2nd 25 dpv 53% 20 dpc Rout et al. (2007)
VP28 DNA constructs pVAX1 P. monodon Injc. Injc. 1st-(4 d)-2nd 35 dpv 50% 20 dpc Rout et al. (2007)
VP28 DNA constructs pVAX1 P. monodon Injc. Injc. 1st-(4 d)-2nd 50 dpv 30% 20 dpc Rout et al. (2007)
VP28 DNA constructs pVAX1 L. vannamei Injc. (20 lg sh.
1) Injc. Once 7 dpv 53% 5 dpc Li et al. (2010)
VP28 DNA constructs pVAX1 L. vannamei Injc. (20 lg sh.
1) Injc. Once 14 dpv 20% 5 dpc Li et al. (2010)
VP28 DNA constructs pVP28-LH P. monodon Injc. (35 lg sh.
1) Injc. Once 1 dpv 75% 15 dpc Das et al. (2010)
VP28 DNA constructs pCMV-VP28-LH P. monodon Injc. (25 lg sh.
1) Injc. Once 24 hpv 58% 20 dpc Krishnan et al. (2009)
VP28 DNA constructs pCMV-VP28-LH P. monodon Injc. (35 lg sh.
1) Injc. Once 24 hpv 75% 20 dpc Krishnan et al. (2009)
VP28 DNA constructs pCMV-VP28-LH P. monodon Injc. (50 lg sh.
1) Injc. Once 24 hpv 79% 20 dpc Krishnan et al. (2009)
VP19 DNA constructs pCMV-VP19-LH P. monodon Injc. (10 lg sh.
1) Injc. Once 24 hpv RPS 29% 20 dpc Krishnan et al. (2009)
VP28 DNA constructs pcDNA3.1 P. monodon Injc. (30 lg sh.
1) – Once 7 dpv 0% 14 dpc Kumar et al. (2008)
VP28 DNA constructs pcDNA3.1 P. monodon Injc. (30 lg sh.
1) – Once 14 dpv 0% 14 dpc Kumar et al. (2008)
VP28 DNA constructs pcDNA3.1 P. monodon Injc. (30 lg sh.
1) – Once 21 dpv 0% 14 dpc Kumar et al. (2008)
VP28 DNA constructs pcDNA3.1 P. monodon Injc. (30 lg sh.
1) – Once 30 dpv 0% 14 dpc Kumar et al. (2008)
VP28 DNA constructs plasmid DNA-VP28 P. monodon Injc. (30 lg sh.
1) – Once 7 dpv 90% 14 dpc Kumar et al. (2008)
VP28 DNA constructs plasmid DNA-VP28 P. monodon Injc. (30 lg sh.
1) – Once 14 dpv 77% 14 dpc Kumar et al. (2008)
VP28 DNA constructs plasmid DNA-VP28 P. monodon Injc. (30 lg sh.
1) – Once 21 dpv 67% 14 dpc Kumar et al. (2008)
VP28 DNA constructs plasmid DNA-VP28 P. monodon Injc. (30 lg sh.
1) – Once 30 dpv 57% 14 dpc Kumar et al. (2008)
VP28 DNA constructs pCMV-VP28 P. japonicus Injc. (10 lg sh.
1) Imm. Once 7 dpv RPS 62% 12 dpc Kono et al. (2009)
VP35 DNA constructs pVAX1 P. monodon Injc. Injc. Once 25 dpv NP 20 dpc Rout et al. (2007)
VP281 DNA constructs pVAX1 P. monodon Injc. Injc. Once 25 dpv 50% 20 dpc Rout et al. (2007)
VP281 DNA constructs pVAX1 P. monodon Injc. Injc. Once 35 dpv 47% 20 dpc Rout et al. (2007)
VP281 DNA constructs pVAX1 P. monodon Injc. Injc. Once 50 dpv 34% 20 dpc Rout et al. 2007;
VP28 DNA constructs pCMV-VP28-LH P. monodon Injc. (10 lg sh.
1) Injc. Once 24 hpv RPS 50% 20 dpc Krishnan et al. (2009)
VP28, VP19 DNA constructs pVP28-LH:pVP19-LH P. monodon Injc. (17.5:17.5 lg sh.
1) Injc. Once 1 dpv 75% 15 dpc Das et al. 2010;
VP28, VP19 DNA constructs pVP28-LH:pVP19-LH P. monodon Injc. (25:10 lg sh.
1) Injc. Once 1 dpv 58% 15 dpc Das et al. (2010)
VP28, VP19 DNA constructs pleVP28, pleVP19 P. clarkii Injc. (20 lg an.
1) Injc. Once 5 dpv 86, ~35% 15 dpc Mu et al. (2012)
VP28, VP24 antisense constructs pH3-VP28, pH3-VP24 P. monodon Transfection (1 lg g
1) Injc. Once 2 dpv 80%, 80% 15 dpc Ahanger et al. (2014)
VP28 DNA constructs with Transgenic SISK cells line P. monodon Oral Oral 7d 7 dpv RPS 85% 14 dpc Rajeshkumar et al. (2009)
chitosan nanoparticle
VP28 DNA constructs with Transgenic SISK cells line P. monodon Oral Oral 7d 15 dpv RPS 65% 14 dpc Rajeshkumar et al. (2009)
chitosan nanoparticle
VP28 DNA constructs with Transgenic SISK cells line P. monodon Oral Oral 7d 30 dpv RPS 50% 14 dpc Rajeshkumar et al. (2009)
chitosan nanoparticle
Combination of VP28 and pVAX1 P. monodon Injc. Injc. Once 25 dpv 43% 20 dpc Rout et al. (2007)
VP281 DNA constructs
Combination of VP28 and pVAX1 P. monodon Injc. Injc. Once 35 dpv ~46% 20 dpc Rout et al. (2007)
VP281 DNA constructs
Combination of VP28 and pVAX1 P. monodon Injc. Injc. Once 50 dpv ~40% 20 dpc Rout et al. (2007)
VP281 DNA constructs
S. typhimurium expressing VP28 Transgenic S. typhimurium C. clarkii Oral Injc. – 7 dpv RPS 83% 15 dpc Ning et al. (2009)
S. typhimurium expressing VP28 Transgenic S. typhimurium C. clarkii Oral Injc. – 15 dpv RPS 67% 15 dpc Ning et al. (2009)
S. typhimurium expressing VP28 Transgenic S. typhimurium C. clarkii Oral Injc. – 25 dpv RPS 57% 15 dpc Ning et al. (2009)
Baculovirus displaying VP28 Recombinant Bac-VP28-dsrr2 L. vannamei Injc. – 1st-(4 d)-2nd 3 dpv 77% 14 dpc Rattanarojpong et al. (2016)

© 2019 Wiley Publishing Asia Pty Ltd

Reviews in Aquaculture, 1–44
 White spot syndrome virus in shrimp

al. (2011)
al. (2011)
al. (2011)
al. (2011)
Rattanarojpong et al. (2016)
Rattanarojpong et al. (2016)
1998; Stentiford et al. 2012), quarantine measures were ini-

an., Animal; C. clarkia, Cambarus clarkia; d, Day; dpc, Day post challenge; dpv, Day post vaccination; h, Hour; hpc, Hour post challenge; Imm., Immersion; Injc., Injection; NP, No protection; P. clarkia,
tiated to prevent the transmission of the pathogen. The pri-

Pathan et al. (2013)

References

mary purpose of quarantine is to prevent entrance of

Khimmakthong et
Khimmakthong et
Khimmakthong et
Khimmakthong et
Mu et al. (2012) pathogens like WSSV to areas where such a pathogen has
not been reported, and thus, to reduce the risk of diseases
caused by the pathogens.(Arthur et al. 2008). Currently,
quarantine measures has been suggested by the World
Organisation for Animal Health (OIE), and this measures
Within 14 dpc

may be applied to animals which are being moved within

checked
Survival

(between farms, villages, etc.) or between countries (OIE

14 dpc

15 dpc
14 dpc

15 dpc
15 dpc
15 dpc
15 dpc

2018). Quarantine procedures usually start by evaluating

the health status of the shrimp stocks and other susceptible
~85, ~41%
Protection

WSSV carriers and/or vectors to be imported; and the pro-

rate

cedures help to assess the risk of importation of certain

36%

83%

63%
87%
57%
0%

0%

commodities from a given area or source (Cock et al.

2015). To maintain quarantine measures at the national
Challenge

level, Geering et al. (1999) suggested to prevent uncon-

10 dpv
30 dpv
3 dpv
3 dpv
5 dpv
1 dpv

3 dpv
7 dpv

trolled entry of animals, animal products and other poten-

tially dangerous goods through the national boarder, and
1st-(4 d)-2nd
1st-(4 d)-2nd
Vaccination

to initiate a legal method to ensure sound animal health

period

certification and pre- and post-quarantine measures upon

Once

Once
Once
Once
Once

release at the boarder. Quarantine conditions should be

–

negotiated with the exporter countries to maintain pre-ex-

Challenge
mode

port testing and quarantine, animal health certification and

Injc.
Injc.

Procambarus clarkia; RPS, Relative percentage survival; S. typhimurium, Salmonella typhimurium; sh., Shrimp.

other necessary post-arrival inspections, testing and quar-

–
–

–
–
–
–

antine. Even, quarantine inspection of people along with

goods upon arrival at international airports and seaports;
Administration

and safe disposal of international aircraft and ship food-

Injc. (20 lg an.
1)

sh.
1)
sh.
1)
sh.
1)
sh.
1)
mode

waste through burning or deep burial (Geering et al. 1999).

Injc. (40 lg
Injc. (40 lg
Injc. (40 lg
Injc. (40 lg

In international aspect, the importing country may use risk

assessment to identify the level of possible risk of importing
Oral
Injc.
Injc.

animals from a given source or area; and the impact of dif-

ferent risk mitigation measures should be considered (OIE
Experimental

monodon
vannamei
vannamei

vannamei
vannamei
vannamei
vannamei
species

2018). Arthur et al. (2008) elaborately described various

clarkii

aspects of quarantine measures for aquatic animals (see

P.
P.

Arthur et al. 2008). Several countries across the planet

L.
L.

L.
L.
L.
L.

including EU (Karunasagar & Ababouch 2012) have been

Recombinant Bac-VP28-dsegfp

noticed to be strict to the introduction of new species and

Expression vector

stocks of shrimps; and strict quarantine services have been

Recombinant Bac-VP28

established in different regions to reduce the risk of WSSV

Transgenic E. coli
pCMV-His-PmAV

introduction via transportation of infected live animals and

shrimp products between two different geographical loca-
phMGFP
phMGFP
phMGFP
phMGFP

tions (Cock et al. 2015).

Specific pathogen-free (SPF) shrimps

E. coli expressing VP28, VP19
Baculovirus displaying VP28
Baculovirus displaying VP28

PmAV DNA constructs with

Specific pathogen-free (SPF) shrimps are those do not carry

Table 8 (continued)

chitosan nanoparticle

the specified pathogens like WSSV. There are many options

PAP DNA constructs
PAP DNA constructs
PAP DNA constructs
PAP DNA constructs

to get SPF populations, and to determine whether a given

Details of vaccine

shrimp population is free of WSSV. To check these condi-

tions, the shrimps may have been produced in an area or
zone free of the disease or the populations may have been
quarantined and tested by sensitive techniques such as PCR

Reviews in Aquaculture, 1–44

© 2019 Wiley Publishing Asia Pty Ltd 27
B. K. Dey et al.

Table 9 Application of RNA interference technology for anti-WSSV infection

dsRNA target Experimented Administration Vaccination Challenge Challenge Protection Survival References
gene species mode period mode rate checked

VP28 P. japonicus Injc. (8 lg sh.
1) – Injc. Simultaneously ~70% 14 dpc Xu et al. (2007)
VP28 M. japonicus Injc. Once Injc. 24 hpv 85% 25 dpc Sudhakaran et al.
(2011)
VP28 L. vannamei Injc. (4 lg sh.
1) Once Injc. Consecutive: 2, 87, 67, 50% 10 dpc Mej
ıa-Ru
ız et al.
12, 22 dpv (2011)
VP28 L. vannamei Injc. (4 lg sh.
1) Once Injc. 10, 20 dpv 37%, 13% 10 dpc Mej
ıa-Ru
ız et al.
(2011)
VP28 P. vannamei Oral 5d Injc. 0 dpv ~ 70% 7 dpc Thammasorn et al.
(2015)
VP28 P. monodon Oral 5d Oral 0 dpv 68% 15 dpc Sarathi
et al. (2008)
VP28, P. monodon Injc. (6 lg g
1) Once Injc. 24 hpv 90, 66.7, 93.3% 14 dpc Sanjuktha
VP281, rr1 et al. (2012)
VP28, VP19 L. vannamei Injc. Once Oral Simultaneously 85, 81% 10 dpc Robalino
et al. (2005)
VP26 L. vannamei Injc. (4 lg sh.
1) Once Injc. Consecutive: 79, 45, 31% 10 dpc Mej
ıa-Ru
ız et al.
2-12-22 dpv (2011)
VP26 L. vannamei Injc. (4 lg sh.
1) Once Injc. 10, 20 dpv 20, 0% 10 dpc Mej
ıa-Ru
ız et al.
(2011)
VP9, GFP M. rosenbergii Injc. (5 lg an.
1) Once Injc. 1st: 24 hpv; 1st: 80, 70%; 25; 18 dpc Feng et al. (2018)
2nd: 30 dpv 2nd: 67, 0%
VP9, GFP P. monodon Injc. (5 lg an.
1) Once Injc. 24 hpv 70, 10% 7 dpc Feng et al. (2018)
VP9, GFP M. japonicus Injc. (5 lg an.
1) Once Injc. 24 hpv 65, 20% 7 dpc Feng et al. (2018)
WSSV051 P. vannamei Oral 5d Injc. 0 dpv 40% 7 dpc Thammasorn et al.
(2015)
PmRab7 + rr2 P. monodon Injc. (5 + 0.6 5 d (once Injc. 3rd day of 1st ~95% 8 dpc Attasart
lg sh.
1) day
1) vaccination et al. (2009)
Non-coding L. vannamei Injc. Once Injc. Simultaneously 75% 10 dpc Robalino
region of et al. (2004)
BAC6

an., Animal; d, Day; dpc, Day post challenge; dpv, Day post vaccination; h, Hour; hpc, Hour post challenge; Imm., Immersion; Injc., Injection;
M. japonicas, Marsupenaeus japonicas; M. rosenbergii, Macrobrachium rosenbergii; P. vannamei, Penaeus vannamei; sh., Shrimp.

for the presence of the virus. The other option is the SPF
population may have been raised for several generations in
high biosecurity facilities declared to be free of WSSV
(Cock et al. 2015). Despite the multiple ways of generating
SPF populations, the FAO clearly states that the genuine
shrimps are those which are produced from high biosecu-
rity facilities, have been repeatedly examined and found
free of WSSV using intensive surveillance protocols. This
SPF population should originate from broodstock devel-
oped with strict founder population protocols. According
to FAO rules and regulations, if SPF shrimps are put any-
where else, for instance into a non-biosecure maturation
unit, hatchery or farm units, these animals can no longer be
named as SPF shrimps as they are now exposed to a high
risk of infection (Briggs et al. 2005).
The United States of Marine Shrimp Farming organiza-
tion has developed protocol to define SPF animals based
on OIE notifiable disease list, for instance like WSSV and
yellow head virus (YHV). According to this organization,
the SPF programme was not primary intended for quaran-
tine purposes. However, the programme was planned to
produce broodstock free of specific pathogens like WSSV
that could be distributed to producers. This SPF popula-
tion should be coupled with high level of bio-secure pro-
duction system (Moss et al. 2003; Lightner et al. 2009;
Kumara & Hettiarachchi 2018). In Colombia, different
studies have indicated that the offspring from SPF brood-
stock were highly susceptible to WSD, whereas farm mass-
selected populations repeatedly challenged by the disease
were highly resistant. This may indicate that SPR popula-
tions may have developed resistance genes to WSSV as
compared with SPF shrimps. Furthermore, several authors
have also found that in the absence of disease pressure the
offspring of the SPF broodstock grow faster than SPR
population. Thus, populations that are selected in the
presence of WSSV are more likely carry resistance genes to
the disease (Rassoulzadegan et al. 2006; Slotkin &
Martienssen 2007).

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28 © 2019 Wiley Publishing Asia Pty Ltd
 White spot syndrome virus in shrimp

Table 10 MicroRNAs related to immune response against WSSV in shrimp

Shrimp microRNA Role in immune response References

M. japonicus miR-7 Virus–host interaction by targeting the 30 UTR of mRNA from viral early gene wsv477 Huang and Zhang (2012)
M. japonicus miR-965 Inhibition of viral infection by targeting the viral wsv240 gene Shu et al. (2016)
M. japonicus miR-71, miR-13b Regulation of autophagy and WSSV infection He et al. (2017)
M. japonicus miR-12 Promote phagocytosis, apoptosis and antiviral immunity Shu and Zhang (2017)
through down-regulation of PTEN and BI-1 expression and the viral gene wsv024
M. japonicus miR-100 Regulation of the superoxide dismutase activity, haemocyte Wang and Zhu (2018)
phagocytosis and phenoloxidase activity to inhibits the progression of WSSV infection

M. japonicas, Marsupenaeus japonicus.

Ecuador exported 115 000 metric tonnes of shrimp in

Specific pathogen resistance (SPR) shrimps
Specific pathogen resistance (SPR) shrimps are populations
that are resistance to a single or multiple pathogens. SPR
shrimps usually result from a specific breeding programme
designed to increase resistance to a particular pathogen like
WSSV. Much work has been done on the selective breeding
of shrimp such as P. vannamei and P. stylirostris aiming at
increased growth rate and resistance to a variety of diseases,
with many positive results. However, most of the reports
discussed the resistance against TSV in shrimp. Most of the
TSV-resistant stocks currently available have documented
survival rates of 80–100% in laboratory challenge studies
with four TSV isolates (Briggs et al. 2005). However, the
survival rate differs between laboratory experiment and
field experiment. For instance in some experiments, SPR
strains of P. vannamei challenged with different isolates of
TSV showed survival rates of 55–100% where the survival
was 82% in ponds (Briggs et al. 2005). In another labora-
tory experiment, TSV resistance generation obtained
through selective breeding showed 18.4% increase in sur-
vival and additionally 21.2% increase in weight gain com-
pared with unselected control group (Argue et al. 2002).
Aiming at WSSV resist shrimp production, some work had
been done upon a strain of P. chinensis that is SPR for
WSSV, where survival rate was increased <45% in pond,
whereas laboratory challenge tests revealed up to 60%
improvement in survival for 3rd and 4th generation sur-
vivors (Kong et al. 2003). To the best of our knowledge, to
date WSSV-resistant P. monodon and P. vannamei, the
most commercially important shrimp species, are not com-
mercially available all over the world. But, successful pro-
duction of WSSV resistance shrimp would bring a
revolutionary change in shrimp culture especially in areas
where WSSV is endemic. For example 3–4 years of genetic
selection work on domesticated stocks of P. vannamei sur-
viving WSSV outbreaks resulted in enhanced resistance to
WSSV in Ecuador. Thus P. vannamei culture industries in
Central and South America were slowly recuperated after
the emergence of WSSV epidemic in 1999. For instance
1998, which dropped to only 38 000 metric tonnes in 2000
after the emergence of WSSV in 1999. Subsequently, in
2003, Ecuador exported an estimated 50 000 metric tonnes,
indicating a recovery to export (Briggs et al. 2004), perhaps
due to the success in SPR shrimp production those are
resistant to WSSV. As such, disease management measures
should be targeted more with SPR populations rather than
SPF populations. Epidemics of WSD may never develop in
populations that have a certain threshold level of resistance
even if the disease is introduced to the system. However,
the Epidemics of WSD can occur with SPF populations if
the disease is introduced to them (Chakrabarty et al. 2015;
Cock et al. 2015). For instance in Asia, in contrast to the
Americas, most shrimp’s growers do not have access to
SPR populations to WSD (Gitterle & Diener 2014). This
lack of SPR stocks has reduced both the effectiveness of
local exclusion and eradication programmes of WSD. This
problem also frequently exposes shrimp growers to the risk
of outbreaks of the disease (Gitterle & Diener 2014).
Several authors have suggested that good management
approach of WSD is to exclude it for as long as possible in
the production cycle or to slow the development of the dis-
ease outbreaks. Application of SPR populations which have
already developed resistance genes to WSSV infections is
one of the most effective means of slowing the development
of WSD epidemics (Gitterle & Diener 2014; Cock et al.
2015; Mallik et al. 2016). The studies by Olesen et al.
(2015) have indicated that the breeding programmes in
Asia should focus on breeding of SPR populations which
have resistance genes to WSSV. These SPR populations play
an important role for controlling and managing of WSD
epidemics (Olesen et al. 2015). The other studies by Briggs
et al. (2005) observed that Latin America is currently
almost entirely using SPR P. vannamei populations due to
their better performance in maturation, hatcheries and
grow-out ponds. Furthermore, these studies have also
revealed that these SPR populations may have more resis-
tance to a combination of diseases including WSD com-
pared with SPF populations that are more susceptible to

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© 2019 Wiley Publishing Asia Pty Ltd 29
B. K. Dey et al.
WSSV infection (Briggs et al. 2005). In Asia, some growers
have also started using disease-resistant or SPR populations
to WSSV aiming avoidance loss due to WSD (Briggs et al.
2005). Although the production of SPR population through
selective breeding has come with benefits in terms of dis-
ease protection, but cost-benefit report in this regard is not
available. Additionally, in the recent time, emergence of
AHPND is an extra pressure on shrimp farms. Hence, per-
haps it is a big challenge to successfully produce SPR
shrimp to both WSSV and AHPND causing Vibrio spp.
Stocking density
Lower stocking densities in shrimp culture can reduce
stress which leading to low disease incidence in aquaculture
industries. In the Pacific Coast of South America, according
to the studies of Cock et al. (2015), shrimp growers have
started raring shrimp at lower stocking densities comparing
with those they used to maintain before, aiming lessen the
prevalence of WSD. Indeed, lower stocking density finally
yields lower, but perhaps the farmers tend to compensate
the production by lowering the frequency and intensity of
devastating WSD by maintaining lower stocking density; in
fact, it seems impossible to exclude WSD completely from
the farm premises where WSD is endemic. However, the
production system in Asia is moving towards more inten-
sive systems with high stocking densities. This situation
often likely to make the animal stressed, and occurs dis-
tressing WSD incidence. Thus, generally low stocking den-
sity and WSD occurrence are reversely correlated,
particularly in the study of WSSV epidemics (Cock et al.
2015). Moreover, low stocking density contributes to
reduce the chance of host–pathogen interaction in addition
to reduced stress, thus reducing the risk of infection (Lucas
et al. 2019). Hence, combining good sanitary practices and
optimum stocking density might enhance the effectiveness
of policies on prevention and control of WSD (Cock et al.
2015).
Application of biofloc technology
Biofloc technology mainly follows the principle of waste
nutrients recycling, nitrogen in particular, into microbial
biomass that can be fed in situ by the cultured animals or
be harvested and processed into feed ingredients (Avnim-
elech 2009; Kuhn et al. 2010). This technology is a recent
aquaculture farming technique in aquaculture regarding as
not only a direct food source, but also immunity booster,
consequent detoxification of the system for cultured organ-
isms and others (Panigrahi et al. 2018) such as better water
quality. In an experiment, biofloc have been found to
enhance the transcription of immune-related genes in L.
vannamei (Soto-Alcal
a et al. 2018).
30
On the other hand, good management practices like low
water exchange, use of filters may also reduce the chances
of re-infection of ponds that have already been disinfected.
The application of biofloc technology (Hargreaves 2013;
Cock et al. 2015; Pilotto et al. 2018) with aeration systems
in aquaculture can reduce the water exchange rate to a
minimum level which can limit the chances for introduc-
tion of pathogens like WSSV during water exchange (Harg-
reaves 2013; Cock et al. 2015). In South East Asia, better
management of water exchange has been mainly used to
limit the epidemics of yellow head virus (YHV) and WSSV.
In this region, not only minimal water exchange was prac-
ticed, but also the sources of water intakes were located in
order to avoid the contaminated water with the viruses
from nearby farms (Lebel et al. 2002). Similarly, the other
studies by Hameed et al. (2003) indicated carrier vectors of
WSSV can enter shrimp ponds via pumped water, and
these vectors transmit the pathogen to the farmed shrimps.
Thus, management strategies that reduce water exchange
rates or rely in closed cycles and recirculation can reduce
the risk of occurring of the disease outbreak (Hameed et al.
2003). Moreover, biofloc technology has also been reported
to control bacterial diseases as well. For example in an
experiment, this technology conferred protection to P. van-
namei challenged with an AHPND strain of V. para-
haemolyticus, and survival rate was recorded as 86.7%,
whereas the survival was 86.7% in clear water (Shinn et al.
2018). Hence, biofloc technology has emerged as a promis-
ing technique towards disease control in aquaculture. Fur-
thermore, co-culture of shrimp with two species of
macroalgae Gracilaria vermiculophylla and Dictyota dichot-
oma enhanced several immune parameters such as superox-
ide dismutase (SOD) and catalase (CAT) after infection
with V. parahaemolyticus and WSSV. In that study,
macroalgae provided a nutritional benefit to the shrimps;
and improved ammonium concentration in co-culture sys-
tem (Anaya-Rosas et al. 2019).
Conclusion and recommendations
White spot syndrome virus is still a most significant patho-
gen in shrimp production and a major threat to shrimp
farming industry across the world. Nowadays, WSD has
received significant scientific attentions which are driven by
the commercial impacts of the disease. Globally, research is
being carried out aiming at a better understanding on biol-
ogy and pathology of WSSV and sub-sequential proper
treatment and prevention. However, further studies should
be focused in areas of more resistant host organisms to
understand their resistance mechanisms, and these knowl-
edge might be helpful to find a solution against WSD. In
endemic areas of the disease, SPR shrimps are more recom-
mended in farming compared with SPF animals. Thus,
Reviews in Aquaculture, 1–44
© 2019 Wiley Publishing Asia Pty Ltd

further research should also be carried out in breeding pro-
gramme to produce SPR animals against WSSV. Interest-
ingly, several studies have been conducted in the areas of
vaccination against WSSV, however, their field level appli-
cation is still at the grass root level. Therefore, another aim
could be the development of commercial application of
vaccine. However, the applications of new technologies
such as minimal water exchange, greenhouse and polycul-
ture techniques could be the promising practices towards
WSD management.
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