Fish & Shellfish Immunology 35 (2013) 1598e1603

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Fish & Shellfish Immunology

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Field trial tests of FKC vaccines against RSIV genotype Megalocytivirus

in cage-cultured mandarin fish (Siniperca chuatsi) in an inland
reservoir
Youyong Dong a, Shaoping Weng a, Jianguo He a, b, Chuanfu Dong a, *
a
MOE Key Laboratory of Aquatic Food Safety/State Key Laboratory for Bio-control, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275,
People’s Republic of China
b
School of Marine Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Megalocytiviruses are one of the most important causative agents in finfish industry in China, Japan and
Received 23 June 2013 South East Asia. The viruses are mainly composed of ISKNV, RSIV and TRBIV genotypes. Among them,
Received in revised form ISKNV genotype isolate is the most important causative agent in mandarin fish industry in South China.
28 August 2013
Since its first occurrence in mid-1990s in China, no effective drug has been developed to prevent and
Accepted 2 September 2013
Available online 11 September 2013
control this virus until our recent work. In this study, unusual RSIV genotype Megalocytivirus was
validated as the causative agent in natural mass mortality of cage-cultured mandarin fish in an inland
reservoir. One isolate was obtained using MFF-1 cells from natural mass mortality of mandarin fish and
Keywords:
Megalocytivirus
designated as Megalocyti-LJ2012. Based on two previous megalocytiviral isolates, formalin-killed cell
Infectious spleen and kidney necrosis virus (FKC) vaccines were prepared to immunize 2000 and 9000 cage-cultured mandarin in October 2011 and
(ISKNV) August 2012, respectively. As results, greater than 70% protective effects were observed in vaccination
RSIV genotype Megalocytivirus group in both individual field tests. Adjuvant-emulsified FKC vaccine provided even greater than 99%
Formalin-killed cell (FKC) vaccine protective effect (N ¼ 1000). In contrast, almost all fish died in non-vaccination group (N ¼ 1000).
Field trial test Immuno-protection test under laboratory condition showed that 100% relative percent survival was
obtained in surviving fish from vaccination group after challenge with Megalocyti-LJ2012 at 4 months
post vaccination. Taken together, the present study shows that FKC vaccine is also efficient in preventing
RSIV genotype Megalocytivirus in cage-cultured mandarin fish in two field tests.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction viral agents in both freshwater and marine finfish industry around
the Pacific region of Asia area in the past decade [10,11]. Phyloge-
Megalocytivirus, a large double-strands DNA virus with icosa- netic analysis showed that all members of megalocytiviruses could
hedral symmetry, is the newly defined piscine iridovirus within the be classified into three main genotypes, which were represented by
family Iridoviridae [1]. The first report of megalocytivirus-associ- ISKNV, RSIV and TRBIV [9,10,12]. ISKNV genotype Megalocytivirus
ated disease was documented in cage-cultured red sea bream mainly infected freshwater fish such as mandarin (Siniperca
(Pagrus major) in Japan in early-1990s and designated red sea chuatsi) and marbled sleepy goby (Oxyeleotris marmoratus) in
bream iridovirus disease (RSIVD) [2]. Then outbreaks of RSIVD-like mainland China [13,14], murray cod (Maccullochella peelii peelii) in
diseases in finfish were successively recorded in Australia [3], Australia [15], and some small-sized tropical ornamental fish
Singapore [4], mainland China [5], Chinese Taiwan [6], South Korea worldwide [16,17]. TRBIV genotype Megalocytivirus mainly infec-
[7] and other Southeast Asian regions or countries [8,9]. Literatures ted flatfish species such as flounder (Paralichthys sp.) and turbot
showed that megalocytivirus has become one of the most alarming (Scophthalmus maximus) [18e20]. RSIV genotype Megalocytivirus
mainly infected marine Perciformes fish species, the largest
member of marine fish species, known as red sea bream (Pagrus
major), rock bream (Oplegnathus fasciatus) and all kinds of groupers
(Epinephelus sp.) etc. [9].
* Corresponding author. School of Life Sciences, Sun Yat-sen University, No. 135,
Xingang Road West, Guangzhou 510275, People’s Republic of China. Tel.: þ86 20
Mandarin fish (Siniperca chuatsi), also known as Chinese perch,
84113792; fax: þ86 20 84113229. is a precious and economically valuable freshwater food fish species
E-mail address: [email protected] (C. Dong). nationwide in China, especially in South and East China. Infectious

1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fsi.2013.09.005
 Y. Dong et al. / Fish & Shellfish Immunology 35 (2013) 1598e1603 1599

spleen and kidney necrosis virus (ISKNV) is the most important identification. However, no pathogenic bacteria and parasite were
causative agent in mandarin fish aquaculture industry in Guang- detected from moribund fish (data not shown). In October 2011 and
dong province since its first record in pond cultured mandarin fish August 2012, when mass mortalities of mandarin fish occurred,
in 1994 [14]. To date, ISKNV was defined as the type species of the diseased fish were collected and sent to our laboratory in
genus Megalocytivirus because it was the first megalocytivirus for Guangzhou city for megalocytivirus detection.
which the full genomic sequence was determined and published
[1,21]. Besides ISKNV, mandarin fish was also evidenced experi- 2.3. Identification of megalocytivirus by PCR detection and
mentally to be a high susceptible host fish species to marine fish- phylogenetic analysis
originated RSIV genotype Megalocytivirus under artificial infec-
tion [22]. However, natural outbreak of RSIV genotype Mega- Total DNAs from 100 mg spleen tissues each from ten diseased
locytivirus in cultured mandarin fish is still rare case [12]. Based on mandarin fish were prepared using OMEGA tissue DNA extraction
a highly susceptible cell line for ISKNV, a formalin-killed cell (FKC) kit according to the kit protocol (OMEGA, China), and used as DNA
vaccine has been developed and shown high immuno-protection templates for PCR detection of megalocytivirus. Universal primer
effect against challenge with virulent ISKNV genotype Mega- set (MCP-F: 50 -TCA TTG TCA TCA TCA TGT CTG C-30 ; MCP-R: 50 -AGA
locytivirus in immunized mandarin fish under laboratory condition CAC ACG GGG CAA TC-30 ) specific for both ISKNV genotype and
[23]. In this study, RSIV genotype Megalocytivirus was demon- RSIV genotype megalocytiviral MCP gene was used to amplify the
strated for the first time as the causative agent in natural mass target gene similar to our previous description [22]. The amplifi-
mortalities of cage-cultured mandarin fish in an inland reservoir. cation products were selected to be cloned into pGEM-T Easy vector
Then FKC vaccines were assessed to prevent this disease in two and sequenced in ABI-377 DNA sequencer. The obtained sequence
individual field trial tests since October 2011. fractions were assembled using DNASTAR software and then run
BLAST program using NCBI nr database. Nucleotide data from
2. Materials and methods several selected typical megalocytiviral strains were firstly aligned
by using ClustalW program [26], and phylogenetic tree was con-
2.1. Cell line, viruses and antibody structed by neighbor-joining method with MEGA4.0 program [27].
Bootstrap sampling was resampling 1000 replicates.
MFF-1 cell line was developed and kept in our laboratory [24].
Megalocytivirus strains ISKNV-NH060831 and SKIV-ZJ07 were 2.4. Isolation and identification of Megalocyti-LJ2012 in MFF-1 cells
previously isolated from natural mass mortalities of pond cultured
mandarin fish (Siniperca chuatsi) in August 2006 and cage-cultured Virus isolation was performed similar to our recent isolation of
spotted knifejaw (Oplegnathus punctatus) in July 2007, respectively. OSGIV-HN11 [28]. Briefly, spleen and kidney tissues from diseased
Both viruses propagated well in MFF-1 cells and stored in 80  C fish were homogenized with 9 volumes of sterile phosphate buff-
until use [22,24]. MFF-1 cells were maintained in complete Dul- ered saline (PBS, pH7.4). The suspension was centrifuged at
becco’s modified Eagle’s medium (DMEM) containing 10% fetal 6000 g for 10 min at 4  C and then filtered through 0.45 mm filter
bovine serum (Invitrogen, USA), 100 IU/ml penicillin, 100 mg/ml membrane (Millipore, USA). Total 200 ml filtered suspension was
streptomycin, and 0.25 mg/ml amphotericin B (Invitrogen, USA) at added into a 25-cm2 confluent monolayer MFF-1 growing flask
27  C. Mouse anti-ISKNV-VP23 serum was prepared, characterized contained 5 ml DMEM medium plus with 10% fetal bovine serum
by Dr. Xu et al. [25] and kept in our laboratory. (Invitrogen, USA), 100 IU/ml penicillin, 100 mg/ml streptomycin, and
0.25 mg/ml amphotericin B (Invitrogen, USA). After absorption for
2.2. Mandarin fish samples 2 h at 25  C, the supernatant was removed and 5 ml maintaining
medium (DMEM plus 5% FBS, 200 IU/ml penicillin, 200 mg/ml
Cage-cultured mandarin fish in one fish farm in an inland streptomycin, and 0.5 mg/ml amphotericin B) was added. The
reservoir started in 2008, and 30,000, 4000 and 3000 mandarin fish infected MFF-1 cells were kept at 25  C for 5 days and observed
were cultured in 2008, 2009 and 2010, respectively (Table 1). Prior daily to monitor the cell morphological change. Two rounds of viral
to be transferred into cage culture in reservoir, mandarin fish were passages in MFF-1 cells were required to yield stable viral titer.
maintained in common pond culture to body weight of 13e20 g/per Indirect immunofluorescence assay (IFA) was carried out to assess
fish (approximate 500 g/30 fish) with live commercial fish bait. the viral replication in MFF-1 cells. Briefly, MFF-1 cells were grown
Water temperature maintained at 25e28  C. However, mass mor- in a 48-well tissue culture plate and then infected with Megalocyti-
talities always occurred during 20e30 days post culturing in cage. LJ2012 at an MOI of 5. When advanced CPE appeared, the infected
Thereafter, almost all fish died in the next 2 months. The diseased cell culture medium was removed, and the virus-infected cells were
fish were sent to a local fish disease laboratory for pathogen fixed with pre-cooled methanol (20  C) for 15 min at RT, followed
by washing twice with sterile PBST. The fixed cells were allowed to
air dry at RT. Mouse anti-ISKNV-VP23 serum at a dilution of 1:1000
Table 1
Culture data of cage-cultured mandarin fish since 2008 to 2012.
were applied to these wells and incubated at 37  C for 1 h. After
washing the wells twice with sterile PBST for 3 min each time,
Year Fish V or non-V Survival numbers Survival (%) in 3 Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen,
numbers in 3 months months post
post vaccination vaccination
USA) at a dilution of 1:200 was added and incubated for 1 h at 37  C.
2008 30 000 Non-V 0 0 After rinse twice with sterile PBST and once with sterile PBS for
2009 4000 Non-V 0 0 5 min each time, the Alexa Fluor 488-stained MFF-1 cells were
2010 3000 Non-V 0 0 observed and photographed under an inverted fluorescence mi-
2011 1000 Non-V 0 0
croscope imaging system (Nikon, Japan).
2000 V z1400 z70
2012 1000 Non-V 0 0
8000 V z6300 z78 2.5. Vaccine preparation and immunization
1000 V-adjuvant z990 z99

V, Vaccination group; Non-V, Non-vaccination group; V-adjuvant, adjuvant- Vaccine preparation was conducted similar to our recent report
emulsified vaccine. with some modification [23]. Briefly, confluent MFF-1 cells were
1600 Y. Dong et al. / Fish & Shellfish Immunology 35 (2013) 1598e1603

infected with ISKNV-NH060831 or SKIV-ZJ07 with a multiplicity of Twenty non-vaccination mandarin fish with similar body sizes from
infection (MOI) of 1 and incubated at 27  C. When advancement a local pond-cultured mandarin fish farm were used as an infection
CPE appeared, the whole cell suspensions were collected and viral control. MFF-1 cell-cultured Megalocyti-LJ2012 was diluted to
titer was determined by 50% tissue culture infective dose (TCID50) 105TCID50/0.1 ml and administered to challenge mandarin by intra-
assay as described earlier [29]. The cell suspension was stored peritoneally injection with 0.2 ml each fish. Water temperature were
at 80  C and kept for at least 24 h. After thawing at room tem- kept at 22e23  C. The fish were fed with commercial live fish bait and
perature (RT), formalin with a final concentration of 0.2% (v/v) was observed daily for 20 days. Dead or moribund fish were collected for
added to the cell suspension, mixed thoroughly and kept at 4  C for megalocytivirus detection by PCR method as above mentioned.
10 days. The formalin-containing cell suspensions with different
dilutions were inoculated with confluent MFF-1 cells in a 96-well 3. Results
culture plate to assess the viral activity. The inactivated virus sus-
pensions were diluted with sterile 0.85% NaCL solution to the 3.1. Validation of causative agents of mass mortality of cage-
equivalent of 106 TCID50/0.1 ml and kept 4  C until use. The virus is cultured mandarin fish in reservoir
no longer infectious at this stage. For adjuvant vaccine preparation,
the inactivated virus suspension was added 3% volume of tween-80 Diseased fish samples from mass mortalities of caged-
and mixed thoroughly. Then 2 fold volume of white oil adjuvant cultured mandarin fish in 2011 and 2012 were collected and
MARCOL 52 (ESSO, France) was added and emulsified thoroughly at sent to our laboratory for ISKNV-like virus detection. PCR
8000 rpm for 10 min at RT using an IKA homogenizer (IKA, investigation showed that all detected samples were strong
German). The prepared vaccines were stored at 4  C until use. positive (data not shown). The viruses from 2011 to 2012
Mandarin fish with body weight of about 500 g/30 fish were were designated as Megalocyti-LJ2011 and Megalocyti-LJ2012,
administered to immunize by intramuscular injection with 0.1 ml respectively. PCR products were sequenced and assembled
per fish. Before immunization, the fish were fasted for one day. The and the complete MCP genes (1362 bp) of Megalocyti-LJ2011
immunized fish were cultured in pond for 7 days and then trans- and Megalocyti-LJ2012 were obtained and deposited in Gen-
ferred into culture-cages in reservoir. The cage-cultured mandarin Bank nucleotide database (access numbers KC775380 and
fish were feed with fresh ice-stored fish bait. Total 2000 and 9000 KC775381, respectively). Blast comparison showed that MCP
mandarin fish were vaccinated in October 2011 and August 2012, genes of both Megalocyti-LJ2011 and Megalocyti-LJ2012 share
respectively (Table 1). The cage size (length, width and depth) is over 99% identities to that of SKIV-ZJ07 (access number
5  5  3 m3 and the largest design breeding capacity is 3000 kg GQ202216), a typical RSIV genotype Megalocytivirus from marine
fish for each cage. In first test, 1000 non-vaccination fish and 2000 cage-cultured spotted knifejaw [30]. In contrast to the MCP gene
vaccination fish were cultured in two neighbored cages, respec- of SKIV-ZJ07, one and two nucleotides substitutions were found
tively. In second test, 8000 non-adjuvant FKC vaccine immunized existence in those of Megalocyti-LJ2011 and Megalocyti-LJ2012,
fish were cultured in two cages with 4000 fish per cage. Non- respectively. However, on protein levels, LJ2011-MCP was
vaccination and adjuvant-emulsified FKC vaccine vaccination fish completely identical to SKIV-ZJ07-MCP, and only one amino acid
(N ¼ 1000, respectively) were cultured in other two cages, substitution was observed between Megalocyti-LJ2012-MCP and
respectively. When fish grew to over 250 g per fish, they were re- SKIV-ZJ07-MCP (Fig. 3). Furthermore, based on full-length MCP
assigned to 1000e1200 fish per cage until they were bred to nucleotide sequences, phylogenetic analysis also shows that both
commercial fish for sale. All fish were administrated under the Megalocyti-LJ2011 and Megalocyti-LJ2012 belong to RSIV geno-
same breeding management. type Megalocytivirus cluster (Fig. 4).

2.6. Artificial challenge using Megalocyti-LJ2012 3.2. Isolation and culturing of Megalocyti-LJ2012 in MFF-1 cells

After 4 months, the surviving fish in vaccinated group grew to Tissue sample from mass mortality of cage-cultured mandarin
200e350 g per fish. Twenty immunized fish (approximate 250 g/fish) fish in 2012 was performed for virus culture in MFF-1 cells. The CPE
were sent to our laboratory in Guangzhou city for immune- induced by Megalocyti-LJ2012 in MFF-1 cells (Fig. 1) is in high
protection assessment as previous description [23]. Prior to chal- accordance to those of other members of megalocytivirus growing
lenge, the fish were acclimated to laboratory conditions for 10 days. in MFF-1 cells [22,24,28]. Using mouse anti ISKNV-VP23 serum as

Fig. 1. Isolation and CPE profile of Megalocyti-LJ2012 in MFF-1 cells. A, Mock infected MFF-1 cells; B, Megalocyti-LJ2012 infected MFF-1 cells at 60 h post infection, characterized by
increasing enlarged rounding cells.
 Y. Dong et al. / Fish & Shellfish Immunology 35 (2013) 1598e1603 1601

Fig. 2. Monitor of Megalocyti-LJ2012 propagation in MFF-1 cells by indirect immunofluorescence assay (IFA). Entire CPE appeared at 72 h post infection. Mouse anti ISKNV-VP23
serum was used as the first antibody; Alexa fluor 488-coupled donkey anti-mouse IgG was used as second antibody.

first antibody, IFA showed that numerous enlarged rounding MFF-1 vaccine. SKIV-ZJ07 was used as vaccine strain. Another 1000 fish
cells can be well recognized and be stained with strong green were set as non-vaccination control. The surface water temperature
fluorescence under an inverted fluorescence microscope (Fig. 2). All maintained 30  2  C. Outbreak of ISKNV-like disease occurred at
these data indicated that Megalocyti-LJ2012 was successfully 21 days post cage culture. Afterwards all non-vaccinated fish died in
cultured in MFF-1 cells. 20 days. About 1300 non-adjuvant FKC vaccine immunized fish
died during the outbreak in 1 month. However, less than 10
3.3. Efficacy of FKC vaccines against RSIV genotype megalocytiviral adjuvant-emulsified FKC vaccine-immunized fish died during the
agent outbreak. A new RSIV genotype Megalocytivirus was isolated from
diseased fish using MFF-1 cells and designated as Megalocyti-
Two individual field tests were carried out to assess the pro- LJ2012. Megalocyti-LJ2012 could propagate well in MFF-1 cells
tection effects of FKC vaccines in cage-cultured mandarin fish since and showed high virulent to mandarin fish under experimental
October 2011 and August 2012, respectively. Although ISKNV-like infection (data not shown). To date, the surviving fish have been
megalocytivirus was highly suspected to be the causative agents cultured to large-sized commercial fish with body weight of 1000e
for mass mortalities of cage-cultured mandarin fish in reservoir 1500 g per fish since initial implement of the cage culture in August
since 2008, the previous diseased fish samples were not retained 2012. Artificial challenge showed that all immunized fish survived
for virus detection. In first field test in October 2011, ISKNV geno- against Megalocyti-LJ2012 infection under laboratory condition,
type strain ISKNV-NH060831 was used as a vaccine strain for but 100% non-vaccinated fish died due to challenge with
preparation of FKC vaccine and 3000 fish were administrated in this Megalocyti-LJ2012 (Fig. 5).
field test. Among them, 2000 fish were immunized with FKC vac-
cine, and 1000 fish were set as non-vaccination control. The surface 4. Discussion
water temperature maintained 27  2  C. Mass mortality of man-
darin fish started at 27 days post cage culture. After then, almost all Mandarin fish was previously evidenced a high susceptible fish
non-vaccinated fish (N ¼ 1000) died in 20 days. By contrast, about species to both ISKNV genotype and RSIV genotype Megalocytivirus
1400 vaccinated fish survived after counting at 3 month post under artificial infection [22,24,31]. A recent molecular investiga-
vaccination. The surviving vaccinated fish were cultured continu- tion also showed that RSIV genotype Megalocytivirus was detected
ously to Dec 2012 and grew to large-sized fish with body weight of in pond-cultured mandarin fish [12]. However, RSIV genotype
2000e3000 g per fish. Excluding other causes of death after the Megalocytivirus was never confirmed as the causative agents in
outbreak, about 1200 fish were yielded for sale. During outbreak, natural outbreak of iridovirus-associated diseases in mandarin fish
diseased fish were sent to our laboratory for pathogen identifica- industry. In most cases, freshwater-borne megalocytiviruses always
tion and an RSIV genotype Megalocytivirus was detected in all belong to ISKNV genotype [9,10,12,22]. In present study, we are first
sampled fish. The virus was designated as Megalocyti-LJ2011. to confirm that RSIV genotype Megalocytiviruses are the causative
The second field test was performed in August 2012, and total agents of mass mortality of cage-cultured mandarin fish in an
10,000 mandarin fish were involved in this test (Table 1). Among inland reservoir in 2011 and 2012 by molecular investigation, virus
them, 8000 fish were immunized with non-adjuvant FKC vaccine isolation and artificial challenge. Because diseased fish sample
and 1000 fish were immunized with adjuvant-emulsified FKC before 2010 was not retained for megalocytivirus detection, it is

Fig. 3. Multiple sequence alignment analysis of Megalocyti-LJ2011 and -LJ2012 with SKIV-ZJ07 based on the deduced amino acid sequences of their major capsid proteins (MCP).
The deduced MCP amino acid sequence of Megalocyti-LJ2011 is completely identical to that of SKIV-ZJ07. Comparison to Megalocyti-LJ2011 and SKIV-ZJ07, one amino acid mutant is
observed in the MCP sequence of Megalocyti-LJ2012. Arrow indicates the mutant amino acid site.
1602 Y. Dong et al. / Fish & Shellfish Immunology 35 (2013) 1598e1603

contrast, greater than 78% and 99% fish survived in non-adjuvant

FKC vaccination group and adjuvant-emulsified FKC vaccination
group, respectively. A megalocytivirus was isolated from the
diseased fish and two nucleotides variation were observed in MCP
gene comparison with that of SKIV-ZJ07. These results showed that
SKIV-ZJ07 was also a suitable vaccine strain for protection against
RSIV genotype Megalocytivirus.
To further determine the protectiveness, twenty survival fish in
non-adjuvant vaccinated group were sent to our laboratory for
artificial challenge test after 4 months of cage culture. The result
showed that all immunized fish survived against the challenge with
Fig. 4. Phylogenetic analysis of Megalocyti-LJ2011 and -LJ2012 with several other Megalocyti-LJ2012. Blood samples from five fish were prepared for
known typical megalocytiviruses based on the nucleotide sequences of their complete megalocytivirus detection by PCR method at 10 days post chal-
MCP genes. Both strains in this study are clustered into RSIV group. Circles indicate the lenge, and no positive results were obtained (data not shown).
newly detected megalocytiviral strains from cage cultured mandarin fish. Triangle
These data indicated the vaccinated fish have cleared up the
indicates an early RSIV genotype isolate from diseased marine cage-cultured spotted
knifejaw, based on which, FKC vaccines were prepared to immunize mandarin fish for injected Megalocyti-LJ2012. All non-vaccinated fish died in 16 days
the second field trial test and showed effective protectiveness against outbreak of post challenge (Fig. 5). All dead fish were detected strong positive
Megalocyti-LJ2012. results by PCR method (data not shown), which suggested all these
fish died of Megalocyti-LJ2012 infection.
Vaccine candidate strain is a very crucial factor for development
still uncertain whether RSIV genotype Megalocytivirus should be
and application of an effective cell-cultured vaccine [34]. Although
responsible for the early mass mortalities of cage-cultured man-
it is still unclear whether serological difference exists among
darin fish in reservoir.
different megalocytiviral strains, a previous report suggested that
Culture cell based vaccine against RSIV genotype Mega-
phenotypic diversity exists in different viral strains, even that those
locytivirus has been developed and commercially applied in Japan
strains belong to completely the same genotype cluster. The
[10,32]. In our recent report, a formalin-killed cell vaccine against
phenotypic diversity maybe lead to significant differences in viral
ISKNV was also developed and shown high efficacy against virulent
replication in vitro, virulence to host fish and viral antigenicity for
ISKNV [23]. In contrast to non-adjuvant RSIV vaccine in Japan,
vaccine development [34]. For instance, in Shinmoto et al.’s report,
adjuvant-emulsified ISKNV vaccine showed higher effective pro-
RSIV-U-6 based cell vaccine could achieve 69% protective effect in
tection to immunized fish under laboratory condition [23,33].
immunized red sea bream juveniles. However, only 11% protec-
However, the adjuvant-emulsified vaccine is quite inconvenient for
tiveness was done from that of RSIV-KST-Y1 [34]. In our previous
large-scale application by low dosage of injection inoculation in fish
report, ISKNV-NH060831 has been demonstrated to be a suitable
farm due to its enhancement viscosity. In first field test, non-
vaccine candidate viral strain against challenge with homogenous
adjuvant ISKNV vaccine was used to immunize cage-cultured
viral strain under laboratory condition [23]. In present study,
mandarin fish. Mass mortality occurred at 27 days post cage cul-
ISKNV-NH060831 based FKC vaccine also showed effective in pre-
ture in reservoir as well as before. During the outbreak, 70% of
vention of RSIV genotype Megalocytivirus associated disease, as
immunized fish survived. However, all non-vaccinated fish died
well as that of SKIV-ZJ07 did in two individual field trial tests. Be-
due to virus infection. SKIV-ZJ07-close megalocytivirus was
sides viral replication in MFF-1 cells and their virulence to man-
detected from the diseased fish. These results indicated that ISKNV
darin fish [22,24], proteomic profiles of both ISKNV-NH060831 and
vaccine, based ISKNV-NH060831, was effective in protection
SKIV-ZJ07 were also comprehensively determined in our recent
against RSIV genotype Megalocytivirus. In second field trial test,
reports [30,35], which will greatly benefit for better understanding
based SKIV-ZJ07, non-adjuvant and improvement adjuvant-
its infection and immunity of this virus species.
emulsified FKC vaccines were used to immunize 8000 and 1000
In conclusion, outbreaks of RSIV genotype Megalocytiviruses in
fish, respectively. Outbreak of diseases came as expected at 21 days
cage-cultured mandarin fish in reservoir were first to be confirmed.
post cage culture. During the outbreak, all non-vaccinated died. By
The outbreaks were effectively controlled by application of FKC
vaccines in two field trial tests. It is of great interest to perform
more field trial tests to assess the protective effect of FKC vaccine in
other fish species and other aquaculture manner.

Acknowledgments

The authors are grateful for the fish farm owner, Mr. D.M. Wu, to
kindly provide culture data from 2008 to 2010. This research was
supported by the National Basic Research Program of China under
grant No. 2012CB114406; the National Natural Science Foundation
of China under grant No. 31001122; Technology Planning Project
of Guangdong Province under number 2011A020102002 and
2012A020800006.

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