Aquaculture 451 (2016) 340–344

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Aquaculture

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Isolation and identification of a new isolate of lymphocystis disease virus

isolated from black rockfish (Sebastes schlegelii) in China
Fengrong Zheng a, Hongzhan Liu b,⁎, Xiangyun Guo a, Bo Wang a,⁎
a
First Institute of Oceanography SOA, Qingdao 266061, China
b
Marine College of Shandong University, Weihai 264209, China

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

Article history: Iridoviruses consist of five genera as follows: Ranavirus, Lymphocystivirus, Megalocytivirus, Iridovirus and
Received 31 August 2015 Chloriridovirus. Lymphocystis disease virus (LCDV) belongs to Lymphocystivirus, and it has been identified as
Received in revised form 21 September 2015 the causative agent of lymphocystis disease (LCD) in more than 100 different species of seawater and freshwater
Accepted 24 September 2015
fish. In recent years, the types of Lymphocystivirus-infected fish have been rapidly increased. In the present study,
Available online 26 September 2015
we isolated an iridovirus from nodules of LCDV-infected black rockfish, and it was named as LCDV-ss. On the skin,
Keywords:
fins and mouth of infected black rockfish, we observed a large number of irregular white nodules. Histophological
Lymphocystis disease virus analysis indicated that the nodules contained encapsulated hypertrophic cells and an enlarged nucleus. Through
Sebastes schlegelii (black rockfish) transmission electron microscopy, numerous viral particles with a diameter of 200 nm were observed in the
Major capsid protein cytoplasm, showing a hexagonally arranged pattern. Based on the major capsid protein (MCP) and DNA polymer-
Phylogenetic analysis ase genes, our phylogenetic analysis revealed that LCDV-ss was closely related to LCDV from China (LCDV-C),
DNA polymerase followed by LCDV-1. In summary, we, for the first time, provided molecular evidence that LCDV-ss was a novel
Iridovirus emerging iridovirus pathogen in cultured black rockfish.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction the molecular and pathological evidence of LCDV in black rockfish.

Lymphocystis disease virus (LCDV) is a virus with icosahedral sym-
metry and hexagonal profiles, and it has a diameter of 120–300 nm
and a DNA core, consisting of linear single- or double-stranded DNA
(Darai et al., 1983; Tidona and Darai, 1997; Kitamura et al., 2006a).
The Sebastes schlegelii, black rockfish (Scorpaeniformes: Sebastidae), is
widely distributed in China, Japan and North Korea. In addition, black
rockfish is considered as one of the most commercially important
marine fish due to its characteristics of delicious taste, suitableness for
intensive aquaculture and high nutritional value. However, black rock-
fish has been afflicted by serious disease in recent years, leading to se-
vere loss in its aquaculture production (Ju-Won et al., 2011; Chan-Il
et al., 2012). In the present study, we investigated lymphocystis disease
(LCD) in cultured black rockfish from Penglai aquarium farm in Shan-
dong Province, China, and about 20% of studied individuals displayed
symptoms of LCD. Moreover, we identified the pathogen of LCD in
black rockfish, which was designated as LCDV-ss. In addition, viral par-
ticles of LCDV-ss were examined using transmission electron microsco-
py (TEM), and its genotype was determined by DNA sequencing and
phylogenetic analysis. Taken together, we, for the first time, provided
⁎ Corresponding authors at: Ecology Laboratory of the First Institute Oceanography SOA,
6 Xianxialing Rd., Qingdao City, Shandong Province 266061, China.
E-mail addresses: hongzhan2002@163.com (H. Liu), ousun@fio.org.cn (B. Wang).
Moreover, ultrastructural and phylogenetic analyses on LCDV-ss
would bring new insights into the underlying mechanism of LCDV path-
ogenesis in black rockfish.
2. Materials and methods
2.1. Fish samples
The naturally infected black rockfish (mean weight of 500 g, n = 10)
were collected from the aquarium farms in Penglai and Qingdao,
Shandong Province, China. These black rockfish showed numerous nod-
ules on the skin or fins, which is the typical symptom of LCDV. Those
nodules were dissected from the skin and fins for pathological and ultra-
structural analyses.
2.2. Pathological study
The collected nodules were stripped and then fixed with 10% buffered
formalin solution at room temperature for 24 h. The fixed samples were
washed with distilled water and transversely sectioned into approxi-
mately 0.5-cm small pieces. Sections were then embedded in paraffin
and stained with hematoxylin and eosin (H&E) for histopathological
analysis according to a previously described method (Sheng et al., 2007).

http://dx.doi.org/10.1016/j.aquaculture.2015.09.026
0044-8486/© 2015 Elsevier B.V. All rights reserved.
 F. Zheng et al. / Aquaculture 451 (2016) 340–344
2.3. Ultrastructural study
The nodules were cut into 2 × 2-mm thick pieces and fixed with 2.5%
glutaraldehyde in phosphate-buffered saline (PBS) overnight in order to
determine whether viral particles existed in the wart-like lesions.
Subsequently, glutaraldehyde-fixed tissues were washed with PBS and
further fixed in 1% osmium tetroxide and 1% ferricyanide in PBS at
4 °C for 1 h. The specimens were dehydrated in graded series of ethanol
and embedded in Epon epoxy resin. Finally, epoxy-embedded sections
were stained with uranyl acetate and examined with a transmission
electron microscope (JEM-1200EX, JEOL, Japan).
2.4. Experimental infection
LCDV-ss-free black rockfish individuals (n = 30, three of them were
sacrificed to confirm the absence of LCDV-ss by PCR), with a mean
weight of 400 ± 20 g, were used to evaluate experimental infection.
The fish were obtained from an aquarium farm in Qingdao, Shangdong
Province, and kept in a tank with a flow-through, filtered and virus-free
water system. The tank was maintained at approximately 18–22 °C, and
its water quality was daily monitored. Fish of each group (n = 10) were
challenged with 0.1 mL purified virus or PBS (control) and then main-
tained for 50 days. Clinical signs of infection were daily monitored,
and the incidence was determined at 50 days post-challenge (p.c.). At
the end of the experiment, the nodules were dissected and prepared
as described in Section of 2.3 and examined by TEM.
2.5. PCR amplification and phylogenetic analysis
Genomic DNA of skin nodules was isolated for PCR amplification
using Tissue DNA kit (OMEGA, Bio-TEK, China). The expression levels
of major capsid protein (MCP) and DNA polymerase (DNA Pol) were
examined with gene-specific primers according to the sequences of
LCDV-1 and LCDV-C (Tidona & Darai, 1997; Zhang et al., 2004). The
primers employed in this study were as follows: MCP (F 5′-ATGACTTC
TGTAGCGGGTTCG-3′; R 5′-TACAGGAAATCCCATAGATCC-3′) and DNA
Fig. 1. Typical signs of lymphocystis in diseased black rockfish.
341
Pol (F 5′-CACTGCTTACTGGCTTA-3′; R 5′-CTGATATTTTACATGCTAA-3′).
PCR amplification was performed using Taq (Thermo, USA) according
to the manufacturer's instructions. Briefly, after a denaturation step at
95 °C for 3 min, the amplification was carried out with 35 cycles at a
melting temperature of 95 °C for 45 s, an annealing temperature of
53 °C for 45 s, and an extension temperature of 72 °C for 1 min. The
amplicons were purified and then sequenced by Shanghai Sang-gong
Biological Engineering Technology & Services Co., Ltd. (Shanghai,
China).
Homology comparison was conducted using the full-length se-
quence of MCP of LCDV-ss by BLAST of the National Center of Biotech-
nology Information (NCBI) (http://www.ncbi.nlm.nih.gov/BLAST).
Sequence assembly was performed using DNASTAR. The phylogenetic
tree based on MCP, DNA Pol genes and other 26 iridovirus isolates was
constructed using the neighbor-joining method (bootstrap value of
1000) by MEGA 5.0 software.
3. Results
3.1. Signs of LCD in black rockfish
Fig. 1A, B, C and D shows that although diffusely distributed through-
out the entire body of diseased black rockfish, the irregular white
nodules were predominantly localized on the caudal and pectoral fins,
opercular region, skin and mouth.
3.2. Pathological findings
Histopathological analysis revealed that many inclusion bodies were
strongly stained by H&E, exhibiting peripheral staining near the mem-
brane. Fig. 2 shows that lymphocystis cells squeezed each other, show-
ing irregular shape and enlarged nucleus. The size of these lymphocystis
cells was approximately 100 times greater than that of normal cells, and
lots of large basophilic inclusion bodies with irregular reticular forma-
tion were found in the cytoplasm.
342 F. Zheng et al. / Aquaculture 451 (2016) 340–344

Fig. 2. Histological sections of the nodules from the diseased black rockfish. (a) basophilic intracytoplasmic inclusion bodies, (b) magnification of lymphocystis cell, (c) irregular nucleus.

3.3. Ultrastructure of the viral particles
Fig. 3 reveals that the white nodules in black rockfish were
lymphocystis lesions, evidenced by that numerous hexagonal-shaped
viral particles (diameter of 200 nm) were found in nodules. These dis-
persed particles were localized in the cytoplasm of hypertrophied
cells, exhibiting a well-defined electron-lucent envelope with surface-
associated fibrils and an electron-dense nucleocapsid. In addition, the
viral particles displayed typical characteristics of iridovirus infection,
which were assembled and hexagonally packed into crystalline arrays.
The 200-nm viral particles consisted of three concentric layers (outer
envelope, hexagonal-shaped protein shell and inner membrane) and a
central DNA core.
3.4. Experimental infection
Experimental infection showed that lymphocystis cells were firstly
observed on the fins at 20 days p.c., and white particles were found in
the lesions. Moreover, TEM results indicated that size and shape of the
viral particles were the same as those of natural viral particles at
42 days p.c. Furthermore, the incidence of LCD reached 53% at 42 days

Fig. 3. Transmission electron micrographs of ultra-thin sections of skin nodules. The 200 nm-viral particles composed of three concentric layers: outer envelope, hexagonal profiles protein
shell (a) inner membrane (b) and central DNA core (c).
 F. Zheng et al. / Aquaculture 451 (2016) 340–344 343

p.c., while no signs were observed in the control group. Taken together,
we proposed that LCDV was the causative agent of LCD in black rockfish.
3.5. Phylogenetic analysis of LCDV-ss
To further ascertain the phylogenetic relationship between LCDV-ss
and other iridovirus isolates, we constructed the phylogenetic tree
based on the MCP and DNA Pol genes (Fig. 4). We cloned the full-
length sequence of MCP gene, and the accession numbers of MCP and
DNA Pol genes were KT630271 and KT438163, respectively. According
to the MCP-based phylogenetic tree, our newly identified LCDV-ss
from black rockfish was largely correlated with LCDV from China
(LCDV-C), KLDV-1 and LCDV-jf, followed by LCDV-1 and LCDV-rf
(Fig. 4A). Fig. 4B shows that based on DNA Pol gene, LCDV-ss, LCDV-1
and LCDV-C were clustered into one branch, and they were significantly
different from other Iridoviridae. Therefore, we hypothesized that
LCDV-ss could be a novel LCDV isolate in family Iridoviridae.
4. Discussion
As the causative agent of LCD, LCDV belongs to the genus
Lymphocystivirus (Alonso et al., 2005; Huang et al., 2007; Sheng et al.,
2007). LCD is widely spread in 125 different fish species from 34 differ-
ent genera of seawater and freshwater fish, resulting in serious
economic losses in aquaculture and severe decline in wild populations
of these species (Noga, 2010; Pirarat et al., 2011; Sheng et al., 2007;
Alonso et al., 2005). LCDV isolates have been increasingly identified
over recent decades from different fish species, such as Lateolobrax
japonic, Lutjanus argentimalulatus, Sparus aurata, Amphiprion ocellaris,
Rachycentron canadus and epinephelus (Chang et al., 2006; Zhang et al.,
1997; Hagit et al., 2008; Pirarat et al., 2011; Kitamura et al., 2006b)]. Re-
cent studies have identified LCDV-PF from the paradise fish Macropodus
opercularis (Xu et al., 2014) and isolated GLCDV from cultured grouper
(Huang et al., 2015). Moreover, LCDV-1 and LCDV-C have been
completely sequenced and annotated to explore the underlying molec-
ular mechanisms of LCDV pathogenesis (Tidona & Darai, 1997; Zhang
et al., 2004). In the present study, we found numerous nodules on the
skin or fins of diseased black rockfish. Furthermore, many inclusion
bodies were observed in the nodules of the diseased black rockfish by
histopathology. The typical characteristics of LCD mainly show
intracytoplasmic inclusions in the lymphocytic cells (Alonso et al.,
2005; Sheng et al., 2007). Moreover, our findings supported that viral
particles of LCDV-ss were the causative agents, morphologically evi-
denced by that numerous hexagonally arranged viral particles with a di-
ameter of 200 nm were observed in the nodules of infected fish. Based
on two core genes, our phylogenetic analysis indicated that LCDV-ss
was a novel isolate, like megalocytivirus, and it could cause disease in
black rockfish. In summary, we, for the first time, provided the

Fig. 4. Phylogenetic analysis of MCP (A) and DNA Pol (B) from different iridovirus isolates using MEGA 4.0. A. Phylogenetic analysis of MCP from different iridovirus isolates using MEGA 4.0.
GenBank accession numbers of the sequences were used for the MCP gene tree. LCDV-ss: KT630271, LCDV-rc: ABN71582, LCDV-sb: BAE78579, LCDV-1: AAC24486, LCDV-jf: AAW48180,
LCDV-rf: BAE46552, LCDV-C: AAS47819, LCDV-pf: AHW84100, LCDV-sa: ABP38209,LCDV-cb: BAF57228, LCDV-yp: ADF4257, LCDV: -tl: BAF57229, KLDV-1: AAP84612, RSIV: BAK14277,
STIV: ACF42314, RGIV: AFG73139, FV3: AAT09750, CMTV: AFA44920, TFV: AAL77814, ATV: AAP33191, EHNV: ACO25204, ECV: AFJ52305, GIV: AAV91066, SGIV: AAS18087, RBIV:
AAS44554, ISKNV: ADU25251, TRBIV: ADE34351, OSGIV: AAX82316, CGSV: AET51835, OFIV: ABY16712. B. Phylogenetic analysis of DNA Pol from different iridovirus isolates using
MEGA 4.0. GenBank accession numbers of the sequences were used for the DNA Pol gene tree. LCDV-ss: KT438163, LCDV-C: YP_073706, LCDV-1: NP_078724, STIV: ACF42281, RGV:
AFG73105, TFV: AAL77804, FV3: AAT09720, CMTV: AFA44952, ATSV: AAP33223, EHNV: ACO25234, ECV: AFJ52345, GIV: AAV91098, ADRV: AGV20578, OSGIV: AAX82331, RSIV:
O70736, RBIV: AGG37900, ISKNV: NP_612241, TRBIV: ADE34365, SGIV: AAS18143.
344 F. Zheng et al. / Aquaculture 451 (2016) 340–344
molecular evidence of LCDV in black rockfish, and our findings would
greatly contribute to diagnosis and control of viral diseases in black
rockfish.
Acknowledgments
This work was supported by the National Hightech Research and Devel-
opment (863) Program of China (No. 2012AA10A408, 2012AA10A413)
and the National Natural Science Foundation of China (No.41106147).
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