JOURNAL OF VIROLOGY, Jan. 2009, p. 347–356 Vol. 83, No.

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0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00707-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Novel C-Type Lectin from the Shrimp Litopenaeus vannamei

Possesses Anti-White Spot Syndrome Virus Activity䌤
Zhi-Ying Zhao,1† Zhi-Xin Yin,1 Xiao-Peng Xu,1 Shao-Ping Weng,1 Xia-Yu Rao,1 Zong-Xian Dai,1
Yong-Wen Luo,1 Gan Yang,1 Zong-Sheng Li,1 Hao-Ji Guan,1 Se-Dong Li,3 Siu-Ming Chan,4
Xiao-Qiang Yu,2* and Jian-Guo He1*
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingang Road West,
Guangzhou 510275, People’s Republic of China1; Division of Cell Biology and Biophysics, School of Biological Sciences,
University of Missouri—Kansas City, Kansas City, Missouri 641102; Fisheries Research Institute of Zhanjiang,
Zhanjiang 524039, Guangdong, China3; and Department of Zoology, the University of
Hong Kong, Pokfulam Road, Hong Kong SAR, China4
Received 29 March 2008/Accepted 6 October 2008

C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we
report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity
against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type
carbohydrate recognition domain with an EPN (Glu99-Pro100-Asn101) motif that has a predicted ligand binding
specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically
expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating
activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV
and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1
to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV
infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins
of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that
has direct anti-WSSV activity.

Lectins are a group of nonimmunogenic proteins possessing
at least one noncatalytic domain that binds reversibly to spe-
cific carbohydrates and usually agglutinates cells (16, 35). They
exist ubiquitously in animals, plants, and bacteria. Due to their
ability to bind to specific carbohydrates on the surfaces of
microorganisms, lectins have been regarded as primary candi-
dates for pattern recognition receptors (PRRs) in animal in-
nate immunity. Calcium-dependent (C-type) lectins, which
contain a characteristic carbohydrate recognition domain
(CRD) with two or three pairs of disulfide bonds, are believed
to mediate pathogen recognition and play an important role in
the clearance of pathogens in innate immunity (6, 15, 42, 47).
C-type lectins can be further classified into a number of sub-
groups based on their functions and structures (5, 7). Mam-
malian mannose-binding lectin (MBL), a liver-derived pluri-
potent serum C-type lectin, belongs to the collectin subgroup
and has been well studied. MBL is a PRR that can activate the
complement system through the lectin pathway (11). It has
been suggested that MBL plays an important role in host
defense as a primary immune response to pathogens (9, 38). In
* Corresponding author. Mailing address for Jian-Guo He: State
Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen
(Zhongshan) University, Guangzhou 510275, People’s Republic of
China. Phone: 86 20 8411 0976. Fax: 86 20 8411 3819. E-mail: lsshjg
@mail.sysu.edu.cn. Mailing address for Xiao-Qiang Yu: Division of
Cell Biology and Biophysics, School of Biological Sciences, University
of Missouri—Kansas City, Kansas City, MO 64110. Phone: (816) 235
-6379. Fax: (816) 235-1503. E-mail: Yux@umkc.edu.
† Present address: Hainan Provincial Fisheries Research Institute,
Haikou 570206, Hainan, China.
䌤
Published ahead of print on 22 October 2008.
addition, MBL has also been shown to link the innate immune
system to the acquired immune system (2, 10).
C-type lectins in invertebrates are also involved in innate im-
mune responses, including the promotion of phagocytosis (18,
28), nodule formation, encapsulation, melanization (20, 48), and
the activation of prophenoloxidase (48, 49). A number of C-type
lectins in invertebrates, particularly in insects and shrimps, have
been isolated and characterized (20, 25, 28, 29, 31, 36, 46–49). In
the shrimp Penaeus monodon, two C-type lectins containing a
single CRD have been characterized (28, 29). In Penaeus japoni-
cus, an N-acetylglucosamine (GlcNAc)-specific lectin and another
lectin have also been reported (21, 46). A C-type lectin in Litope-
naeus vannamei with two CRDs (LvCTL) is expressed, but only in
the hepatopancreas (31). In Fenneropenaeus chinensis, two C-type
lectins (Fclectin [FcCLec-1] and Fc-hsL [FcCLec-2]) have been
reported (25, 36). FcCLec-1 contains dual CRDs and is expressed
in hemocytes, while Fc-hsL contains only one CRD and is ex-
pressed specifically in the hepatopancreas. These shrimp C-type
lectins have ligand binding specificities for carbohydrates such as
N-acetylglucosamine and lipopolysaccharide, and they can agglu-
tinate erythrocytes and exhibit antimicrobial activity against some
bacteria and fungi. Moreover, many expressed sequence tags
(ESTs) encoding lectins have been identified in cDNA libraries
from healthy shrimps (P. japonicus, Litopenaeus setiferus, and L.
vannamei) (12, 14), and partial cDNAs for C-type lectins are also
obtained by subtraction using shrimps (L. vannamei) infected
with white spot syndrome virus (WSSV) and healthy controls
(51). However, almost all shrimp C-type lectins exhibit activity
mainly against bacteria or fungi, and little is known about the
effects of C-type lectins in response to viruses such as WSSV.

347
348 ZHAO ET AL.
The Pacific white shrimp, L. vannamei, is a commercially
important species of cultured penaeid shrimps in China, as well
as worldwide. With the rapid production and development of
L. vannamei worldwide, shrimp diseases have become wide-
spread and, in recent years, have threatened the shrimp indus-
try. WSS, which is caused by WSSV, is the most severely
damaging disease of shrimps and other crustaceans around the
world (13, 24, 26). The outbreak and spread of WSS have
resulted in the high mortality of farmed penaeid shrimps and
huge economic losses in many regions of the world, especially
in Southeast Asia (13, 24, 26, 39). The viral pathogen, WSSV,
is a member of a new genus, Whispovirus, in the Nimaviridae
family (41), with double-stranded DNA (about 300 kb) and
envelope proteins (39, 45). Since the first report of WSSV in
1993, major concerns in the world aquaculture industry have
been raised, and great effort has been made in preventing and
controlling the disease in recent years (24, 26, 39, 45). Even so,
shrimp defense mechanisms, particularly those against viruses,
are poorly understood.
The shrimp defense system is believed to rely largely on
innate immunity. The innate immune systems of shrimps con-
sist of PRRs that recognize and bind to specific patterns on the
surfaces of pathogens (3, 22). Previously, we employed sup-
pression subtractive hybridization technology to differentially
screen cDNA libraries from normal and WSSV-infected L.
vannamei shrimps by using WSSV-resistant L. vannamei as the
tester and WSSV-susceptible L. vannamei as the driver (51).
More than 1,000 qualified ESTs were obtained from the pos-
itive clones of the cDNA libraries. Many ESTs encoding C-
type lectins are upregulated in virus-resistant shrimps com-
pared to their levels of regulation in susceptible shrimps,
particularly in virus-resistant shrimps at 48 h postinfection
(51). Subsequently, several of these lectins have been cloned
and characterized.
In this study, we report a novel anti-WSSV C-type lectin,
CTL1, from the shrimp L. vannamei (LvCTL1). LvCTL1 con-
tains a single CRD. It exhibits hemagglutinating and sugar
binding activities as well as a strong affinity for WSSV. LvCTL1
binds to several envelope proteins from WSSV virions. More
importantly, LvCTL1 exhibits high antiviral activity against
WSSV both in vitro and in vivo.
MATERIALS AND METHODS
Cloning of full-length LvCTL1 cDNA. A partial cDNA sequence of LvCTL1
was obtained from the clone RS4E82, which was isolated from the subtraction
cDNA library described previously (51); the sequence was homologous to a
C-type lectin from P. monodon (PmAV; GenBank accession no. AAQ75589)
(29). Based on the identified EST sequences, gene-specific primers (GSP1 and
GSP2) were designed for the rapid amplification of 3⬘ and 5⬘ cDNA ends
(RACE) to amplify the full-length cDNA of LvCTL1. Then, gene-specific prim-
ers GSP3 and GSP4 were designed to confirm the full-length cDNA of LvCTL1
from 3⬘- and 5⬘-end-overlapping fragments (Table 1). RACE was carried out
using a BD SMART RACE cDNA amplification kit (Clontech, CA), using the
adapter-ligated cDNA, which was synthesized from total RNA of the hepato-
pancreas according to the manufacturer’s instructions. DNA was sequenced with
an ABI 3700 DNA sequencer (Perkin-Elmer/Applied Biosystems), and the re-
sulting sequences were subjected to cluster and homology analyses.
Sequence analysis. The similarity analysis of the nucleotide and amino acid
sequences of LvCTL1 was carried out using the BLAST programs at the NCBI
(http://www.ncbi.nlm.nih.gov/blast). Translation of the cDNA was performed
using the Expert Protein Analysis System (http://au.expasy.org/). Signal sequence
and CRD domain predictions were conducted using SMART software. Shrimp
C-type lectins used for comparisons include PmCLec-1 (GenBank accession no.
J. VIROL.
TABLE 1. Primers used for this experiment
Primer name Sequence (5⬘–3⬘)
LvCTL1 cDNA cloning
GSP1......................................CTGTCCTTACCCCTACGAGCCC
TTGG
GSP2......................................GCTCCTAACCAGAAGTGCTTGCC
GSP3......................................TGTCGACACTTGACGCAAGACC
GSP4......................................CAGTACTATTCAATATTTCTT
GAGG
RT-PCR
LvCTL1-F .............................TCTGTCGCCAGTGTTCGTTC
LvCTL1-R.............................CGTATTCCCACTATTAGGCTC
␤-actin-F................................AGATGTGTGACGACGAAGTA
GCCG
␤-actin-R ...............................TCACGAACGATTTCTCGCTCG
Recombinant expression
Re-F.......................................CGG/GATCCAATGACTGTCCTTA
CCCCTACa
Re-R ......................................TCCCC/CCGGGTTAATATTTCTTG
AGGCAAATGb
a
The BamHI restriction site is underlined.
b
The XmaI restriction site is underlined.
ABI97373), PsCLec from Penaeus semisulcatus (accession no. ABI97372), Lv-
CLec (accession no. ABI97374), FcCLec-1 from F. chinensis (accession no.
AAX63905), FcCLec-2 (accession no. ABA54612), PmAV from P. monodon
(accession no. AAQ75589), and PmCLec-2 (accession no. AAZ29608). Multiple-
sequence alignment of the LvCTL1 CRD with CRDs from other shrimp C-type
lectins was performed with the ClustalW multiple-alignment software.
Expression of LvCTL1 in tissues. Total RNA was prepared from the hepato-
pancreases, hemocytes, gills, eyestalks, muscles, brains, hearts, pyloric ceca,
nerve cords, intestines (midgut), spermaries, and stomachs of healthy WSSV-
susceptible L. vannamei shrimps. All of the RNA samples were treated with
DNase (Promega) to remove contaminating DNA, and the single-stranded
cDNAs were synthesized using Moloney murine leukemia virus reverse trans-
criptase (RT; Promega) with an oligo(dT)18 primer by following the manufac-
turer’s instructions. Equal amounts of cDNAs from the shrimp tissues were used
as templates for PCR. The LvCTL1 cDNA fragment was amplified with primers
LvCTL1-F and LvCTL1-R (Table 1), using the following conditions: initial
denaturation at 94°C for 3 min and then 30 cycles of 94°C for 30 s, 55°C for 30 s,
and 72°C for 50 s, followed by a final extension at 72°C for 8 min. The consti-
tutively expressed ␤-actin gene was amplified with the specific primers ␤-actin-F
and ␤-actin-R (Table 1) and used to normalize PCRs. The PCR products were
analyzed on a 1% agarose gel and sequenced to confirm the identity of the
LvCTL1 cDNA.
Expression and purification of recombinant LvCTL1. A cDNA fragment en-
coding a mature LvCTL1 protein (residues 21 to 156) (Fig. 1A) was amplified by
PCR using Taq polymerase (Promega) with the specific primers Re-F and Re-R
(Table 1). BamHI and XmaI restriction sites were added to the 5⬘ ends of Re-F
and Re-R (after the stop codon), respectively (Table 1). The PCR fragment was
cloned into the pGem-T easy vector (Promega), completely digested with BamHI
and XmaI (NEB, United Kingdom), and then cloned into the BamHI/XmaI sites
of the expression vector pQE-30 (Qiagen, Germany). The recombinant plasmid
(pQE-30-LvCTL1) was transformed into competent Escherichia coli M15(pREP4)
(Qiagen, Germany) cells for expression of recombinant proteins. Bacterial cells
expressing recombinant proteins were then harvested and sonicated, and the inclu-
sion bodies were resuspended in 1⫻ phosphate-buffered saline (PBS) containing 8
M urea. Recombinant LvCTL1 protein was purified by affinity chromatography with
Ni-nitrilotriacetic acid-agarose (Qiagen, Germany) under denaturing (8 M urea)
conditions according to the manufacturer’s instructions (Qiagen, Germany). Purified
recombinant protein was dialyzed against 1⫻ PBS-glycerol buffer (PBS with 5%
glycerol, pH 7.0) at 4°C overnight. The resulting protein was analyzed by 15%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visu-
alized with Coomassie brilliant blue R-250.
Production of antibodies. The polyclonal antibody against recombinant
LvCTL1 was produced by immunizing BALB/c mice according to the conven-
tional method (34). The titer of the antiserum was then determined by an
enzyme-linked immunosorbent assay (ELISA) to determine the optimal working
 FIG. 1. L. vannamei LvCTL1 cDNA and amino acid sequences. (A) Full-length cDNA and deduced amino acid sequences. The amino acid sequence
is represented by a single capital letter below the nucleotide sequence (GenBank accession no. DQ858900). The putative signal peptide sequence is
underlined; four conserved cysteine residues that define the C-type lectin domain are shaded, and four additional cysteine residues are shaded and
underlined. The EPN motif for ligand binding specificity is boxed. The polyadenylation signal sequence AATAAA is double underlined. (B) Multiple-
sequence alignment of the CRD of LvCTL1 with CRDs of other shrimp C-type lectins. The CRDs of LvCTL1 and four other shrimp C-type lectins were
aligned using ClustalX. Conserved cysteine residues that define the C-type lectin domain are shaded and indicated by asterisks. The EPN or QPD motif
is also shaded. Shown are LvCLec-1 (accession no. DQ858899, lec5D), PmCLec-1-CRD1 (accession no. ABI97373), PmCLec-1-CRD2 (accession no.
ABI97373), PsCLec-CRD1 (accession no. ABI97372), PsCLec-CRD2 (accession no. ABI97372), LvCTL-CRD1 (accession no. ABI97374), LvCTL-
CRD2 (accession no. ABI97374), FcCLec-1-CRD1 (accession no. AAX63905), FcCLec-1-CRD2 (accession no. AAX63905), FcCLec-2 (accession no.
ABA54612), PmAV (accession no. AAQ75589), and PmCLec-2 (accession no. AAZ29608).

349
350 ZHAO ET AL.
concentration. Recombinant LvCTL1 protein samples were separated using 15%
SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane
(Roche Biosciences, Germany). The membrane was blocked with blocking buffer
containing 5% nonfat milk powder in PBS with 0.1% Tween 20 (PBS-T) for 2 h
and then incubated with mouse anti-LvCTL1 antibody (or monoclonal antibody
against the His tag; Novagen) for 2 h, followed by incubation with peroxidase-
conjugated secondary antibody (1:10,000 dilution in PBS-T; Promega) for 1.5 h.
LvCTL1-specific signals were visualized after the addition of diaminobenzidine
colorimetric chromogen (Boster, China).
Expression of LvCTL1 protein in the hemolymph of shrimps after WSSV
infection. To detect LvCTL1 protein in the hemolymph of shrimps after WSSV
infection, healthy WSSV-susceptible L. vannamei shrimps were challenged with
WSSV as described previously (51), and hemolymph was collected from the
pericardial sinus located at the first abdominal segment of each shrimp at 3, 6, 12,
24, 36, 48, and 72 h postinjection, using a 1-ml sterile syringe. The hemolymph
was then immediately centrifuged for 10 min at 5,000 ⫻ g at 4°C, and the cell-free
plasma was collected. Plasma from unchallenged shrimps was also prepared by
the same method. The plasma samples were first diluted with distilled water (1:5,
vol/vol) and then subjected to 15% SDS-PAGE, and proteins were transferred to
a nitrocellulose membrane (Roche Biosciences, Germany). The membrane was
blocked with 5% nonfat milk powder in Tris-buffered saline (TBS) (10 mM
Tris-HCl, pH 7.2, 150 mM NaCl) for 4 h. After being washed three times with
TBS with 0.1% Tween 20 (TBS-T), the membrane was then incubated with the
mouse antiserum against LvCTL1 for 2 h. Antibody binding was visualized by a
colorimetric reaction catalyzed by peroxidase-conjugated goat anti-mouse anti-
body (1:10,000 dilution in TBS; Promega).
ELISA-based assay of LvCTL1 expression in the hemolymph of WSSV-resis-
tant and -susceptible shrimps. WSSV-resistant and -susceptible L. vannamei
shrimps were obtained by means of a selective-breeding program (51). Two
families were selected to perform an ELISA-based assay for LvCTL1 expression.
Family A has a survival rate of up to 70% after WSSV infection, while family B
has a mortality rate of 100% after WSSV infection. Hemolymph samples were
collected in tubes containing 10% saline solution (0.9%, vol/vol, NaCl) from both
WSSV-resistant and -susceptible L. vannamei shrimps at 3, 6, 12, 24, 36, 48, and
72 h postinjection. Hemolymph from unchallenged shrimps was also prepared by
the same method. Hemolymph (200 ␮l) was mixed with commercial extraction
reagent (KeyGen, China) (300 ␮l) on ice for 20 min and then centrifuged at
12,000 ⫻ g at 4°C for 5 min. The supernatant (diluted plasma) was removed to
new tubes, and phenylmethylsulfonyl fluoride was added to each diluted plasma
sample. The protein concentration in the diluted plasma was determined using a
bicinchoninic acid protein assay kit (Pierce), and the plasma was then stored at
⫺80°C for further use.
For ELISA, the 96-well microtiter plates (Costar) were coated overnight at
4°C with the diluted plasma (100 ␮l per well, ⬃20 ␮g total protein per well).
Wells coated with bovine serum albumin (BSA) were used as negative controls.
The plates were washed three times and blocked with blocking buffer containing
0.5% BSA in PBS-T for 2 h at 37°C. The plates were then incubated with mouse
anti-LvCTL1 antibody (1:500 dilution with blocking buffer) for 2 h, followed by
incubation with peroxidase-conjugated secondary antibody (goat anti-mouse im-
munoglobulin G antibody, 1:2,000 dilution in PBS-T; Promega) for 2 h. After the
plates were rinsed with PBS-T, the bound peroxidase activity was determined by
a reaction with TMB (3,3⬘,5,5⬘-tetramethylbenzidine), and the optical density
(OD) value was measured by spectrophotometry, using 450-nm filters. All
ELISAs were performed in triplicate, with the data given in mean values.
Hemagglutination and hemocyte aggregation assays. The hemagglutinating
activity of recombinant LvCTL1 was tested using 2% (vol/vol) erythrocytes from
rabbits, mice, chickens, and fish according to the method described by Luo et al.
(28). Erythrocytes were washed three times with TBS-Ca buffer (50 mM Tris-
HCl, 100 mM NaCl, 10 mM CaCl2, pH 7.0) and then suspended at 2% (vol/vol)
in TBS-Ca buffer. Twofold serial dilutions (25 ␮l) of recombinant LvCTL1 in
TBS-Ca buffer were mixed with 25 ␮l of the erythrocytes in a microtiter U plate.
The plate was incubated for 1 h at room temperature, and the hemagglutination
was observed under a microscope (Nikon, Japan). Erythrocytes mixed with serial
dilutions of BSA (Sigma) in TBS-Ca buffer were processed in parallel with the
above-described erythrocytes to serve as controls.
To test the aggregation of shrimp hemocytes by recombinant LvCTL1, hemo-
cytes of L. vannamei shrimps were cultured in modified Leibovitz’s L-15 medium
(Gibco) in vitro at 24 ⫾ 0.5°C with 5% ⫾ 0.2% CO2, used for the hemocyte
aggregation assay (for hemocyte culture, see “In vitro anti-WSSV activity of
recombinant LvCTL1” below). After 6 days of culture in a 48-well microplate,
the hemocytes were removed by treatment with trypsinase (Promega) at 37°C for
5 min, collected, and washed three times with saline solution (0.9% NaCl) by
centrifugation at 200 ⫻ g for 10 min. Hemocytes were then thoroughly resus-
J. VIROL.
pended in saline solution and seeded in a 48-well microplate at about 4 ⫻ 105
cells per well. Then, 50 ␮l of the hemocyte suspension in each well of a 48-well
microplate was mixed with 50 ␮l of twofold serial dilutions of recombinant
LvCTL1 in saline solution and incubated for 1 h at 25°C. Agglutination of
hemocytes was observed under a microscope (Nikon, Japan). Hemocytes mixed
with serial dilutions of BSA (Sigma) were processed in parallel as controls.
To test whether calcium is required for the hemagglutination activity of
LvCTL1, 12.5 ␮l of serial dilutions of EDTA in TBS was mixed with 12.5 ␮l of
recombinant LvCTL1 (⬃12 ␮g/ml), 25 ␮l 2% trypsin-treated mouse erythrocytes
was added, and the mixture was incubated for 30 min at 25°C. Hemagglutination
of erythrocytes was observed under a microscope (Nikon, Japan). Assays were
performed in triplicate.
Sugar binding specificity of LvCTL1. The sugar binding specificity of recom-
binant LvCTL1 was determined by an inhibitory agglutination assay. Serial di-
lutions (12.5 ␮l) of various carbohydrates (Sigma) in TBS-Ca, including D-
mannose, D-fructose, D-galactose, lactose, D-glucose, and sucrose, were mixed
with 12.5 ␮l of recombinant LvCTL1 (⬃12 ␮g/ml) and incubated for 30 min at
4°C. Then, 2% trypsin-treated mouse erythrocytes were added, and the mixture
was incubated for 30 min at 25°C. An inhibitory effect was expressed as the
minimum concentration of a carbohydrate required for complete inhibition of
the hemagglutinating activity of LvCTL1. This assay was performed in triplicate.
Binding of recombinant LvCTL1 to WSSVs. Intact WSSVs were purified from
the gills of infected L. vannamei shrimps according to the method described by
Xie et al. (44). The virus samples were examined using electron microscopy for
purity. Purified WSSV was dotted onto a nitrocellulose membrane (Roche Bio-
sciences, Germany), and the membrane was blocked with a blocking buffer
containing 5% milk powder in TBS-T overnight. The membrane was washed
three times in TBS-T and then probed with recombinant LvCTL1, Lypn (a fish
protein also expressed using the pET-32a vector [unpublished]), or pET-32a-
His-tag. (Expression of the empty pET-32a vector [Novagen] was performed in
Escherichia coli BL21 cells according to the manufacturer’s instructions, and
pET-32a-His-tag was purified according to the same method described under
“Expression and purification of recombinant LvCTL1” above.) Then, Western
blotting was performed with monoclonal antibody against the His tag to confirm
the presence of the polypeptides in TBS-T containing 0.1% BSA for 2 h at room
temperature. The binding of recombinant proteins to WSSV was detected by
immunoblotting, using mouse monoclonal antibody to the His tag (Novagen) or
mouse polyclonal antibody specific for LvCTL1 for 2 h, followed by incubation
with goat anti-mouse immunoglobulin G conjugated to peroxidase as a secondary
antibody (1:10,000 dilution in PBS-T; Promega) for 1.5 h at room temperature.
Then, a 3,3-diaminobenzidine solution (Boster, China) was added to develop the
brown product for 5 min, and the reaction was stopped with water.
Interaction of recombinant LvCTL1 with WSSV proteins by protein pull-down
assay. The cDNA fragment encoding mature LvCTL1 (residues 21 to 156) (Fig.
1A) was cloned into the pMAL-c2X plasmid (NEB, United Kingdom) to express
a maltose-binding protein (MBP)-lectin fusion protein in E. coli BL21 cells. The
MBP or MBP-lectin fusion protein was fixed to amylose resins (NEB, United
Kingdom). The purified WSSV particles were lysed with radio immunoprecipi-
tation buffer supplemented with protease inhibitor (Calbiochem). The viral ex-
tract was centrifuged at 14,000 rpm for 15 min at 4°C, and the supernatant was
diluted five times with TBS containing 1% Triton X-100, 10 mM EDTA, and
protease inhibitor. Then, the viral protein extract was added to the MBP or
MBP-lectin fusion protein-fixed amylose resins, and the mixture was incubated
for 3 h at 4°C. The amylose resins were then washed with TBS buffer containing
1% Triton X-100, and the pull-down proteins were analyzed by SDS-PAGE. The
bands pulled down by MBP-lectin but not by MBP were excised from gels, cut
into pieces, and transferred to a 1.5-ml microcentrifuge tube.
Protein identification by mass spectrometry. The in-gel enzymatic digestion
and mass spectrometry analysis were performed by the Shanghai Institutes for
Biological Sciences, Research Center for Proteome Analysis. The digested pro-
tein was separated and identified with a Finnigan LTQ mass spectrometer (Thermo-
Quest, San Jose, CA), coupled with a Surveyor high-performance liquid
chromatography system (ThermoQuest). First, a Microcore reverse-phase col-
umn (C18, 0.15 mm by 120 mm; Thermo Hypersil, San Jose, CA) was used to
separate the protein digests. Solvent A was 0.1% (vol/vol) formic acid, and
solvent B was 0.1% (vol/vol) formic acid in 100% (vol/vol) acetonitrile. The
solvent B gradient was held at 2% for 15 min and increased linearly to 98% in 90
min. The peptides were eluted from the C18 microcapillary column at a flow rate
of 150 ␮l per min and then electrosprayed directly into the mass spectrometer,
with the application of a spray voltage of 3.2 kV and with a capillary temperature
of 170°C. The full scan ranges from m/z 400 to 2,000. Protein identification using
raw data from tandem mass spectrometry was performed with SEQUEST soft-
ware (University of Washington, licensed to Thermo Finnigan) based on se-
VOL. 83, 2009 C-TYPE LECTIN FROM L. VANNAMEI HAS ANTI-WSSV ACTIVITY
quences in the Swiss-Prot database. The species is WSSV. A relative molecular
mass of 57 Da was added to the average molecular mass of cysteines in tandem-
mass spectrometry data searching. Both b ions and y ions were included in the
database search.
In vitro anti-WSSV activity of recombinant LvCTL1. To test whether recom-
binant LvCTL1 has anti-WSSV activity, a coculture assay was performed. This
assay is based on the percentages of cytopathic effects (CPE) and dead cells
induced by WSSV infection relative to those of normal cells. Hemocytes from
healthy L. vannamei shrimps were collected as the primary cells and challenged
with WSSV according to the method described by Jiang et al. (17). The L.
vannamei hemocytes were cultured in vitro at 24 ⫾ 0.5°C with 5% ⫾ 0.2% CO2
in a 96-well microplate in the modified Leibovitz’s L-15 medium (80% 1⫻
Leibovitz’s L-15; Gibco) and supplemented with 15% fetal bovine serum (Hy-
Clone), 1.0 g/liter glucose, 0.3 g/liter glutamine, 0.1 mg/liter vitamin C, 12.0 g/liter
NaCl, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin sulfate (pH 7.2). The
cells were observed daily with an inverted phase-contrast microscope (Nikon,
Japan), and half of the culture medium was replaced with fresh culture medium
every 3 days. The number of cells was also counted before inoculating them into
96-well microplates at about 3 ⫻ 106 cells per well for WSSV challenge exper-
iments. The WSSV inoculum was prepared according to the method described in
our previous work (51). WSSV dilutions (50 ␮l for each well) were preincubated
with the same volume of serial dilutions of LvCTL1 (the ultimate LvCTL1
concentrations were 10 ␮g/ml, 20 ␮g/ml, 40 ␮g/ml, and 60 ␮g/ml) or BSA (as a
control) in serum-free culture medium for 20 min at 4°C and then added to the
confluent hemocytes, which had been cultured in a 96-well microplate for 2
weeks. This procedure gave a final WSSV dilution of 10⫺3 virion after the
addition of the hemocyte suspension. After 2 h of incubation, the viral solutions
were removed, and completely fresh medium was added. The cells were incu-
bated at 25°C and monitored for 5 days. CPE and the number of death cells
induced by WSSV were recorded. Assays were performed in triplicate.
In vivo anti-WSSV activity of recombinant LvCTL1. The anti-WSSV activity of
recombinant LvCTL1 was also tested in healthy L. vannamei shrimps challenged
with WSSV. Healthy Pacific white shrimps with an average weight of 15 g were
kept in separate 500-liter tanks (each tank contained 30 shrimps) of well-aerated
seawater (at 26 ⫾ 1°C) and acclimatized to laboratory conditions for 1 week
before experiments. Shrimps were fed a commercial diet three times daily, and
approximately 50% of the seawater was changed per day. Twelve tanks of the
healthy shrimps were divided into four groups (three tanks for each group).
Shrimps were injected intramuscularly with sterile 1⫻ PBS solution (as a nega-
tive control; group 1), WSSV inoculum (as a positive control; group 2), or WSSV
inoculum preincubated with LvCTL1 (60 ␮g/ml; group 3) or pET-32a-His-tag
(group 4) for 20 min at 4°C (50 ␮l per shrimp). During shrimp culture, shrimp
mortality was monitored daily for 9 days and calculated, and the dead shrimps
were examined by PCR to confirm infection with WSSV.
Statistical analysis. Student’s t test was used for statistical comparison of the
mean cumulative mortality rates across groups in the antiviral activity experiment
in vivo. P values of ⬍0.05 were considered statistically significant.
RESULTS
Cloning of the LvCTL1 cDNA from L. vannamei. Based on
the initial partial cDNA sequence of LvCTL1, gene-specific prim-
ers (GSP1 and GSP2) (Table 1) were then designed and used for
RACE reactions to obtain both the 5⬘ and 3⬘ ends of the cDNA,
and the full-length cDNA was assembled from multiple overlap-
ping clones. Then, the sequence was verified by reamplifying the
complete sequence, using gene-specific primers GSP3 and GSP4.
The full-length LvCTL1 cDNA was 638 bp, with an open reading
frame of 471 bp encoding a protein of 156 amino acids (GenBank
accession no. DQ858900) (Fig. 1A).
The deduced amino acid sequence of LvCTL1 contains a
putative signal peptide of 20 residues and a single C-type CRD.
The CRD of LvCTL1 contains an EPN motif (Glu99-Pro100-
Asn101), which has a predicted ligand binding specificity for
mannose in mammalian C-type CRDs (8). This result suggests
that LvCTL1 may bind to mannose. The calculated molecular
mass of the mature LvCTL1 protein (residues 21 to 156) is
15.86 kDa, with an estimated pI of 5.20.
351
Comparison of LvCTL1 with other shrimp C-type lectins.
Database searches with the deduced amino acid sequence of
LvCTL1 showed that LvCTL1 is most similar to the C-type
lectin FcCLec-2 (GenBank accession no. ABA54612) (77%
identity). The CRD of LvCTL1 was also highly similar to that
of FcCLec-2 (accession no. ABA54612) (79% identity), and it
is also similar to CRDs of PsCLec (accession no. ABI97372)
(34% identity), LvCTL (accession no. ABI97374) (34%),
PmCLec-1 (accession no. ABI97373) (32%), and PmAV (ac-
cession no. AAQ75589) (28%). Based on the BLASTP analy-
sis, several shrimp C-type lectins were selected and aligned
with LvCTL1 (Fig. 1B). These shrimp lectins contain at least
one C-type CRD. The alignment showed that the CRD of
LvCTL1 contains all 14 invariant amino acid residues and 16 of
18 highly conserved amino acid residues that define a C-type
CRD (6), including the conserved cysteine residues that stabi-
lize the C-type lectin domain and the EPN motif for the ligand
binding specificity of mannose (Fig. 1B).
LvCTL1 mRNA is specifically expressed in the hepatopan-
creas. Reverse transcription-PCR (RT-PCR) was performed
to determine tissue-specific expression of LvCTL1 mRNA.
The LvCTL1 transcript was highly expressed in the hepatopan-
creas, and a much lower expression of LvCTL1 was detected in
the stomach. However, the LvCTL1 transcript was not de-
tected in the hemocytes, eyestalks, muscles, brains, gills, intes-
tines (midgut), pyloric ceca, nerve cords, spermaries, or hearts
of healthy L. vannamei shrimps (Fig. 2A and B). These results
suggest that the LvCTL1 transcript is specifically expressed in
the hepatopancreases of shrimps.
Expression and purification of recombinant LvCTL1. Com-
petent E. coli M15(pREP4) cells were transformed with the
recombinant expression vector pQE30-LvCTL1, and the re-
combinant protein, which has a six-His-tag addition to the N
terminus, was expressed abundantly after IPTG (isopropyl-␤-
D-thiogalactopyranoside) induction. The recombinant LvCTL1
protein was expressed as inclusion bodies and purified with
Ni-nitrilotriacetic acid agarose under denaturing conditions.
After being refolded by gradient dialysis against PBS buffer,
almost all of the purified LvCTL1 protein became soluble, and
the purity was estimated to be higher than 95% (Fig. 2C, lane
3). Recombinant LvCTL1 has an apparent mass of ⬃16 kDa as
determined by SDS-PAGE, which matched the predicted mo-
lecular mass calculated from the deduced amino acid se-
quence. Recombinant LvCTL1 was specifically recognized by
both monoclonal antibody against the His tag and polyclonal
antibody against LvCTL1 (Fig. 2C, lanes 4 and 5).
Expression of LvCTL1 protein in shrimp hemolymph is
induced after WSSV infection. The hemolymph was collected
from healthy shrimps or shrimps infected with WSSV at dif-
ferent times (3, 6, 12, 24, 36, 48, and 72 h) postinfection, and
LvCTL1 was detected by immunoblotting, using polyclonal
antibody against recombinant LvCTL1 (Fig. 2D). LvCTL1 pro-
tein was not detected in the hemolymph of healthy (unchal-
lenged) shrimps. After WSSV infection, a 24-kDa protein was
detected in the hemolymph at 12 h postinfection, which was
assumed to be the natural glycoprotein of LvCTL1, just like
other shrimp lectin glycoproteins from Fenneropenaeus mer-
guiensis, Penaeus monodon, and Macrobrachium rosenbergii
(32, 33, 50). The concentration of LvCTL1 in hemolymph
increased steadily up to 48 h postinfection (Fig. 2D, lanes 4, 5,
352 ZHAO ET AL. J. VIROL.

FIG. 2. Expression of LvCTL1 in shrimp tissues and production of recombinant LvCTL1. (A) Expression of LvCTL1 in different tissues as
shown by RT-PCR. Total RNA from different tissues of L. vannamei shrimps was reverse transcribed to cDNA, which was used as a template for
RT-PCR. The tissues include hepatopancreas (Hp), hemocyte (Hm), muscle (Ms), eyestalk (Es), brain (Br), pyloric cecum (Pc), nerve cord (Nc),
gill (Gi), intestine (midgut) (Mg), spermary (Sp), heart (Ht), and stomach (St). Expression of ␤-actin was used as a control. (B) Statistical analysis
of the RT-PCR results. The relative expression level of LvCTL1 is shown as the ratio of lectin to ␤-actin. (C) Production and purification of
recombinant LvCTL1. Bacterial lysates without (lane 1) or with (lane 2) IPTG induction, as well as purified recombinant LvCTL1 (lane 3), were
subjected to SDS-PAGE (15% gel). Proteins were stained with Coomassie brilliant blue R-250. Purified recombinant LvCTL1 was also analyzed
by immunoblotting with mouse monoclonal antibody against the His tag (lane 4) and polyclonal antibody specific for LvCTL1 (lane 5). Lane M,
protein molecular standard. (D) Western blot analysis of LvCTL1 protein in the hemolymph of L. vannamei. Hemolymph from healthy
(unchallenged) shrimps or shrimps injected with WSSV at different time points (0, 3, 6, 12, 24, 36, 48, and 72 h) postinjection was subjected to 15%
SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. LvCTL1 was identified by Western blot analysis, with mouse polyclonal
antiserum against recombinant LvCTL1. Lane 1, unchallenged hemolymph; lanes 2 to 8, hemolymph from shrimps challenged with WSSV at 3 h,
6 h, 12 h, 24 h, 36 h, 48 h, and 72 h postinjection. (E) Comparison of LvCTL1 expression levels in WSSV-resistant and -susceptible shrimp
hemolymph by an ELISA-based assay. Total proteins extracted from hemolymph samples from both the WSSV-resistant and -susceptible L.
vannamei shrimps at 0, 3, 6, 12, 24, 36, 48, and 72 h after WSSV injection were assayed by the ELISA procedure. The 96-well microtiter plates
were coated overnight at 4°C with 20.0 ␮g total protein per well in about 100 ␮l of protein extracts. The bound peroxidase activity was determined
by a reaction with TMB (3,3⬘,5,5⬘-tetramethylbenzidine), and the OD value was measured by spectrophotometry, using 450-nm filters. Negative-
control plates included plates coated with BSA. For each sample, the OD of the control well was subtracted from the OD of the hemolymph sample
well. All ELISAs were performed in triplicate, with the data given in mean values.

6, and 7). However, LvCTL1 in hemolymph decreased dramat-
ically at 72 h postinjection (Fig. 2D, lane 8). These results
suggest that LvCTL1 protein in hemolymph was induced after
WSSV infection.
Expression of LvCTL1 in the hemolymph of WSSV-resistant
and -susceptible shrimps. To compare the expression levels of
LvCTL1 in the hemolymph of WSSV-resistant and -susceptible
shrimps, an ELISA-based assay was performed. Cell-free
plasma samples were prepared from both the WSSV-resistant
and -susceptible L. vannamei shrimps at 0, 3, 6, 12, 24, 36, 48,
and 72 h after WSSV injection and used for the ELISA. ELISA
results showed that LvCTL1 in the hemolymph of both the
WSSV-resistant and -susceptible shrimps was recognized by
the mouse antiserum against recombinant LvCTL1. The mean
OD values from the hemolymph samples at 12, 24, 36, and 48 h
after WSSV injection were gradually increased and obviously
higher than those of the hemolymph samples from the healthy
control shrimps. The mean OD values from the hemolymph
samples of the WSSV-resistant shrimps after WSSV injection
were significantly higher than those of the hemolymph samples
from the WSSV-susceptible shrimps (Fig. 2E). These results
indicated that the expression levels of LvCTL1 in the hemo-
lymph of the WSSV-resistant shrimps were significantly higher
than those in the hemolymph of the WSSV-susceptible shrimps
after WSSV injection, suggesting that induced expression of
LvCTL1 is a response against the WSSV infection.
Hemagglutinating activity and carbohydrate binding speci-
ficity of recombinant LvCTL1. Refolded recombinant LvCTL1
protein was used to perform a hemagglutination assay with
erythrocytes from different animals. Among the animal eryth-
VOL. 83, 2009 C-TYPE LECTIN FROM L. VANNAMEI HAS ANTI-WSSV ACTIVITY
TABLE 2. Sugar specificities of LvCTL1
Sugar name MIC (mM)
D-Mannose ..................................................................................... 5
D-Glucose ....................................................................................... 15
D-Fructose ...................................................................................... 35
D-Galactose .................................................................................... 95
Lactose............................................................................................⬎100
Sucrose............................................................................................⬎100
rocytes tested, LvCTL1 agglutinated mouse and chicken eryth-
rocytes more effectively than rabbit erythrocytes with a mini-
mal agglutination concentration of 6.25 ␮g/ml, and it
agglutinated fish and rabbit erythrocytes at a concentration
higher than 12.5 ␮g/ml. Recombinant LvCTL1 also aggluti-
nated shrimp hemocytes at a concentration higher than 80.0
␮g/ml. However, no agglutination could be observed in the
BSA control under the same conditions. When mouse hemo-
cytes were agglutinated in the presence of EDTA, agglutina-
tion was inhibited when the EDTA concentration was higher
than 2.0 mM (agglutination occurred at EDTA concentrations
of 0, 0.5, 1.0, and 1.5 mM), indicating that the agglutination of
animal erythrocytes by recombinant LvCTL1 is calcium depen-
dent.
To test the carbohydrate binding specificity of LvCTL1, an
inhibitory hemagglutination assay was performed. Carbohy-
drates were preincubated with recombinant LvCTL1, and the
mixtures were then added to mouse erythrocytes. Among
the carbohydrates tested, D-mannose and D-glucose inhibited
the hemagglutinating activity of recombinant LvCTL1 most
effectively, with minimal concentrations of 5 and 15 mM, re-
spectively (Table 2). D-Fructose inhibited the hemagglutinating
activity of LvCTL1 at a higher concentration (35 mM), while
D-galactose, lactose, and sucrose had little inhibitory activity
(Table 2). These results suggest that LvCTL1 has ligand bind-
ing specificity for mannose/glucose, which is consistent with the
predicted ligand specificity of mannose.
Recombinant LvCTL1 binds to WSSVs. To test whether
LvCTL1 can directly bind to WSSVs, immunostaining was
performed. WSSV virions were spotted onto a nitrocellulose
membrane and then incubated with recombinant LvCTL1,
Lypn (a His-tagged control protein from fish [unpublished]), or
pET-32a-His-tag. Binding of recombinant proteins to WSSVs
was detected by monoclonal antibody against the His tag or
polyclonal antibody specific for LvCTL1. As shown in Fig. 3a,
binding of recombinant LvCTL1 to WSSV was detected by
both anti-His tag and LvCTL1-specific antibodies (Fig. 3aA
and B). No Lypn or pET-32a-His-tag bound to WSSVs (Fig.
3aC and D). These results suggest that LvCTL1 may bind to
proteins on the surfaces of WSSVs.
Recombinant LvCTL1 interacts with WSSV envelope pro-
teins. To further confirm that recombinant LvCTL1 can inter-
act with proteins from WSSVs, a pull-down assay was per-
formed. WSSV proteins pulled down by MBP-lectin but not by
MBP were analyzed by mass spectrometry and identified as
VP95, VP28, VP26, VP24, VP19, and VP14 (Fig. 3b), with
sequence coverage of 21%, 28%, 62%, 53%, 41%, and 46%,
respectively. These results suggest that LvCTL1 may bind to
these WSSV envelope proteins.
353
Recombinant LvCTL1 has anti-WSSV activity both in vitro
and in vivo. To test whether recombinant LvCTL1 has direct
anti-WSSV activity, an in vitro assay was first performed with
a primary culture of L. vannamei hemocytes. Hemocytes from
healthy shrimps were collected and cultured in 96-well micro-
plates. Next, the primary hemocytes were infected with WSSV
that had been preincubated with recombinant LvCTL1 or BSA
(as a control), and the cells were monitored for 5 days. These
hemocytes were viable after 2 days, but after 3 days, clumps of
infected cells and lysed cells were found in wells that were
inoculated with BSA-treated WSSV (data not shown). There
were no significant changes, however, in the hemocytes inoc-
ulated with recombinant LvCTL1-treated WSSV (data not
shown). At 5 days postinfection, CPE were apparent in the
hemocytes inoculated with BSA-treated WSSV; most cells be-
came detached from the wells, and cell debris was observed
(Fig.4aB). On the other hand, only small clumps of infected
cells and very little cell debris were found in the hemocytes
inoculated with LvCTL1 (40 ␮g/ml)-treated WSSV (Fig.4aC).
When the concentration of recombinant LvCTL1 was 20 ␮g/ml
or 60 ␮g/ml, the appearance of infected cell clumps was also
delayed, and the amounts of cell clumps and CPE induced by
WSSV were decreased (data not shown). The hemocytes with-
FIG. 3. Interaction of recombinant LvCTL1 with WSSV. (a) Bind-
ing of recombinant LvCTL1 to the WSSV virions. Purified WSSV was
dotted onto a nitrocellulose membrane and probed with recombinant
LvCTL1, Lypn (a fish protein that was also expressed with the pET-32a
vector), or pET-32a-His-tag. Binding of recombinant proteins to the
viruses was detected by mouse monoclonal antibody to the His tag or
mouse polyclonal antibody specific for LvCTL1. (A) The membrane
was probed with recombinant LvCTL1 and detected with a mouse-
specific antibody for LvCTL1. (B) The membrane was probed with
recombinant LvCTL1 and detected with anti-His tag antibody. (C) The
membrane was probed with pET-32a-His-tag and detected with anti-
His tag antibody. (D) The membrane was probed with Lypn and
detected with anti-His tag antibody. (b) Protein pull-down assay for
WSSV proteins that interact with the lectin LvCTL1. The cDNA frag-
ment encoding mature LvCTL1 (Fig. 1A, residues 21 to 156) was
cloned into the PMAL-C2X plasmid (NEB, United Kingdom) to ex-
press an MBP-lectin fusion protein in E. coli BL21 cells. The MBP or
MBP-lectin fusion protein was fixed to amylose resins. The purified
WSSV particles were lysed and centrifuged at 14,000 rpm for 15 min at
4°C, and the supernatant was diluted five times with TBS containing
1% Triton X-100, 10 mM EDTA, and protease inhibitor. Then, the
viral protein extract was added to the MBP or MBP-lectin fusion
protein-fixed amylose resins, and the mixture was incubated for 3 h at
4°C. The amylose resins were then washed with TBS buffer containing
1% Triton X-100, and the pull-down proteins were analyzed by SDS-
PAGE. Lane 1, proteins pulled down by MBP; lane 2, proteins pulled
down by MBP-lectin; lane M, protein molecular standard.
354 ZHAO ET AL. J. VIROL.

FIG. 4. Anti-WSSV activity of recombinant LvCTL1. (a) In vitro anti-WSSV activity of recombinant LvCTL1 in the primary hemocyte culture
of L. vannamei. Primary hemocytes were inoculated with recombinant LvCTL1-treated WSSV (C) or BSA-treated WSSV (B) or not inoculated
(A). Cells were then incubated for 5 days. Scale bar ⫽ 100 ␮m. (b) In vivo anti-WSSV activity of recombinant LvCTL1 in shrimps. Healthy shrimps
were injected with PBS (negative control), untreated WSSV (positive control), recombinant pET-32a-His-tag-treated WSSV, or LvCTL1-treated
WSSV. The cumulated mortality of shrimps was recorded up to 9 days postinjection. Each column represents the means from triplicates, with
standard deviations. The significance of differences was calculated by the t test (P ⬍ 0.05).

out WSSV inoculation grew well throughout the experimental DISCUSSION

period (Fig.4aA). However, the anti-WSSV activity of LvCTL1
was not significant when the concentration of LvCTL1 was 10 C-type lectins exist in almost all animals. They are capable of
␮g/ml (data not shown). These results suggest that LvCTL1 can binding to specific carbohydrates in a Ca2⫹-dependent manner
protect shrimp hemocytes from WSSV infection. and play an important role in nonself-recognition and the
To determine whether LvCTL1 also has anti-WSSV activity clearance of invading microorganisms (6, 8, 42, 47). In this
in shrimps in vivo, healthy L. vannamei shrimps were infected study, we isolated a cDNA for a novel C-type lectin (named
with untreated WSSV (positive control), recombinant LvCTL1) from the Pacific white shrimp, L. vannamei, by using
LvCTL1- or pET-32a-His-tag-treated WSSV, or PBS (negative ESTs and RACE. LvCTL1 contains a single CRD that is sim-
control), and the mortality of shrimps was monitored daily for ilar to PmAV (black tiger shrimp P. monodon) (29) and Fc-hsL
9 days. Initial mortality was observed after the first or second
day postinjection in all three groups of shrimps infected with
WSSV as well as in the negative-control group (first day). On TABLE 3. Cumulative mortalities of the shrimp L. vannamei after
the injection of PBS, WSSV, or WSSV preincubated with
the fifth day postinfection, the mortality of the shrimps in- recombinant LvCTL1 or pET-32a-His-tag protein
fected with recombinant LvCTL1-treated WSSV (15.6%) was
significantly lower (P ⬍ 0.05) than that of the shrimps infected Initial no. Mortality postinfection (%)a
of
with pET-32a-His-tag-treated WSSV (43.3%) or untreated Treatment
shrimps
WSSV (58.9%), and no mortality was observed in the negative- (n ⫽ 360) Day 3 Day 5 Day 9
control shrimps (Fig. 4b and Table 3). On the ninth day postin- PBS 90 0 0 0
fection, the mortality of shrimps infected with LvCTL1- and WSSV 90 13.3 ⫾ 1.00 58.9 ⫾ 1.53A 100 ⫾ 0.00A
pET-32a-His-tag-treated WSSV reached 47.8% and 100%, re- WSSV ⫹ 90 11.1 ⫾ 0.57 43.3 ⫾ 1.00A 100 ⫾ 0.00A
spectively, and no mortality was observed in the negative-con- PET-32a
WSSV ⫹ 90 6.7 ⫾ 1.00 15.6 ⫾ 0.58B 47.8 ⫾ 1.15B
trol shrimps, whereas the cumulated mortality of the shrimps
LvCTL1
infected with untreated WSSV had already reached 100% on
a
the eighth day postinfection (Table 3 and Fig. 4b). These Results are means from three independent experiments with standard devi-
ations. P values were determined using analysis of variance, and the significance
results suggest that recombinant LvCTL1 can protect shrimps of differences was determined by the t test (P ⬍ 0.05). Values in the same column
from WSSV infection. with different letters (A and B) are significantly different.
VOL. 83, 2009 C-TYPE LECTIN FROM L. VANNAMEI HAS ANTI-WSSV ACTIVITY
(FcCLec-2) (Chinese shrimp F. chinensis) (36), but it is differ-
ent from LvCTL, which has two putative CRDs (31). In the
CRD of LvCTL1, there is an EPN motif (Glu99-Pro100-Asn101)
that has a predicted ligand binding specificity for mannose (or
mannose-related sugars) (Fig. 1A), and recombinant LvCTL1
did bind to mannose and glucose with high affinity (Table 2),
indicating that LvCTL1 is mannose specific. LvCTL contains
two CRDs, one with an EPN motif for mannose and the other
with a QPD motif for galactose (31). The two L. vannamei
lectins share about 34% identity in the CRDs.
LvCTL1 mRNA was specifically expressed in the hepato-
pancreas (Fig. 2A and B), similarly to LvCTL and PmAV (29,
31). LvCTL1 protein was not detected in the hemolymph of
healthy shrimps, but it was present and its concentration was
increased in the hemolymph of shrimps injected with WSSV
(Fig. 2D and E), suggesting that expression of LvCTL1 was
induced by WSSV infection. Expression of other shrimp lec-
tins, such as PmAV, LvCTL, and FcCLec-1, was also induced
by WSSV infection (29, 25, 31); however, expression of these
C-type lectins in virus-infected shrimps showed different pat-
terns. The transcription level of FcCLec-1 reaches a maximum
expression at 3 h postinfection and then decreases from 6 h to
48 h postinfection (25). The mRNA level of PmAV is slightly
decreased from 6 h to day 1 postinfection and then starts to
increase from day 2 and reaches a peak at day 4 postinfection
(30). Similarly, the transcription level of LvCTL decreases
initially in the first 2 h and then increases to a much higher
level at 4 h postinfection (31). The LvCTL1 protein appeared
in the hemolymph at 12 h postinfection, increased steadily
thereafter, and then remained at a high level until 48 h postin-
fection (Fig. 2D and E). A delay in the progression of mortality
in the shrimps infected with recombinant LvCTL1-treated
WSSV was also observed from day 3 postinfection until the
end of the challenge (Table 3; Fig. 4b). These results suggest
that LvCTL1 in high concentrations in shrimp hemolymph may
bind to WSSV and neutralize the viruses, protecting the
shrimps from WSSV infection. However, the concentration of
LvCTL1 in the hemolymph decreased dramatically at 72 h
postinfection. It is possible that LvCTL1 recognizes and binds
to the viruses; thus, the free protein in hemolymph decreases.
Another possibility is that the decrease of LvCTL1 in the
hemolymph might be due to the decrease in shrimp activity at
the moribund stage, resulting from an increase in the amount
of viruses.
Shrimp C-type lectins have been shown to have antibacterial
activity and promote phagocytosis. For example, PmCLec-2,
FcCLec-1, and FcCLec-2 have been reported to possess anti-
bacterial activity (25, 28, 36), and a C-type lectin from P.
japonicus can promote phagocytosis (46). However, only one
C-type lectin, PmAV, which contains a QPD motif in the CRD,
has thus far been reported to have antiviral activity by inhib-
iting CPE induced by grouper iridovirus in fish cells in vitro
(29). PmAV was thought to have nonspecific antiviral activity
and may act as an intermediate rather than a recognition factor
during the antiviral reaction, since no PmAV protein was
found to bind WSSV by an immunohistological assay (29). We
showed that recombinant LvCTL1 directly bound to the WSSV
virions to exert its antiviral activity (Fig. 3 and 4). LvCTL1
interacted with WSSV envelope proteins, including VP95,
VP28, VP26, VP24, VP19, and VP14 (Fig. 3b). In mammals,
355
C-type lectins, such as MBL, have been shown to be able to
bind envelope glycoproteins of human immunodeficiency virus
(4) and envelope proteins of the influenza A virus (19), and
dendritic cell-specific intercellular adhesion molecule 3-grab-
bing nonintegrins can interact with envelope glycoproteins of
Ebola virus (1) and bind to Dengue (37) and hepatitis C (27)
viruses. Thus, it is likely that LvCTL1 may bind to the surface
glycoproteins of WSSV to inhibit the infection.
So far, approximately 58 structural proteins, including 9 nu-
cleocapsid proteins and 32 envelope proteins, have been iden-
tified in the WSSV virions (23), and they may play key roles in
the initial infection of WSSV in shrimps (40). These data
facilitate us further in studying antiviral activity. It was shown
that even a single WSSV envelope protein, such as VP28, can
prolong the survival of shrimps following WSSV challenge
when it is injected intramuscularly or administered orally, pos-
sibly by competitive saturation of receptor sites of the host cells
relevant for viral adhesion and viral penetration (43). Like-
wise, antibodies raised against VP28 can also neutralize and
inhibit WSSV infection (40).
We showed that recombinant LvCTL1 protein, when bound
to WSSV, significantly increased the survival of shrimps against
WSSV challenge and also protected shrimp hemocytes from
WSSV infection (Table 3; Fig. 4). LvCTL1 may serve as a PRR
to recognize and bind envelope proteins of WSSV, to block the
virus from entering cells, or to neutralize the virus. Our future
goal is to investigate how the binding of LvCTL1 to WSSVs
inhibits the infection.
ACKNOWLEDGMENTS
This work was supported by the National High Technology Re-
search and Development Program of China (863 Program) under
grants 2001AA601030 and 2006AA09Z445, the Guangdong Province
Natural Science Foundation under grant 20023002, and the Science
and Technology Bureau of Guangdong Province.
We thank Chuan-Fu Dong for his advice on cell culture and Hua-
Shui Ai for technical assistance.
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