Virology 391 (2009) 315–324
Contents lists available at ScienceDirect
Virology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o
Localization of VP28 on the baculovirus envelope and its immunogenicity against
white spot syndrome virus in Penaeus monodon
S. Syed Musthaq a, Selvaraj Madhan a, A.S. Sahul Hameed b, Jimmy Kwang a,c,⁎
a
b
c
Animal Health Biotechnology, Temasek Lifesciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore
OIE Expert, OIE Reference Laboratory for WTD, C. Abdul Hakeem College, Melvisharam 632 509, India
Department of Microbiology, Faculty of Medicine, National University of Singapore, Singapore
a r t i c l e
i n f o
Article history:
Received 5 May 2009
Returned to author for revision 24 May 2009
Accepted 3 June 2009
Available online 14 July 2009
Keywords:
WSSV
WSSV ie1
Recombinant baculovirus
Sf9 cells
Penaeus monodon
Surface display
In vivo
a b s t r a c t
White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and
other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral
infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus
with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells.
Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by
immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy
revealed that rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant
baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus
successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus
displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and
73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR
also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post
challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant
baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance
in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.
© 2009 Elsevier Inc. All rights reserved.
Introduction
White spot syndrome virus (WSSV) is a rod-shaped enveloped
dsDNA virus ∼275 × 120 nm in size with a tail-like appendage at one
end. It belongs to a new virus family Nimaviridae under the new genus
Whispovirus — (www.ncbi.nih.gov/ICTVdb/Ictv/index.htm). WSSV
has a wide host range that includes freshwater prawn, lobsters,
freshwater crabs and several species of marine crabs (Sahul Hameed et
al., 2001, 2002, 2003; Syed Musthaq et al., 2006b). WSSV is highly
pathogenic to shrimp and causes up to 100% mortality within 2 to
5 days from the onset of clinical signs (Chou et al., 1995; Lightner,
1996). Whole genome sequencing of 3 different WSSV isolates from
different geographical regions has revealed that the virus has a genome
of about 300 kb in size, with approximately 184 open reading frames
(Tsai et al., 2000; van Hulten et al., 2001a; Yang et al., 2001). Till date 39
structural proteins of WSSV have been identified, 22 of which are
envelope proteins (Tsai et al., 2004). The viral particle consists of at
least five major structural proteins: VP28, VP19, VP26, VP24 and VP15.
⁎ Corresponding author. Animal Health Biotechnology, Temasek Lifesciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore.
Fax: +65 68727007.
E-mail address: [email protected] (J. Kwang).
0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2009.06.017
VP28 and VP19 are associated with the virion envelope while VP26 acts
as a tegument protein that links the nucleocapsid associated proteins
VP24 and VP15 with the envelope (Tsai et al., 2006).
Envelope structural proteins play a crucial role in viral infections,
and are often involved with viral entry, assembly and budding. VP28 is
one of the major envelope proteins of WSSV and is involved in the
systemic infection of shrimp (van Hulten et al., 2001b; Syed Musthaq
et al., 2006a). During the course of infection, VP28 acts as a viral
attachment protein to the shrimp cells (Yi et al., 2004) and interacts
with host cellular proteins such as PmRab7 (Sritunyalucksana et al.,
2006), heat shock cognate protein 70 (Xu et al., 2009) and signal
transducer and activator of transcription (STAT) (Liu et al., 2007).
Along with the ongoing detailed investigations in the structure and
the biological nature of VP28, we have studied, for the first time, the
localization of VP28 in insect cells and thereby giving an insight into
the morphogenesis of WSSV.
Several reports demonstrated that VP28-based vaccines have
protected or at least increased the survival rate of WSSV challenged
shrimp, as compared to unvaccinated shrimp (Witteveldt et al., 2004a,
2004b; Namikoshi et al., 2004; Jha et al., 2006). In this study, we were
also able to develop a novel baculovirus-based vaccine against WSSV.
A baculovirus vector was constructed with WSSV immediate early
promoter 1 (ie1), which is capable of expressing VP28 at an earlier
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stage of infection in insect cells. The novel nature of the baculovirus to
transduce several cell lines from different sources (Shoji et al., 1997)
and the ability of WSSV ie1 promoter (Gao et al., 2007; Lu et al., 2007)
to act as a shuttle promoter between insect and shrimp cells were
taken into consideration in this study. We confirmed with our results
that the baculovirus expressed rVP28 was able to translocate to the
plasma membrane of infected insect cells. Recombinant baculovirus
was also able to harbour the rVP28 on its envelope during the budding
process. Our results showed that infection of shrimp with wild type
baculovirus alone is not able to cause any abnormality to shrimp.
Hence, we used this baculovirus displaying rVP28 as a vaccine
candidate against WSSV and were able to achieve a high level of
protection against WSSV infection in vaccinated shrimp. The ability of
recombinant baculovirus to express VP28 in vivo in shrimp was also
assessed and the expression of VP28 in shrimp tissue was confirmed.
Results and discussion
A recombinant baculovirus was constructed with an ie1 promoter
derived from the WSSV genome (Bac-wt) (Fig. 1A). WSSV ie1
Fig. 1. Schematic illustration of VP28 gene construct in baculovirus transfer vector and expression of rVP28 analysed by Immunofluorescence assay and Western blot using antimouse VP28 polyclonal antibodies. (A) VP28 gene (205 amino acid) of WSSV with Signal peptide (SP) and transmembrane (TM) domain located at the N-terminal region spanning
∼30 amino acids. VP28 gene was cloned downstream of WSSV ie1 promoter and upstream of Tsv40 of pFASTBac HT A vector and named as Bac-VP28 and the above construct without
VP28 gene was named as Bac-wt. (B) Expression of rVP28 in Sf9 cells. The cells were infected with Bac-VP28 and Bac-wt at an MOI of 0.5 and at 48 h post infection, expression of VP28
was detected by Immunofluorescence Assay. (C) Western blot analysis of purified WSSV virions compared with baculovirus expressed rVP28. Lane M — broad range protein
molecular weight marker; Lane 1 — purified wild type WSSV virions from WSSV-infected shrimp; Lane 2 — supernatant of WSSV-uninfected shrimp; Lane 3 — Bac-VP28 infected cell
lysate; Lane 4 — Bac-wt infected cell lysate.
S. Syed Musthaq et al. / Virology 391 (2009) 315–324
promoter has been previously demonstrated to be highly active in
insect and mammalian cells. Baculovirus vector engineered with
WSSV ie1 promoter was also shown to efficiently express avian
influenza hemagglutinin (HA) in both insect and mammalian cells (Lu
et al., 2007). In this study, we have used a WSSV ie1 based
recombinant baculovirus to express VP28 (Bac-VP28) in insect cells.
Immunoflourescence assay indicated that rVP28 expressed by the
recombinant baculovirus was able to maintain its structural and
antigenic conformity compared to its original counterpart (Fig. 1B).
The molecular mass of rVP28 present in the baculovirus infected cell
lysate and purified WSSV virions was directly compared by the
western blot analysis, which detected a band both with the similar
size of 28 kDa (Fig. 1C) with anti-VP28 polyclonal antiserum raised in
mouse. This data is consistent with the results obtained by van Hulten
et al. (2000, 2001b) during an attempt to identify major WSSV
proteins. Also it has been demonstrated that VP28 from both insect
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cells and intact WSSV particles were not glycosylated despite the
presence of multiple N and O glycosylation sites. These observations
indicate that the posttranslational modification of rVP28 in insect cells
is similar to that occurring in shrimp cells.
Previous reports have studied the biochemical properties of
baculovirus expressed VP28 but none of them have investigated the
cellular localization of the protein. Hence, we attempted to further
examine the localization of rVP28 in Sf-9 cells infected with
recombinant baculovirus. Interestingly, our confocal microscopy
results revealed that rVP28 was directed to the plasma membrane of
the infected Sf-9 cells (Fig. 2). Influenza hemagglutinin (HA), being a
viral envelope protein, follows a similar manner of translocation to the
plasma membrane of insect cells infected with recombinant baculovirus (Yang et al., 2007). To make sure that the baculovirus proteins
were not involved in this phenomenon, plasmid vectors containing
only the VP28 gene placed downstream of WSSV ie1 promoter were
Fig. 2. Localization of rVP28 on the plasma membrane of infected insect cells. Cells were grown in sterile cover slip and infected with Bac-VP28 at an MOI of 0.5. Cells were fixed at
48 h post infection for confocal microscopic image analysis. Bac-wt infected Sf9 cells served as a negative control.
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transfected into the Sf-9 cells and the expression of rVP28 was
confirmed to be on the cell surface by IFA (Data not shown). It is
evident from the literature that most of the integral membrane
proteins that are targeted to the host cell surface contain a short signal
peptide (SP) sequence and a hydrophobic transmembrane domain
(TM). Computational analysis of VP28 amino acid sequence by SignalP
3.0 server and TMAP also predicted a potential SP and TM region (1–
30 amino acids) at the N-terminal end of VP28 with significant
number of overlapping amino acids (Fig. 1A). This overlapping of SP
and TM region could account for the reason why the signal peptide of
VP28 was not cleaved in matured virions, as reported by Tsai et al.
(2004).
Fig. 3. Incorporation of rVP28 into the baculovirus envelope. (Ai) Western blot analysis of rVP28 in the supernatant of infected insect cells. Lane 1 — Bac-wt infected insect cell culture
supernatant; Lane 2 — Bac-VP28 infected insect cell culture supernatant; Lane 3 — Complete Bac-VP28 virions. (Aii) Immunogold electron microscopy of purified Bac-VP28 using
mouse anti-VP28 as primary antibody and anti-mouse IgG conjugated with 10-nm gold particles as secondary antibody. (B) Schematic illustration of budding process of Bac-wt and
Bac-VP28 recombinant baculoviruses. (C) Western blot analysis of membrane association of rVP28 in the complete Bac-VP28 virions. The purified Bac-VP28 virions were treated with
Triton X-100 and the nucelocapsid fraction was separated from envelope by ultracentrifugation and subjected to Western blot analysis. Lane M — broad range protein molecular
weight marker; Lane 1 — complete Bac-VP28 virions; Lane 2 — complete Bac-wt virions; Lane 3 — envelope fraction of purified Bac-VP28 after treatment with TritonX-100; Lane 4 —
nucleocapsid fraction of purified Bac-VP28 after treatment with Triton X-100.
S. Syed Musthaq et al. / Virology 391 (2009) 315–324
Viral envelope proteins with proper SP and TM domain expressed
in insect cells have been shown to be successfully acquired by the
baculovirus from the host cell membrane. E2 envelope glycoprotein of
classical swine fever virus (CSFV) substituted with SP and TM domain
has been displayed on the baculovirus envelope (Xu and Liu, 2008).
Also Feng et al. (2006) reported that SARS coronovirus spike protein
fused with GP64 signal peptide and VSVG membrane anchor domain
was targeted to the baculovirus surface when expressed in insect cells.
Hence, we next investigated the association of rVP28 with the
baculovirus by analysing the infected cell culture supernatant
containing budded baculoviruses. Western blot analysis of infected
cell culture supernatant detected a band with the expected size of
28 kDa with anti-VP28 polyclonal antibodies (Fig. 3Ai). This data
suggested that the recombinant baculovirus was able to harbour the
rVP28 on its surface. The stronger activity of WSSV ie1 promoter in
insect cells along with its immediate early nature would have
supported the high level of rVP28 expression on the baculovirus
surface. To provide more concrete evidence on rVP28 acquisition by
the baculovirus, Bac-VP28 and Bac-wt was concentrated by ultracentrifugation and subjected to immunogold electron microscopy
with anti-mouse VP28 polyclonal antibodies. As expected, we
observed the distribution of gold particles only on the surface of
Bac-VP28 which confirms the incorporation of rVP28 on the
baculovirus envelope (Fig. 3Aii). The schematic illustration of the
recombinant baculovirus budding process and display of rVP28 on the
surface of Bac-VP28 is depicted in Fig. 3B. To further support our
findings, the baculovirus stock was treated with Triton X-100 and the
nucleocapsid fraction was separated from the envelope via ultracentrifugation. Western blot analysis confirmed that rVP28 was exclusively present on the envelope as no bands were detected in the
nucleocapsid fraction (Fig. 3C). This result correlates with the
previous study which demonstrated that influenza HA expressed in
insect cells was also associated only with the baculovirus envelope
fraction and not with the nucleocapsid (Lu et al., 2007).
Recombinant baculoviruses displaying foreign viral proteins have
long been used as a vaccine in animal models against several viral
diseases (Lanford et al., 1989; Mori et al., 1994; Lu et al., 2007; Wang et
al., 2007; Lin et al., 2008). Since the presence of VP28 on the
baculovirus envelope was confirmed in our previous experiments, the
possibility of using baculovirus displaying rVP28 as a vaccine against
WSSV was also assessed. Before using Bac-VP28 as a vaccine, the effect
of wild type baculovirus in normal healthy shrimps was studied by
injecting it at a dose range 50 μl of 1 × 108 pfu/ml. The shrimp were
monitored daily over a period of 3 months to analyse its growth and
behavioural changes. We found no abnormality in the life cycle of
these shrimps. To further support this, we also confirmed that wild
type baculovirus was unable to replicate in shrimp and the transcription and translation of the two major baculovirus proteins, gp64 and
VP39, required for assembly was absent in shrimp tissues (data not
shown). These findings support the work carried out by Gao et al.
(2007), which states that the baculovirus, Autographa californica
Multiple Nuclear Polyhedrosis Virus (AcMNPV) infection is restricted
within Lepidoptera species and its sub classes and does not replicate
in other invertebrate cells.
Baculovirus have also been reported to deliver foreign genes in vivo
in mice. Tani et al. (2003) demonstrated that VSVG-modified
baculovirus was able to transduce a reporter gene into the cerebral
cortex and testis of mice by direct inoculation in vivo. In order to
analyse the in vivo delivery capacity of Bac-VP28, we studied the
expression of VP28 at transcriptional and translational levels in BacVP28 vaccinated shrimps. A 50 μl of 1 × 108 pfu/ml dose of Bac-VP28
was administrated to shrimp and total RNA was extracted from
different tissues (gill, abdominal muscle, pleopods and hemolymph)
of Bac-VP28 immunized shrimp. RT-PCR results revealed that
transcription of VP28 was present in all analysed shrimp tissues at
various time points (Fig. 4A). However, beyond 40 days post
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vaccination (dpv), transcription of VP28 gene was found to occur
only in the abdominal muscle and pleopods of shrimp. This is in
agreement with earlier data obtained with DNA vaccine encoding
VP28 gene, which showed that VP28 is not consistently expressed in
all examined tissues at different time points (Rajeshkumar et al.,
2008; Ning et al., 2009). Our Immunohistochemistry analysis also
revealed that the VP28 protein was expressed on Bac-VP28 immunized shrimp tissues: gill, muscle and eyestalk on 7 dpv (Fig. 4B).
To assess the vaccination efficiency, a proper dilution of 10 μl of
1 × 10− 5 dilution of WSSV was chosen, which could cause 90%
mortality at a period of 8 to 9 days and 100% mortality was observed
10 days after inoculation of the virus. Our results of vaccination studies
showed 86.3% and 73.5% survival rates in Bac-VP28 immunized
shrimps at 3 and 15 dpv respectively (Figs. 5A and B). In support to the
protection conferred by Bac-VP28, we found that WSSV viral copy
numbers was significantly reduced from 7.1 × 107 WSSV copies to
4.97 × 105 WSSV copies mg− 1 total DNA on 7 days post infection (dpi)
and reached to less than 0.4 × 101 WSSV copies mg− 1 total DNA on 25
dpi (Fig. 6). Durand and Lightner (2002) observed at least a 5.7 × 1010
WSSV copies g− 1 total DNA of moribund post-larvae. This reduced
viral copy numbers may be possibly due to the activation of the innate
antiviral immunity in the vaccinated shrimp. Bac-VP28 vaccinated
shrimp also showed higher survival rate compared to shrimp
vaccinated with proteins (Witteveldt et al., 2004a, 2004b) or DNA
vaccines (Rout et al., 2007; Rajeshkumar et al., 2008; Ning et al.,
2009). Shrimp vaccinated with subunit vaccines comprising VP28
protein showed only 44% survival rate at 2 dpv and with time,
immunity against WSSV was decreased (Witteveldt et al., 2004b). It
has also been shown from these studies that the protection conferred
by protein vaccine was only for a maximum period of 3 weeks. For
shrimp vaccinated with DNA vaccine encoding the VP28 gene, a
survival rate of 56.6% was obtained. 54.7% survival rate was observed
at 25 dpv (Ning et al., 2009) and the protection against WSSV was also
reduced at 30 dpv (Rajeshkumar et al., 2008). Furthermore, the
survival rate of DNA vaccinated shrimp was comparatively low at 15
dpv and the in vivo presence of VP28 transcript was decreased at 40
dpv. Practical application of such DNA-based vaccines is also limited
since they have been found to be effective only when delivered by
injection, gene gun and scarification (Corbelil et al., 2000) or
application of short-pulse ultrasound (Fernandez-Alonso et al.,
2001). Simple bath immersion, which is effective for delivery of
some traditional inactivated or attenuated vaccines (Corbelil et al.,
2000) has not been found to be effective for DNA vaccines. Hence, the
ability of baculovirus displaying VP28 to confer increased protection
rate in vaccinated shrimp together with good biosafety profile to both
humans and shrimps makes them an attractive vaccine candidate over
other forms of vaccines against WSSV.
With our findings, we conclude that the signal peptide of VP28 is
functional in insect cells and the protein is translocated to the cell
surface probably via a secretory pathway. It is worth noting here that
gp64, a major envelope protein of baculovirus, attains its envelope
association only at the time of virion budding and exists as a
homotrimer on the viral surface (Oomens et al., 1995). Similarly,
VP28 is also reported to exist as homotrimers on the WSSV envelope
and considered to have a role in the membrane fusion activity of the
virus, a characteristic feature of most viral envelope proteins. Based on
our results along with evidence available in the literature, we propose
here that VP28 may attain its association with WSSV only at the late
stage of assembly due to their natural localization to the host cell
membrane. Furthermore, a novel approach was also made to use
recombinant baculovirus as a vaccine candidate for WSSV. The ie1
promoter of WSSV was efficiently used to express VP28 consistently in
insect cells and we demonstrated that baculovirus were able to
acquire VP28 on its envelope. Recombinant baculovirus is able to
transduce successfully into shrimp cells without having any effect to
the shrimp. In vivo expression of VP28 at the transcriptional and
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Fig. 5. Time–mortality relationship of vaccination experiments. Shrimp were
challenged with WSSV or PBS buffer (negative control) on 3 and 15 days post
vaccination (dpv) with Bac-VP28, Bac-wt or PBS. The final cumulative mortality of the
Bac-VP28 vaccinated shrimps were 13.7% and 26.5% respectively, when compared with
100% cumulative mortality of the positive control groups (immunized with PBS or Bacwt and challenged with WSSV).
translational level during various time points in shrimp cells was also
confirmed. As observed in the present study, Bac-VP28 immunized
shrimp showed a higher survival rate when compared with other
forms of vaccination. It could be possible that recombinant baculovirus have acted as a vectored as well as a protein vaccine. We are
further evaluating the mechanism by which the baculovirus mediated
immunity confers protection to shrimp against WSSV. Oral vaccination will be evaluated in future in order to overcome the practical
difficulties associated with injection.
Materials and methods
Collection and maintenance of experimental animals
Shrimp, Penaeus monodon (10–12 g body weight), were imported
from Malaysia and maintained in 1000-l fiberglass tanks with air-lift
biological filters at an ambient temperature of 27–30 °C with salinity
between 20 and 25 ppt. Natural seawater was used in all experiments
and it was pumped from the adjacent sea to Singapore and further
treated as described by Syed Musthaq et al. (2006a). The animals
were kept in tanks for 5 days and acclimatized prior to the
experiments. The animals were fed with artificial pellet feed (BTA
feed, Malaysia). From the experimental animals, 5 from a group of 30
were randomly selected and screened for the presence of WSSV by
polymerase chain reaction (PCR) using the primers designed by
Takahashi et al. (1996) and only healthy shrimp were used for the
experiments.
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Fig. 6. Real-time PCR quantification of WSSV viral copy numbers. WSSV DNA was
extracted from the gill tissue and the samples were collected at 2, 5, 7, 10, 15 and 25 dpi
from the Bac-VP28, Bac-wt immunized shrimp challenged with WSSV. A 211 bp
segment of WSSV VP26 gene served as qPCR primers. Plasmid encoding the full length
ORF of VP26 gene was used to obtain the standard curve for quantifying the WSSV viral
genomic copy numbers. Each column represents the mean of triplicate assay with
standard deviation. Asterisks denote significant differences (P b 0.05) between samples.
fluid was filtered through a 0.4 μm filter. The filtrate was then stored at
−80 °C for experimental studies.
In vivo viral titration of WSSV
The WSSV stock was titrated by in vivo infection experiments as
previously described by Witteveldt et al. (2004a) with minor
modifications. Briefly, shrimp (10–12 g) were injected with different
concentration (1 × 10− 1 to 1 × 10− 8) of 50 μl of inoculum consisting of
10 μl virus and 40 μl of PBS (pH 7.4) on the second abdominal segment
of shrimp using a 26-gauge needle. The mortality was recorded twice
a day and dead shrimp were tested for the presence of WSSV by PCR
(Takahashi et al., 1996). The obtained virus dose–mortality relationship was used to determine the desired challenge pressure for the
injection experiments.
Preparation of anti-VP28 polyclonal antibody
The prokaryotic expression and purification of recombinant VP28
protein was carried out as described by Syed Musthaq et al. (2006a).
Polyclonal antiserum was raised in mice using purified recombinant
VP28 emulsified with equal volume of Freund's complete adjuvant.
Antiserum was collected from the immunized mice after completion
of standard immunization trial. The immunoglobin (IgG) fraction was
purified by protein A-Sepharose (Bio-rad, UK) and stored at −80 °C
for further experiments.
Preparation of WSSV viral inoculum
Generation of recombinant baculovirus
The laboratory WSSV stock (Liu et al., 2002; Wang et al., 2002)
used in this study was propagated by intramuscular injection into
healthy shrimp. The hemolymph was drawn directly from the hearts
of moribund shrimp using sterile syringes followed by centrifugation
(3000 × g for 20 min at 4 °C). The supernatant fluid was then recentrifuged (8000 ×g for 30 min at 4 °C) and the final supernatant
For the construction of recombinant baculovirus Bac-VP28, the full
length ORF of VP28 gene was amplified from WSSV genome using the
primers Bac-VP28F 5′-CGCCGGTCCGATGGATCTTTCTTTCACTCTTTC-3′
and Bac-VP28R 5′-CCGAAGCTTTTACTCGGTCTCAGTGCCAG-3′ with
RsrII and HindIII restriction enzymes. The original pFASTBacHT A
Fig. 4. In vivo expression analysis of VP28 from Bac-VP28 and Bac-wt vaccinated shrimp tissues. (A) In vivo expression analysis at transcriptional level, different tissue samples were
collected on day 2 (a), 7 (b), 15 (c), 25 (d) and 40 (e) after vaccination and subjected to RT-PCR. Lane M: 100 bp Marker; Lanes 1 to 5: RT-PCR product (615 bp) with RNA template
from hemolymph, gill, hepatopancrease, abdominal muscle and pleopods of shrimps immunized with Bac-VP28. Lane 6: negative control (RT-PCR product with RNA template from
abdominal muscle of shrimp immunized with Bac-wt). (B) In vivo expression analysis of VP28 at translational level detected by immuno histochemistry in Bac-VP28 and Bac-wt
vaccinated shrimp tissues at 7 dpv from (a) Gill, (b) Muscle, (c) Eyestalk.
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(Invitrogen, San Diego, CA, USA) baculovirus transfer vector was
modified by deleting the polyhedrin promoter sequence including His
tag, ATG and multiple cloning sites with RsrII and HindIII restriction
enzyme before the insertion of VP28 expression cassette. The ie1
promoter was amplified from WSSV DNA using the primers
WSSVie1F-5′-CCTACGTATCAATTTTATGTGGCTAATGGAGA-3′and
WSSVie1R-5′-CGCGTCGACCTTGAGTGGAGAGAGAGCTAGTTATAA-3′
then inserted into pFASTBacHT A using AccI and RsrII restriction site.
The amplified VP28 gene was placed upstream of the SV40 terminator
in pFASTBacHT A vector and downstream of ie1 promoter and named
as Bac-VP28 (Fig. 1A). The above construct without VP28 gene is Bacwt and served as negative control (Fig. 1A).
For the generation of recombinant baculoviruses the constructs
were integrated into the baculovirus genome within DH10Bac™
(Invitrogen, USA) through site-specific transposition according to
the protocol of Bac-To-Bac system (Invitrogen, USA). Then recombinant baculovirus were then propagated in SF-900II SFM (Gibco BRL,
USA) at 27 °C by infecting 200 ml of Sf9-cells in suspension at a cell
density of 2 × 106cells/ml with the multiplicity of infection (MOI) of
0.5. The budded virus particles released into the media were
harvested at 4 dpi and filtered through 0.22 μm filter for infection
experiments. The virus titre was determined by plaque assay and
the virus stock was stored at 4 °C. A large scale amplification of BacVP28 was carried out and the virus particles were purified by two
rounds of sucrose gradient following the standard protocols (O'Reilly
et al., 1992).
Immunofluorescence assay to detect the expression of rVP28 in
insect cells
To detect the Immunofluorescence signals, Sf-9 cells were grown
in 24 well plates and infected with Bac-VP28 and Bac-wt baculovirus
at a MOI of 0.5. After 48 h post infection, the cells were fixed with
paraformaldehyde for 20 min and blocked with 1% gelatin for 30 min
at room temperature. The fixed cells were then incubated with antimouse VP28 polyclonal antibody at a dilution of 1:100 for 1 h at 37 °C.
FITC-conjugated rabbit anti-mouse (DakoCytomation, Denmark) at a
dilution of 1:100 was subsequently incubated with the cells for 1 h .
The fluorescence signal was detected with an inverted fluorescence
microscope (Olympus, UK) and the images were captured by a digital
imaging system (Nikon, USA).
Western blot analysis of rVP28
Bac-VP28 infected cells or cell lysate were mixed with Laemmli
sample buffer and SDS-PAGE was performed (Laemmli, 1970). VP28
from purified WSSV virions served as a reference while Bac-wt was
included as a negative control. The gel was transferred to nitrocellulose membrane and Western blot was performed by the method
described by Talbot et al. (1984). The anti-mouse VP28 polyclonal
antibodies at a dilution of 1:2000 were used as primary antibody and
rabbit anti-mouse IgG (DakoCytomation, Denmark) at a dilution of
1:1000 were used as secondary antibody to detect the baculovirus
expression of rVP28.
Confocal image analysis
Sf-9 cells were cultured on sterile cover slips (placed in six-well
plates) and infected at an MOI of 0.5. Two days post infection, the cells
were fixed by paraformaldehyde for 20 min and blocked with 1%
gelatin for 30 min at room temperature. The fixed cells were then
incubated with anti-mouse VP28 polyclonal antibody at dilution of
1:100, for 1 h at 37 °C. FITC-conjugated rabbit anti-mouse IgG
(DakoCytomation, Denmark) at a dilution of 1:1000 was subsequently
incubated with the cells for 1 h . Localization of VP28 was visualized
using a confocal microscope (Carl Zesis LSM 510, Germany).
Display of rVP28 on the baculovirus envelope
Transmission electron microscopy (TEM) analysis of Bac-VP28 was
performed as described previously (Hu et al., 2003) with minor
modifications. The carbon-coated grids were floated on 10 μl purified
baculovirus suspension for 30 min. The grids were then incubated
with anti-mouse VP28 polyclonal antibody at 1:500 dilution for
30 min. Later the grids were exposed to 10 nm gold labelled goat antimouse IgG + IgM (H + L), (GE Healthcare, UK) for 30 min. Finally the
grids were stained with 2% phosphotungstic acid and examined under
TEM (Joel, USA).
In order to confirm the surface display of rVP28 on the baculovirus,
the Bac-VP28 infected cell culture supernatant was concentrated and
purified by ultracentrifugation. The purified virions were treated with
0.5% Triton X-100 for 15 min and the viral membrane was solubilized.
The nucleocapsid fraction was separated from the envelope via
ultracentrifugation and the fractions were evaluated by Western
blot as described by Talbot et al. (1984).
Vaccination and challenge experiments
For the vaccination experiment, two batches of shrimp (4 groups
per batch and 20 shrimp per group) were selected. In each batch, the
first and second groups were injected with 50 μl of 1 × 108 pfu/ml of
Bac-VP28 and Bac-wt respectively for two times at 5 days interval,
whereas third and fourth group were administrated with PBS. In batch
I, the first three groups were challenged with 10 μl of 1 × 10− 5 dilution
of WSSV at 3 dpv, whereas in batch II, the first three groups were
challenged with the same concentration of virus at 15 dpv. The fourth
group in each batch was administrated with PBS during 3 dpv and 15
dpv respectively. The experiments were carried out in quadruplicate.
In vivo expression of VP28
To assess the in vivo expression of VP28 at transcriptional level, RTPCR was carried out. Briefly 50 μl of 1 × 108 pfu/ml of Bac-VP28 and
Bac-wt was administrated to the two groups of shrimp (20 shrimp
each group) and samples were collected at 2, 7, 14, 25 and 40 dpv,
intervals and analysed. The total RNA was extracted from various
organs (hemolymph, gill, eyestalk, abdominal muscle and pleopods)
by using Trizol following the manufacturer's protocol (Invitogen, USA).
The RNA quality and quantity was determined by Nanodrop spectrophotometer ND-1000. DNA contamination was removed by using
RNase free DNase I (Fermentas Inc, USA) and synthesis of cDNA was
performed with oligo dT primer (Roche, USA) and cDNA conversion
mix (Promega, USA) as per manufacturer's instructions. The cDNA was
amplified by using the primers Bac-VP28F and Bac-VP28R followed by
analysis of PCR products in 1.2% agarose gel electrophoresis.
Immuno histochemistry analysis was performed to evaluate the in
vivo expression of VP28 at the translational level. The shrimp tissues
(gill, muscle and eye stalk) were collected from Bac-VP28 and Bac-wt
vaccinated shrimp at 7 dpv and was fixed in neutral 10% buffered
formalin pH 7.0 (Formalin 100 ml, Sodium phosphate dibasic 6.5 g,
Sodium phosphate monobasic 4.0 g, distilled water 900 ml). After
48 h , the samples were processed and embedded in paraffin wax
using Embedder (Leica, Germany), then sectioned into 4 μm thickness
using microtome (Leica Autocut microtome model 2255, Leica
Microsystems, Wetzlar, Germany). The sections were de-paraffinized
using Histo-choice (Amersco, Solon, Ohio, USA) and rehydrated in
sequentially graduated ethanol baths to distilled water. The sections
were treated with trypsin (0.1% w/v in PBS) for 10 min and washed
twice with PBS-Tween 20 (0.01% v/v with PBS). Slides were blocked in
0.1% non fat milk in PBS for 30 min followed by incubation with antimouse Pr-VP28 polyclonal antibody at a dilution of 1:100 for 1 h at
room temperature. Slides were then washed 3 times in PBS-T and
incubated with FITC-conjugated rabbit anti-mouse (DakoCytomation,
S. Syed Musthaq et al. / Virology 391 (2009) 315–324
Denmark) at a dilution of 1:50 for 30 min. After washing, the sections
were mounted using glycerol. The slides were observed under a
fluorescent microscope (Olympus, UK) and the images were captured
by digital imaging system (Nikon, USA).
Safety profile of wild type baculovirus in shrimp
Shrimp, P. monodon were divided into three groups (20 shrimp per
group). The first and second groups were injected with 50 μl of
1 × 108 pfu/ml of Bac-VP28 and Bac-wt respectively while the third
group was injected with PBS control. All shrimp were monitored daily
for their behavioural changes and body weight up to 3 months.
Quantitative real-time PCR
Quantitative real-time PCR was performed to determine the WSSV
viral loads in shrimp tissues samples collected at 2, 5, 7, 10, 15 and 25
dpi. The viral copy number was estimated by LightCycler ® real-time
PCR machine (Roche Applied Sciences, Indianapolis, IN, USA) using
DyNAmo™SYBR® Green qPCR Kit (Finnzymes, Espoo, Finland). Briefly,
the primers QVP26F-5′-ATCTCTACCGTCACACAGCC-3′ and QVP26R-5′GAAGATTTTAATGTCCTTGCTCG-3′ were used to amplify a 211-bp
fragment from the VP26 gene of WSSV. Viral DNA was isolated from
50 mg of gill tissue by using the QIAamp DNA tissue kit (Qiagen Inc.,
USA) according to the manufacturer's instructions. DNA was resuspended in 50 μl of DNase-RNase-proteinase-free water. The PCR
parameters consisted of 40 cycles of denaturation at 94 °C for 10 s,
annealing at 60 °C for 5 s, and extension at 72 °C for 10 s. A standard
curve was obtained using serial dilutions of plasmid pVP26 (full
length ORF of VP26 gene of WSSV was cloned into pQE-30 vector) and
was used to quantify the WSSV viral genomic copy number. The
geometric mean of viral genomic copies per reaction was calculated
for each group after setting results for negative samples to one copy
per sample. Each assay was carried out in triplicate.
Statistical analysis
The values are expressed as arithmetic mean ± SD. The level of
significance was expressed as P b 0.05. The protection against
WSSV after vaccination was calculated as the relative percent survival (RPS) (1 − mortality of vaccinated group / mortality of control
group) × 100 (33).
Acknowledgments
The authors are grateful for the financial support received from
Temasek Life Sciences Laboratory, Singapore. We are also thankful for
technical assistance of Mr. Ramaiah Rajkapoor, Mr. Govindarajan and
Mr. Subramanian Kabilan in animal maintenance.
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