Application of YHV-protease dsRNA for protection and therapeutic treatment against yellow head virus infection in Litopenaeus vannamei.

Abstract

While farming of the Pacific white shrimp Litopenaeus vannamei is well established in North and South America, the industry has more recently been introduced to Asia, and the Pacific white shrimp is now the most commonly farmed species in Thailand. However, outbreaks of yellow head virus (YHV) disease in the Pacific white shrimp have caused severe economic losses and currently there is no effective prevention or treatment of YHV infections. The YHV-protease double-stranded RNA (YHV-Pro dsRNA) can act as both a prophylactic agent and as a treatment to inhibit YHV replication in infected black tiger shrimp Penaeus monodon. The utility of this methodology to other shrimp species has not, however, been established. The purpose of this study was to determine whether YHV-Pro dsRNA can be applied to the Pacific white shrimp. To assess prophylactic efficiency, YHV-Pro dsRNA was injected into juvenile shrimp 24 h prior to challenge with YHV. Subsequent YHV replication was inhibited by YHV-Pro dsRNA as compared with injection of an unrelated dsRNA. For therapeutic treatment of YHV-infected shrimp, shrimp were challenged with YHV before dsRNA injection. Injection of YHV-Pro dsRNA up to 6 h post-infection resulted in the almost complete elimination of YHV replication. These results suggest that YHV-Pro dsRNA can also be broadly applied as a prophylactic agent to inhibit YHV replication and therapeutic treatment of YHV-infected Pacific white shrimp.

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DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 84: 167–171, 2009
doi: 10.3354/dao02044
Published April 6
INTRODUCTION
Farming of Pacific white shrimp Litopenaeus van-
namei is a common industry in North and South Amer-
ica and the industry has more recently become estab-
lished in Asian countries such as China, Taiwan,
Vietnam and Thailand (Briggs et al. 2005), primarily
because of the shrimp’s tolerance to a wide range of
salinities (0.5 to 45 ppt) and temperature (15 to 33°C).
However, it grows particularly well in low salinities of
around 10 to 15 ppt and at temperatures between 23
and 30°C (Wyban & Sweeney 1991). Additionally, spe-
cific pathogen free (SPF) and specific pathogen resis-
tant (SPR) broodstock are available. These positive
characteristics have resulted in a rapid expansion of
Pacific white shrimp farming in Asia with L. vannamei
© Inter-Research 2009 · www.int-res.com*Email: wanchai.a@chula.ac.th
NOTE
Application of YHV-protease dsRNA for protection
and therapeutic treatment against yellow head
virus infection in Litopenaeus vannamei
Wanchai Assavalapsakul1,*, Wanlop Chinnirunvong2, Sakol Panyim2,3
1Department of Microbiology, Faculty of Science, Chulalongkorn University, Phyathai Road, Pathumwan,
Bangkok 10330, Thailand
2Institute of Molecular Biology and Genetics, Mahidol University, Phutthamonthol Sai 4 Road, Phutthamonthol,
Nakhonpathom 73170, Thailand
3Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Phyathai, Bangkok 10400, Thailand
ABSTRACT: While farming of the Pacific white shrimp Litopenaeus vannamei is well established in
North and South America, the industry has more recently been introduced to Asia, and the Pacific
white shrimp is now the most commonly farmed species in Thailand. However, outbreaks of yellow
head virus (YHV) disease in the Pacific white shrimp have caused severe economic losses and cur-
rently there is no effective prevention or treatment of YHV infections. The YHV-protease double-
stranded RNA (YHV-Pro dsRNA) can act as both a prophylactic agent and as a treatment to inhibit
YHV replication in infected black tiger shrimp Penaeus monodon. The utility of this methodology to
other shrimp species has not, however, been established. The purpose of this study was to determine
whether YHV-Pro dsRNA can be applied to the Pacific white shrimp. To assess prophylactic effi-
ciency, YHV-Pro dsRNA was injected into juvenile shrimp 24 h prior to challenge with YHV. Sub-
sequent YHV replication was inhibited by YHV-Pro dsRNA as compared with injection of an unre-
lated dsRNA. For therapeutic treatment of YHV-infected shrimp, shrimp were challenged with YHV
before dsRNA injection. Injection of YHV-Pro dsRNA up to 6 h post-infection resulted in the almost
complete elimination of YHV replication. These results suggest that YHV-Pro dsRNA can also be
broadly applied as a prophylactic agent to inhibit YHV replication and therapeutic treatment of YHV-
infected Pacific white shrimp.
KEY WORDS: Litopenaeus vannamei · Viral disease · Yellow head virus · Double-stranded RNA ·
RNAi
Resale or republication not permitted without written consent of the publisher

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Dis Aquat Org 84: 167–171, 2009
now being the most commonly farmed shrimp species
in Thailand.
Despite the success of a few farms, culturing Pacific
white shrimp in low salinity inland waters has resulted
in a variety of problems that need to be addressed
(Davis et al. 2002). One of these issues is severe out-
breaks of yellow head virus (YHV) disease in Thailand
(Limsuwan et al. 2008). The symptoms and clinical
signs of YHV disease in Pacific white shrimp are simi-
lar to those of the black tiger shrimp Penaeus monodon
(Boonyaratpalin et al. 1993).
Previously, our group (Yodmuang et al. 2006, Tira-
sophon et al. 2007) investigated the YHV-protease
double-stranded RNA (YHV-Pro dsRNA), which can
act as a prophylactic agent as well as a treatment
designed to inhibit YHV replication in infected black
tiger shrimp. The purpose of this study is to determine
whether this methodology can be applied to the Pacific
white shrimp.
MATERIALS AND METHODS
Shrimp culture. Healthy Pacific white shrimp post-
larvae were purchased from commercial farms in Thai-
land. These shrimps were cultured in 80 l tanks con-
taining artificial seawater at 10 ppt salinity with
aeration. The shrimp were acclimated for 2 to 3 d at
28 ± 1°C before use in the experiments.
Yellow head virus stock. YHV titer was determined
using tissue culture infectious dose 50 (TCID50) as
described by Reed & Muench (1938) using a primary
lymphoid organ cell culture of Penaeus monodon as
described in Assavalapsakul et al. (2003). The YHV
stock used in this study was 2 × 109TCID50ml–1.
Double-stranded RNA production. Recombinant
clones expressing the stem-loop of the YHV-protease
(Pro) RNA (Fig. 1) or the stem-loop of green fluores-
cence protein (GFP) RNA were used to produce
dsRNA for injection into shrimp as described in Yod-
muang et al. (2006). The dsRNA concentration was
estimated by spectrophotometry at OD260and stored at
–80°C until use.
dsRNA injection and YHV challenge. For inhibition
of YHV in Pacific white shrimp, healthy Pacific white
shrimp (3 g) were injected with 50 µl of control saline
(150 mM NaCl) or with 2.5 µg YHV-Pro dsRNA or GFP
dsRNA per gram of shrimp in 150 mM NaCl into the
haemolymph through the arthrodial membrane of the
fifth walking leg by means of a 1 ml syringe with a 29
gauge needle. At 24 h post-injection, shrimp were
injected with 5 × 103TCID50ml–1of YHV in 150 mM
NaCl into the haemolymph through the arthrodial
membrane of the fifth walking leg. This amount of
virus had been previously titrated to cause complete
mortality in 2 to 4 d. To evaluate YHV presence in
shrimp, gill tissues were collected 48 h after YHV chal-
lenge from individual shrimp for RNA extraction and
RT-PCR analysis.
For mortality assays, healthy Pacific white shrimp
(3 g) were used (8 shrimp per group). Shrimp were
injected with 2.5 µg dsRNA g–1shrimp in 150 mM
NaCl. At 24 h post-injection of dsRNA 5 × 103TCID50
ml–1YHV was injected into the haemolymph through
the arthrodial membrane of the fifth walking leg. Mor-
tality was recorded twice a day for 14 d after the YHV
challenge. Four independent experiments were con-
ducted.
To test the therapeutic inhibition ability of YHV-Pro
dsRNA in Pacific white shrimp, initially shrimp were
challenged with 5 × 103TCID50ml–1of YHV in 150 mM
NaCl, and then injected with control saline, GFP
dsRNA or YHV-Pro dsRNA at 3, 6, 9, 12 and 24 h after
being challenged. After the 48 h challenge, the gills of
each shrimp were collected and used for RNA extrac-
tion and YHV genome analysis by multiplex RT-PCR.
Statistical analysis. All data were analyzed using the
GraphPad Prism program (GraphPad Software). All
data obtained from the experiments were analyzed
using 1-way ANOVA (p < 0.05 as significance level).
168
Fig. 1. Schematic representation of the YHV genome. Organization of the YHV genome is shown and the position of the YHV-Pro
dsRNA (nucleotides 8667 to 9089) is indicated, as is the position of the helicase RT-PCR product (nucleotides 16661 to 17515)

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Assavalapsakul et al.: Application of YHV-Pro dsRNA in Litopenaeus vannamei
RT-PCR analysis. Either 2 or 3 pieces of gill tissue
were collected from individual shrimp for RNA isola-
tion using TRI Reagent solution (Molecular Research
Center). Total RNA (1 µg) was used for cDNA synthe-
sis using Imprompt II reverse transcriptase (Promega)
and oligo dT18primer as described by the manufac-
turer. Detection of YHV genomic RNA was performed
by multiplex PCR. Primer pairs for YHV helicase gene
(Fig. 1) (5’-CAA GGA CCA CCT GGT ACC GGT AAG
AC-3’ and 5’-GCG GAA ACG ACT GAC GGC TAC
ATT CAC 3’) (Yodmuang et al. 2006, Tirasophon et al.
2007) were used to monitor YHV level whereas primer
pairs for Litopenaeus vannamei actin, based on the
gene sequence available in GenBank (accession no.
AF300705), (5’-GAC TCG TAC GTC GGC GAC GAG
G-3’ and 5’-GCA GCG GTG GTC ATC TCC TGC
TCG-3’) (Yodmuang et al. 2006, Tirasophon et al. 2007)
were used for RNA normalization and internal control.
The multiplex PCR conditions used were as follows:
94°C for 3 min, followed by 25 cycles of 94°C for 30 s,
53°C for 30 s and 72°C for 45 s, with a final extension at
72°C for 10 min. The PCR products were analyzed by
1.5% agarose gel electrophoresis followed by staining
in 0.5 µg ml–1ethidium bromide.
RESULTS
Prophylactic inhibition of YHV replication in Pacific
white shrimp
To investigate whether dsRNA could provide prophy-
lactic protection against YHV replication in Pacific white
shrimp, YHV-Pro dsRNA, GFP dsRNA or control saline
were injected into shrimp 24 h before the YHV challenge
with 5 ×103TCID50ml–1per shrimp and then sampled for
PCR 48 h post-YHV exposure. As illustrated in Fig. 2, the
presence of the YHV genome was detected in all control
saline and GFP-dsRNA-injected shrimp, while no YHV
genome was detected in shrimp injected with YHV-Pro
dsRNA. This suggests that prophylactic YHV-Pro dsRNA
injection effectively inhibited YHV replication.
Prevention of shrimp mortality by dsRNA
From the preceding experiment it was clear that pro-
phylactic injection of YHV-Pro dsRNA was able to re-
duce the YHV load to undetectable levels. To determine
whether this affected shrimp mortality, the above exper-
iment was repeated and this time shrimp were observed
for a period of 14 d post-YHV infection with mortality
recorded twice daily. The experiment was repeated in
quadruplicate, with 8 shrimp per group. Complete mor-
tality (100%) was observed by Day 3 post-YHV infection
in the control-saline injected (p < 0.0001) and GFP-
dsRNA-injected (p < 0.0001) groups (Fig. 3). In contrast,
nearly 100% survival was recorded on Day 14 for the
YHV-Pro-dsRNA-injected shrimp (p < 0.0001).
Therapeutic inhibition of YHV-Pro dsRNA
Analyses of the control saline and GFP-dsRNA-
injected shrimp clearly showed the presence of the
169
Fig. 2. Litopenaeus vannamei. Inhibitory effect of YHV replication in Pacific white shrimp using YHV-Pro dsRNA. Shrimp (~3 g)
were injected with (A) NaCl, (B) GFP dsRNA or (C) YHV-Pro dsRNA 24 h before injection challenge with a saline suspension of
YHV. The YHV load in gills of individual shrimp was determined by RT-PCR. Lane M is 1 kb Plus DNA ladder (Invitrogen)
0
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Day(s)
Percent cumulative mortality
of shrimp
NaCl
GFP dsRNA
YHV-Pro dsRNA
Fig. 3. Litopenaeus vannamei. Percent cumulative mortality
of Pacific white shrimp. Shrimp (~3 g) were injected with
NaCl, GFP dsRNA or YHV-Pro dsRNA for 24 h before in-
jection challenge with a saline suspension of YHV. Shrimp
mortality was recorded twice daily for 16 d. Percent mortality
in each experimental group is presented as the mean of
4 replicate experiments

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Dis Aquat Org 84: 167–171, 2009
YHV genome in all samples from all shrimp tested
(Fig. 4). In contrast, no shrimp injected with YHV-Pro
dsRNA at 3 h post-infection displayed the presence of
the YHV genome, and only 1 shrimp out of 6 injected
with YHV-Pro dsRNA at 6 h post-infection had
detectable levels of the YHV genome. The majority of
shrimp injected at 9 h or later post-infection displayed
the presence of the YHV genome (Fig. 4).
DISCUSSION
YHV is a highly pathogenic agent that causes an
acute and often fatal disease in Penaeus monodon
(Boonyaratpalin et al. 1993). Recently, YHV has caused
mass mortality of cultured Litopenaeus vannamei
resulting in significant economic losses in Thailand
(Limsuwan et al. 2008). RNAi, a sequence-specific
gene-silencing mechanism that has been proposed to
function as a defense mechanism of eukaryotic cells
against viruses and transposons (Haasnoot & Berkhout
2006), was employed to ameliorate mass mortality of
Pacific white shrimp. Robalino et al. (2005) suggested
that low level viral loads in shrimp could be completely
inhibited by injecting a specific dsRNA prior to viral
multiplication. In the present study, an optimized
2.5 µg of dsRNA g–1shrimp and approximately 5 ×
103TCID50ml–1of YHV were used to study prophylac-
tic and therapeutic treatment of YHV replication in
Pacific white shrimp. Using these optimized condi-
tions, non-specific inhibition of YHV by a sequence-
unrelated dsRNA (GFP dsRNA) was not observed. The
results showed that the YHV-Pro dsRNA could effi-
ciently reduce YHV replication when administered
prior to infection.
For therapeutic treatment, inhibition of YHV repli-
cation was most effective at 3 h post-infection and the
effectiveness decreased after longer exposure to the
virus. Thus, at 6 h post-infection approximately 83% of
shrimp showed viral inhibition while inhibition disap-
peared at 24 h post-infection. This phenomenon sug-
gests a competition between YHV replication and the
RNAi pathway in shrimp cells. At 3 h post-infection,
dsRNA should be processed to siRNA (short interfering
RNA) and loaded into RISC (RNA-Induced Silencing
Complex) at a sufficient level to knock out the viral pro-
tease mRNA or the genome. Without the viral protease
the viral polyprotein should not be processed, making
YHV unable to replicate in the shrimp cell. This study
suggests that therapeutic treatment is effective as long as
the infection is not too far advanced. In studies on the
black tiger shrimp, YHV-Pro dsRNA was shown to be
therapeutically effective up to 12 h post-infection (Tira-
sophon et al. 2007). This difference may result from the
significantly larger shrimp used in that study (12 g versus
3 g used in the present study) or may point to species-
specific differences, which would have significant impli-
cations for the therapeutic use of this technology in an
aquaculture setting. The failure to cure YHV-infected
shrimp beyond a certain time post-infection may result
from the ability of YHV to generate antiviral RNAi sup-
pressors including small RNAs or proteins (Silhavy &
Burgyán 2004, Shi et al. 2008). Our results suggest that
YHV-Pro dsRNA can be broadly applied as a prophylac-
tic agent to inhibit YHV replication and as a therapeutic
treatment of YHV-infected Pacific white shrimp. How-
ever, since vaccination of farmed shrimp populations is
not practical, further experimentation is needed to deter-
mine whether oral administration of YHV-Pro dsRNA is
effective in preventing or treating YHV infection.
170
Fig. 4. Litopenaeus vannamei. Time effect for curing YHV-infected Pacific white shrimp using YHV-Pro dsRNA. Shrimp (~3 g)
were injected with a YHV suspension followed by injection with (Panel A) NaCl, (Panel B) GFP dsRNA or (Panel C) YHV-Pro
dsRNA at various time intervals; Lanes 1 to 6: 3 h post-infection; Lanes 7 to 12: 6 h post-infection; Lanes 13 to 18: 9 h post-
infection; Lanes 19 to 24: 12 h post-infection; and Lanes 26 to 30: 24 h post-infection. The YHV load in gills of individual shrimp
was determined by RT-PCR. Lane M is 1 kb Plus DNA ladder (Invitrogen). X in Lane 6 denotes a shrimp that died before the
start of the experiment

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Assavalapsakul et al.: Application of YHV-Pro dsRNA in Litopenaeus vannamei
Acknowledgements. This work was supported by the Thai-
land Research Fund (TRF) and the Commission on Higher
Education (CHE). W.A. is supported by TRF-CHE Grant
MRG4880054. S.P. is a TRF Senior Research Scholar.
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Editorial responsibility: Ken Hasson,
College Station, Texas, USA
Submitted: October 29, 2008; Accepted: February 10, 2009
Proofs received from author(s): April 5, 2009
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