OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
White spot disease
Address of laboratory:
Institute of Zoology,
College of Life Science,
National Taiwan University,
No.1, Sec, Roosevelt Road,
Taipei 106, Taiwan,
CHINESE TAIPEI
Tel.:
(886-2) 2363-3562
Fax:
(886-2) 2363-8179
e-mail address:
website:
gracelow@ntu.edu.tw
http://shrimpwssv.lifescience.ntu.edu.tw/
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Prof/Dr Grace Chu-Fang Lo
Name(including Title and
Position) of OIE Reference
Expert:
Prof/Dr Grace Chu-Fang Lo
Name (including Title and
Position) of writer of this report
(if different from above):
Annual reports of OIE Reference Centres, 2011
1
White spot disease
Part I: Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease/topic at your laboratory
Total
Test
for
Specificity/purpose
Diagnosis
Research
PCR
Viral genome
All known WSSV genotypes
1350
1960
Real-Time RT-PCR
Viral genome
All known WSSV genotypes
0
2654
RT-PCR
Gene transcripts
For specific WSSV/shrimp gene
0
1079
Western blot assay
Antigen
For specific WSSV/shrimp protein
0
1197
Confocal microscopy
Antigen
For specific WSSV/shrimp protein(s)
in viral infected cell
0
4
TEM
Viral particle
All known WSSV genotypes
12
0
2-DE and mass/mass
spectrometry
Shrimp and virus protein
identification
Pathogenesis at molecular and
cellular levels
0
2
Far Western
Protein-protein
interaction
Host-virus interactions
0
7
Co-Immunoprecipitation
Protein-protein
interaction
Host-virus interactions
0
20
Yeast-two hybrid assay
Protein-protein
interaction
Host-virus interactions
0
500
EMSA
Protein -nucleic acid
interaction
Host-virus interactions
0
600
QPCR
Gene/Viral genome copy
number
For specific WSSV/shrimp gene
0
96
Southern blot
Gene transcripts
For specific WSSV/shrimp gene
0
250
2
Annual reports of OIE Reference Centres, 2011
White spot disease
2.
Production and distribution of diagnostic reagents
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied
to other countries
Antibody for P. monodon 1433 protein
~200 μl
0
Antibody for P. monodon IAP (inhibitor of apoptosis
proteins)
~400 μl
0
Antibody for P. monodon Hemocyanin (C-terminal
region)
~400 μl
0
Antibody for P. monodon β-tubulin
~200 μl
0
P. monodon Enolase
~200 μl
0
Antibody for P. monodon Heat shock protein 70
~400 μl
0
Antibody for P. monodon Heat shock protein 90
~200 μl
0
Antibody for P. monodon small heat shock protein
~200 μl
0
Antibody for P. monodon ribosomal protein L23
~800 μl
0
Antibody for P. monodon STAT (Signal Transducer and
Activator of Transcription)
~1ml
0
Antibody for P. monodon thioredoxin
~1ml
0
Antibody for WSSV VP664
~800 μl
200 μl
Antibody for WSSV VP26
~800 μl
200 μl
Antibody for WSSV VP28
~800 μl
200 μl
Antibody for WSSV ICP11
~1mlμl
0
Antibody for WSSV VP51C
~800 μl
200 μl
Antibody for WSSV VP38A
~800 μl
0
Antibody for WSSV VP11
~200 μl
0
Antibody for WSSV VP31
~200 μl
0
Antibody for WSSV VP39B
~200 μl
0
100 ampoules of 10 μl for
WSSV
0
100 chips
25 chips
1. Antibodies against shrimp proteins
2. Antibodies against WSSV proteins
3. Viral inoculum
4. Shrimp & viral gene microarray chip
Annual reports of OIE Reference Centres, 2011
3
White spot disease
Part II: Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
International co-operation with Dr. Lightner’s lab at Department of Veterinary Science, University of Arizona and
an R & D team of GeneReach Biotechnology Corporation (Taiwan) on an interlaboratory evaluation of a new
WSSV kit which has been is developed by GeneReach Biotechnology Corporation and is intended for WSSV
DNA detection from aquaculture specimens based on insulated isothermal PCR technology.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
In the last year, we have prepared standard reference materials including 1) WSSV-infected tissues for positive
control of protein/nucleic acid assays (for more than 1,000 reactions), 2) WSSV nucleic acid for PCR control (for
more than 1,000 reactions), 3) viral proteins for ELISA (for more than 100 reactions/per antigen), 4) viral protein
specific antibodies for western blot analysis (40 ml for each viral protein). We supplied 800μl viral protein specific
antibodies to Belgium, and plasmid DNA containing a WSSV gene fragment (2.5 μg) to Canada.
We also prepared purified WSSV virions/ nucleocapsids and shrimp gene dsDNA chips. We supplied 1μg purified
WSSV virions to Japan and 25 chips to Thailand.
5.
Research and development of new procedures for diagnosis and control
We collaborated with Prof. Wei-Heng Shih's group at Drexel University, USA on label free detection of WSSV
using lead magnesium niobate-lead titanate piezoelectric microcantilever sensors and their application. The
sensitivity of this method has so far not exceeded that of conventional, OIE-certified PCR-based kits.
We have also given comments to GeneReach Biotechnology Corporation on the development of a new WSSV
detection Kit that runs on newly developed compact and portable equipment and is based on the insulated
isothermal PCR (iiPCR) technology. This Kit and its supporting the equipment are specifically designed for
WSSV detection at pond-side.
We investigated the roles of copepods and bivalve mollusks in the transmission of WSSV. The copepod
Apocyclops royi and bivalve mollusk Meretrix lusoria were experimentally challenged with WSSV and then
assayed for both the presence of the virus and for viral gene expression. We showed that the WSSV genome could
be detected and that the viral loads were increased in a time-dependent manner after challenge both in A. royi and
M. lusoria. Reverse transcriptase PCR monitoring of WSSV gene expression showed that WSSV could replicate
in A. royi but not in M. lusoria, which suggested that WSSV, while could infect A. royi, was only accumulated in
M. lusoria. A bioassay further showed that the WSSV accumulated in M. lusoria could be transmitted to
Litopenaeus vannamei and cause severe infection. This is an important information for the development of
measures to control WSD. The details were published in Mar. Biotechnol. (2011) 13:909–917.
In general, research activities of this reference Laboratory continue to aim at the following topics, which are
potentially very useful for the development of measures to control WSD.




6.
The strategies evolved by WSSV to thwart host responses to infection and ensure successful virus replication
in cells
Analysis of the WSSV viral protein interaction networks and the stress response induction mechanisms in
shrimp
Development of platform technologies and resources for studying WSSV itself and WSSV-host interactions
Advances in knowledge for the better understanding of WSSV and its interactions with shrimp
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
None in 2011.
4
Annual reports of OIE Reference Centres, 2011
White spot disease
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
None in 2011
8.
Provision of consultant expertise to OIE or to OIE Member Countries
Drafting the White Spot Disease Chapter of the OIE Aquatic Manual and drafting the response to the comments
from member countries.
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
None in 2011.
10. Provision of diagnostic testing facilities to other OIE Member Countries
Testing of dry shrimp feed pellets for WSSV contamination on behalf of a Vietnamese shrimp feed company. On
request, the protocols and testing procedures were subsequently forwarded to, and adapted by Center for
Veterinary Diagnostics, Regional Animal Health Office No.6 (RAHO6),VIET NAM.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
As Execom Chairperson (2008-2011) of the Fish Health Section (FHS) of Asian Fisheries Society (AFS), I was
involved in organizing the Eighth Symposium on “Diseases in Asian Aquaculture” (DAA8) and worked very
closely with Local Organization Committee and the International Scientific Program Committee. DAA8 was held
in Mangalore on 21-25 November, 2011. The theme of DAA 8 was “Fish Health for Food Security”. Over the
5 days, 4 thematic, 24 key note and 47 contributed papers were presented in 13 technical sessions covering
disease, diagnostics, vaccines, genomics, disease preparedness and national plans. There were also 170 poster
presentations.
12. Participation in international scientific collaborative studies
1.
Collaboration with Assoc. Prof. Amornrat Phongdara’s group at the College of Science, Prince of Songkla
University, Thailand on molecular mechanisms of shrimp fortilin to protect cells from WSSV infection.
Using an insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the
expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an
immediate early gene ie1. The results were published in Developmental and Comparative Immunol. 2011,
35: 469-475.
2.
Collaboration with Dr Saengchan Senapin at National Center for Genetic Engineering and Biotechnology,
National Science and Technology Development Agency, Thailand on shrimp cell receptors for shrimp
viruses. The results was published in Fish Shellfish Immunol. 2011, 31(1): 66-72.
3.
Collaboration with Timothy W. Flegel at Centex Shrimp, Faculty of Science, Mahidol University, Bangkok,
Thailand on Shrimp -WSSV interaction. The overall results suggested that shrimp PmFKBP46 might be
involved in genome packaging by viral VP15 during virion assembly. The results were published in PLoS
ONE. 2011, 6(9): e25420.
4.
Collaboration with Prof. Jan-Ming Ho's team at the Institute of Information Science, Academia Sinica,
Chinese Taipei to constract database of shrimp Shrimp ESTs. We summarized the sixteen major shrimp EST
studies that have been conducted to date. In addition, we analyzed the EST data downloaded from NCBI
dbEST for the four major penaeid shrimp species and constructed a database to host all of these EST data as
well as our own analysis results. This database provides the shrimp aquaculture research community with an
outline of the shrimp transcriptome as well as a tool for shrimp gene identification. All the analysis results
are accessible at the database: http://sysbio.iis.sinica.edu.tw/page/. The paper was published in Mar
Biotechnol. 2011, 13:608–621
Annual reports of OIE Reference Centres, 2011
5
White spot disease
13. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)

Presentations at international conferences and meetings
1.
Lo, C. F.* (2011). Using systems biology approaches to understand shrimp cellular responses during viral
infection. 8th Symposium on Diseases in Asian Aquaculture (DAA8).Mangalore, India. November 21-25,
2011.
2.
Lin, Y. R., Hung, H. C., Leu, J. H., Wang, H. C., Kou, G. H.* and Lo, C. F.* (2011) The role of ALDH and
HSP70 in the suppression of white spot syndrome virus replication at high temperature. 9th Asian Fisheries
& Aquaculture Forum (9AFAF). Shanghai, China. April. 21-25, 2011.

Scientific publications in peer-reviewed journals
1.
Chang, L. K., Huang, P. H., Shen, W. T., Yang, S. H., Liu, W. J., Lo, C. F. (2012) Role of Penaeus monodon
Kruppel-like factor (PmKLF) in infection by white spot syndrome virus. Dev. Comp. Immunol. 36 (1) 121129.
2.
Pakkakul Sangsuriya, Saengchan Senapin, Wei-Pang Huang, Lo C. F., Flegel, T. W.*(2011) Co-Interactive
DNA-Binding between a Novel, Immunophilin-Like Shrimp Protein and VP15 Nucleocapsid Protein of
White Spot Syndrome Virus. PLoS ONE. 2011 6(9):e25420.
3.
Liu, W. J., Chang, Y. S., Huang, W. T., Chen, I. T., Wang, KC H. C., Kou, G. H., Lo, C. F. (2011) Penaeus
monodon TATA box binding protein (PmTBP) interacts with the WSSV transactivator IE1 and promotes its
transcriptional activity. J. Virol. 85(13), 6535-47.
4.
Busayarat N, Senapin S, Tonganunt M, Phiwsaiya K, Meemetta W, Unajak S, Jitrapakdee S, Lo C. F.,
Phongdara A. (2011) Shrimp laminin receptor binds with capsid proteins of two additional shrimp RNA
viruses YHV and IMNV. Fish Shellfish Immunol. 31(1), 66-72.
5.
Lin, Y. R., Hung, H. C., Leu, J. H., Wang, H. C., Kou, G. H. and Lo, C. F. (2011) The role of ALDH and
HSP70 in the suppression of white spot syndrome virus replication at high temperature. J. Virol. 85(7), 35173525.
6.
Chang, Y. S., Chen, T. C., Liu, W. J., Kou, G. H., Lo, C. F. (2011) Assessment of the roles of copepod
Apocyclops royi and bivalve mollusk Meretrix lusoria in white spot syndrome virus transmission. Mar
Biotechnol. 13(5), 909-917.
7.
Nupan B, Phongdara A, Saengsakda M, Leu, J. H., Lo, C. F. (2011) Shrimp pm-fortilin inhibits the
expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model. Dev. Comp.
Immunol. 35(4) 469-475.
8.
Leu, J. H., Chen, S. H., Wang, Y. B., Chen, Y. C., Su, S. Y., Lin, C. Y., Ho, J. M., Lo, C. F. (2011) A review
of the major penaeid shrimp EST studies and the construction of a shrimp transcriptome database based on
the ESTs from four penaeid shrimp. Mar Biotechnol 13(4), 608-21.
9.
Huang, S. W., Lin, Y. Y., You, E. M., Liu, T. T., Shu, H. Y., Wu, K. M., Tsai, S. F., Lo, C. F., Kou, G. H.,
Ma, G. C., Chen, M., Wu, D., Aoki, T., Ikuo Hirono and Yu, H. T. * (2011) Fosmid library end sequencing
reveals a rarely known genome structure of marine shrimp Penaeus monodon. BMC Genomics (12)242.

Other communications
Book Chapters:
1.
Lo, C. F., Aoki, T., Bonami, J. R., Flegel, T. W., Leu, J. H., Lightner, D. V., Stentiford, G., Söderhäll, K.,
Walker, P. W. Wang, H. C.., Xun, X., Yang, F., and Vlak, J. M. Nimaviridae, p.229-234. In Virus
Taxonomy: IXth Report of the International Committee on Taxonomy of Viruses, (A. King, M. Adams, E.
Carstens, and E. Lefkowitz eds.).
_______________
6
Annual reports of OIE Reference Centres, 2011