OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Spring viraemia of carp
1011 Fuqiang Road, Shenzhen
Guangdong province 518045
THE PEOPLE’S REPUBLIC OF CHINA
Tel.:
+86-755 25.58.84.10
Fax:
+86-755 25.58.86.30
e-mail address:
liuhong@szciq.gov.cn
website:
http://www.szciq.gov.cn/dzzx/
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Director Tikang Lu
Name(including Title and
Position) of OIE Reference
Expert:
Dr Hong LIU
Name (including Title and
Position) of writer of this report
(if different from above):
Annual reports of OIE Reference Centres, 2011
1
Spring viraemia of carp
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
Virus isolation is carried out using: EPC, FHM, CO cell lines.
Virus identification and characterization: RT-PCR and gene sequencing, ELISA
2.
Test
For
Specificity
Total
ELISA
Antigen
Group
32
Cell culture
Virus isolation
Virus RNA
241
RT-PCR and sequencing
Virus RNA
Subgroup
231
Real-time RT-PCR
Virus RNA
Virus RNA
32
Production and distribution of diagnostic reagents
The laboratory produced:

Rabbit antiserum against SVCV

Recombinant glycoprotein of SVCV in the system of E. coli

expression of SVCV matrix protein gene and the polyclonal antibody

Monoclonal antibodies of SVCV which will be useful in the ELISA test.
No diagnostic reagents were supplied nationally. The laboratory is using a commercially available SVC ELISA
test kit.
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
OIE Reference Laboratories are mandated to promote and disseminate diagnostic methods validated according to
OIE Standards. They should recommend the prescribed and alternative tests or vaccines as OIE Standards;
a)
Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular inter-laboratory
proficiency testing to ensure comparability of results
Describe separately your roles in the following (or related) activities:
 Organisation by your laboratory of international ring trials (scope, global/regional, number of
participants, outcomes)
 Participation in, or organisation of, interlaboratory test evaluations leading to international
harmonisation and validation (specify the tests, the organisers, participants, outcomes).
 International co-operation on harmonising methods for production and quality control of vaccines.
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Annual reports of OIE Reference Centres, 2011
Spring viraemia of carp
b)
Organisation of inter-laboratory proficiency testing with laboratories other than OIE
Reference Laboratories for the same pathogens and diseases to ensure equivalence of
results
A ring test on SVCV ELISA with Tai Lung Veterinary Laboratory, Agriculture, Fisheries & Conservation
Department, Hong Kong.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
There have been no activities in this area
5.
Research and development of new procedures for diagnosis and control
Development of a real-time RT-PCR assay for detection of spring viraemia of carp virus (SVCV):
A real-time quantitative RT-PCR assay was developed using a Taqman probe to detect and quantify Spring
viraemia of Carp Virus (SVCV) popular in common carp fish. A pair of primers, which amplify a 81bp DNA
fragment, and a Taqman probe were designed that are specific for the recognition of a conservative region in
SVCV G protein. SVCV real-time RT-PCR assay had a detection limit of 40 virus copies, with a dynamic range of
detection between 4×107 ~40 virus copies. Regression analysis showed a significant correlation (R2=0.9916)
between genomic number and threshold cycle (Ct). This quantitative method was found to be highly reproducible,
with the coefficient of variation (CV) of the intra- and inter-assay ranged from 0.16% to 1.93% and 1.09% to
3.76%, respectively. The detection limit of Taqman assay was sen than conventionl PCR and was similar to that of
virus isolation.
Development of a LAMP assay for detection of spring viraemia of carp virus (SVCV)
A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed for
the detection of SVCV. A set of four primers, two outer and two inner primers were designed based on the SVCVM gene for RT-LAMP assay. Under optimized amplification conditions (63C, 60 min), the RT-LAMP was used
to detect SVCV RNA and other RNAs extracted from several RNA viruses including pike fry rhabdovirus
(PFRV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and infectious
hematopoietic necrosis virus (IHNV). Good specificity of the RT-LAMP method was demonstrated as amplified
product was detected only from SVCV RNA but not from other virus RNAs. The test limit was found to be
10×TCID50/100uL in the detection of a serial diluted virus samples. In the detection of 322 clinical samples, the
RT-LAMP showed excellent agreement with RT-PCR. Because it is rapid and easy to use, the RT-LAMP is
suitable for SVCV monitoring and detection, especially for screening large number of samples in the farm.
High-level expression and purification of recombinant glycoprotein of spring viraemia of carp virus (SVCV)
Glycoprotein gene (SVC-g) of spring viraemia of carp virus was cloned. The SVC-g gene was subcloned into
plasmid pET-28 and the resultant plasmid was transformed into E.coli BL-21 (DE3). The total protein was
obtained from E. coli cultures induced by IPTG Western blot using goat anti-SVCV strain. A large quantity of
recombinant glycoprotein can be produced by fermentation of recombinant bacteria.
Preparation and identification of monoclonal antibodies against spring viremia of carp virus
To prepare specific monoclonal antibodies (MAbs) against spring viremia of carp virus (SVCV), mouse myeloma
cells (SP2/0) were fused with spleen cells from BALB/c immunized with purified SVCV. Four hybridoma cell
lines secreting MAbs against SVCV were obtained by indirect ELISA. The titres of ascitic fluids of the MAbs
ranged from 1:160,000 to 1:640,000. Western blot analysis revealed that three MAbs (1F1, 3F5 and 4F9)
specifically recognized to 47 ku of nucleoprotein, while the MAb 3E1 only reacted with 69 ku of glycoprotein.
Epitope reaction analysis demonstrated that 1F1, 3F5, and 4F9 might recognize the same epitope, while 3E1 was
different. Indirect immunofluorescence assay showed that all of these MAbs reacted positively with the virus in
the SVCV-infected cells. These antibodies will be useful tools to establish immunological detection methods of
SVCV.
On-going research
Annual reports of OIE Reference Centres, 2011
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Spring viraemia of carp
To validate the real-time RT-PCR test, LAMP test and indirect ELISA test which are expected in the detection and
diagnostics for SVCV.
To assess the impact of SVCV sub-genotype 1a on major cultured and wild freshwater fish species.
To study on the expression of SVCV structural proteins with insect cell and baculovirus expression vectors.
To study on the molecular epidemiology of SVCV.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
A database of SVCV isolates is being developed, which includes the information of the SVCV isolates published,
the relevant publications for the specific isolate, sequence information, the molecular epidemiological analyse and
geographical information.
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
Provide details.
8.
Provision of consultant expertise to OIE or to OIE Member Countries
Provided information to:
Tai Lung Veterinary Laboratory, Agriculture, Fisheries & Conservation Department, Hong Kong.
Review of aquatic animal code (2011 version).
Review of the Manual of Diagnostic Tests for Aquatic Animals (2012 version) .
Responded to OIE on the chapter of SVC in the Manual of Diagnostic Tests for Aquatic Animals (2012 version)..
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
Two courses of five-day training was arranged for the technicians from Fisheries & Conservation Department,
Hong Kong at the end of September.
10. Provision of diagnostic testing facilities to other OIE Member Countries
There have been no activities in this area.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
No requests for organising scientific meetings were received from OIE or other international bodies.
12. Participation in international scientific collaborative studies
The laboratory participated in the Laboratory Proficiency Test organized by Veterinary Laboratory Quality
Assurance Unit of the U. K. The proficiency test contained two tests in one year.
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Annual reports of OIE Reference Centres, 2011
Spring viraemia of carp
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
None

Scientific publications in peer-reviewed journals

Lan Wengsheng, Liu Hong, Gao Longying, Shi Xiujie, Zheng Xiaocong, Je Junqiang, Lu Tikang, 2011. High
level expression and purification of recombinant glycoprotein of spring viraemia of carp virus (SVCV).
Chinese Journal of Animal Infectious Diseases, 18(2):18-22

ZHANG Peng, LIU Hong, CHEN Xiao-xuan, WU Zhi-xin, YU Jian-min, DU Juan, LAN Wen-sheng, SHI
Xiu-jie, ZHENG Xiao-cong, HE Jun-qiang, JIA Peng, SHEN Sisi, ZHU Jia-zeng, 2011. Preparation and
identification of monoclonal antibodies against spring viraemia of carp virus. Chinese Journal of Preventive
Veterinary Medicine, 33(4): doi: 10.3969/j.issn.1008-0589.2011.04.13

Other communications
Produced the 2011 Annual Report on SVC Reference Laboratory Activities for the OIE
_______________
Annual reports of OIE Reference Centres, 2011
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