RT-PCR

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OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Address of laboratory:
Koi herpesvirus disease
National Research Institute of Aquaculture,
Fisheries Research Agency
Nakatsuhamaura, Minami-Ise,
Watarai, Mie 516-0193,
JAPAN
Tel.:
(+81) 599 66 1830
Fax:
(+81) 599 66 1962
e-mail address:
[email protected]
website:
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Head of Fish diagnostic group
Dr Kei Yuasa
Name(including Title and
Position) of OIE Reference
Expert:
Head of Fish diagnostic group
Dr Kei Yuasa
Name (including Title and
Position) of writer of this report
(if different from above):
Annual reports of OIE Reference Centres, 2011
1
Koi herpesvirus disease
Part I: Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
PCR
DNA
Type
48
TaqMan quantitative PCR
DNA
Type
243
ELISA
antibody
Group
30
KF-1 or CCB cell culture
Virus isolation
RT-PCR
Viral mRNA
15
Type
135
Production and distribution of diagnostic reagents
Type of reagent
Ethanol fixed gills of carp experimentally
infected with KHV isolate (NRIA0301)
Amount supplied nationally
(including for own use)
Amount supplied to
other countries
26 micro-tubes of 0.1g tissue
2 micro-tubes of 0.1g
tissue
DNA extract from the ethanol fixed gills
2 micro-tubes of 0.1 ml
Plasmids DNA inserted with Sph 1-5 region
10 micro-tubes of 0.05 ml
2 micro-tubes of 0.05 ml
Plasmids DNA inserted with 9/5 region
9 micro-tubes of 0.05 ml
2 micro-tubes of 0.05 ml
Viral supernatants obtained from CCB cells
infected with KHV type strain, NRIA0301
24 micro-tubes of 1 ml
Viral supernatants obtained from CCB cells
infected with other 11 KHV isolates
10 micro-tubes of 1 ml for each
isolate
CCB cells
KF-1 cells
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
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
Participated in KHV proficiency test held by Vetqas.
2
Annual reports of OIE Reference Centres, 2011
Koi herpesvirus disease
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
Participated in a worldwide interlaboratory ring-test held by Dr Niels Jorgen Olesen and Nicole Nicolajsen of
EU Reference Laboratory for Fish Diseases.
4.
5.
Preparation and supply of international reference standards for diagnostic tests or vaccines
Type of standards
Quantities available
Quantities supplied
Ethanol fixed gills of carp experimentally
infected with KHV type strain
(NRIA0301)
10 micro-tubes of 0.1g tissue
2 micro-tubes of 0.1g tissue
DNA extract from the ethanol fixed gills
10 micro-tubes of 0.1 ml
2 micro-tubes of 0.1 ml
Plasmids DNA inserted with Sph 15 region
100 micro-tubes of 0.05 ml
2 micro-tubes of 0.05 ml
Viral supernatants obtained from CCB
cells infected with KHV type strain
(NRIA0301)
10 micro-tubes of 1 ml
Research and development of new procedures for diagnosis and control
1. Verification of a RT-PCR assay detecting mRNA of the virus: Viral mRNA specific RT-PCR was developed
and verified. The manuscript on this assay has submitted to Dis. Aquat. Org.
2. Study on fate of the virus in fish and appropriate target tissues for PCR detection of subclinical carrier fish: It is
likelihood that a final habitat of virus is the brain in infected fish. The manuscript on this topic is now preparing.
3. Evaluation of several detection methods for koi herpesvirus (KHV) : PCR with the gills and skin was useful to
diagnose the disease, but additional PCR with the brain and intestine or ELISA is essential to detect latent KHV.
PCR detection from water, cohabitation and RT-PCR using the gills or skin were similarly effective to detect fish
shedding the virus.
4. Epizootiological study on the detection of virus from fish in natural waters: It was found that high anti-KHV
antibody was continuously detected from fish inhabiting rivers where mortalities due to KHV were observed
several years ago.
5. Development of more susceptible cell line for the virus isolation: Several new cell lines from carp were
prepared. Sensitivities of them to the virus will be studies in the future. The manuscript describing procedure for
successful isolation of KHV has submitted to Fish Pathology.
6. Comparison of pathogenicity to carp among year-on-year isolates: Pathogenicity of 12 KHV isolates obtained
between 2003 and 2011 to carp was studied. The result indicates that no change of pathogenicity in each isolate
was observed.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
None
Annual reports of OIE Reference Centres, 2011
3
Koi herpesvirus disease
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
None
8.
Provision of consultant expertise to OIE or to OIE Member Countries
Provided a technical training on diagnosis for KHV disease to Dr. Hyoung-jin kim and Ms. Myoug-hee Sung,
Quality Inspection Division, National Fisheries Products Quality Inspection Service (NFIS), Koria during 13-17,
June 2011.
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
None
10. Provision of diagnostic testing facilities to other OIE Member Countries
None
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
None
12. Participation in international scientific collaborative studies
None
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

Yuasa K, Miwa S, Kawana M, Ito T, Kiryu I, Sano M and Oseko N: Evaluation of several detection methods
for koi herpesvirus (KHV), Book of abstract in 8 th Symposium on Diseases in Asian Aquaculture held on 2125 November 2011 in India, p17.

Scientific publications in peer-reviewed journals
None

Other communications

Yuasa K: Present status of Koi herpesvirus disease (KHV) occurrences and prevention measures in Japan,
Journal of Veterinary Medicine: 64(2), 109-113, 2011 (in Japanese).
_______________
4
Annual reports of OIE Reference Centres, 2011
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