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: KHV-lab@fra.affrc.go.jp 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