OIE Reference Laboratory Reports Activities in 2011
Name of disease (or topic) for Infection with abalone herpes-like virus which you are a designated OIE Reference Laboratory:
Address of laboratory: National Taiwan University School of Veterinary Medicine, 1 Sec. 4 Roosevelt Rd Taipei 10617 CHINESE TAIPEI
Tel.: 886 2 33661296
Fax: 886 2 23661475
e-mail address: [email protected]
website: http://www.vm.ntu.edu.tw/DVM/
Name (including Title and Pen Heng Chang Position) of Head of Laboratory (Responsible Official):
Name(including Title and Pen Heng Chang Position) of OIE Reference Expert:
Name (including Title and Position) of writer of this report (if different from above):
Annual reports of OIE Reference Centres, 2011 1 Infection with abalone herpes-like virus
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
Test For Specificity Total
PCR Viral DNA antigen 120
Real time PCR Viral DNA antigen 30
Histopathology Microscopic lesion lesions 30
Electron microscopy Virus Virus particle 5
2. Production and distribution of diagnostic reagents
Amount supplied nationally Amount supplied to other Type of reagent (including for own use) countries
Positive control DNA 300 µl 300 µl
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
Not.
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
Not.
4. Preparation and supply of international reference standards for diagnostic tests or vaccines
Positive control DNA for real time PCR prepared using TOPO TA cloning kit. The primer sets were provided by Dr Mark Crane, CSIRO Livestock Industries, Australia. The positive control DNA is suggested to do 100X dilution in real time PCR study. We can supply 100 µl cloned positive control DNA per request. And, a total of 300 µl has supplied to other OIE Member Countries.
Positive control DNA derived from DNA polymerase gene of abalone herpes-like virus is ready to supply to other OIE Member Countries.
2 Annual reports of OIE Reference Centres, 2011 Infection with abalone herpes-like virus
5. Research and development of new procedures for diagnosis and control
Development of a polymerase chain reaction for the detection of abalone herpes-like virus infection based on the DNA polymerase gene:
This is a consecutive study. In this study, abalone herpesvirus particles were separated from Taiwanese abalone tissues (AbHV) via a discontinuous sucrose gradient. DNA was extracted from purified virus pellets and sequenced. Analysis of large genome fragments of the Taiwanese isolate using the supermatcher program revealed that the TC04 fragment of the Taiwanese isolate (NCBI accession no. JN083851) had 85.7% (10481/12224) identity to the Victoria/AUS/2007 AbHVscaffold_3172-3200 fragment. The TC02 (NCBI accession no. JF967012) and TC08 fragments (NCBI accession no. HQ890941) of the Taiwanese isolate corresponded to the Victoria/AUS/2007 AbHVscaffold_3197-3033 isolate, and had 66.7% (26,884/40,281) and 61.2% (14,529/23,756) identities, respectively. These results suggested that the Taiwanese isolate, Taiwan/2004, can be interpreted as a different isolate of AbHV-1 (Australia isolate, Victoria/AUS/2007). A PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The protocol employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification with an expected PCR product of 606 bp. Based on concordance of the newly developed PCR protocol and histopathology, this assay can serve as a good proxy for diagnosing herpesvirus infections in abalone suffering mortality. The manuscript has been sent for review in Journal of Virologic Methods.
6. Collection, analysis and dissemination of epizootiological data relevant to international disease control
Epidermiology study of abalone herpes-like virus in maricultured abalone:
A retrospective study using primer sets derived from DNA polymerase gene of abalone herpesvirus to analyze field samples revealed that 32% (6 of 19 batches) of PCR-positive cases had nervous lesions according to the histopathology examination. All PCR-negative batches have unidentified histopathological lesions, and no virions were observed via the electron microscopic examination using direct negative staining of pooled organs consisting of gonads, the hepatopancreas, intestines, gills, muscles, and mantle. Due to the slow rates of abalone mortality noted in the field, the relationship between these cases and herpesvirus infection was analyzed out in this study. Three DNA fragments were cloned from diseased abalone. Amino acid analysis revealed the 1380-bp sequence has 38% (8/21) identity to human herpesvirus-7 DNA polymerase catalytic subunit, the 1406-bp sequence has 34% (14/41) identity to murid herpesvirus 2 and the 1158-bp sequence has 40% (14/35) identy to human herpesvirus-4. All three DNA fragments have no identity to the published DNA sequence including abalone herpesvirus DNA polymerase sequence of Taiwan/2004 and Victoria/AUS/2007. These results suggested a herpes- like virus variant was identified. The study is still on-going. Further analysis of the DNA sequence associated with chronic infection will be conducted next year.
Survey of the biological diversity and abalone herpes-like virus in wild population of of wild mollusks populations in Taiwan:
1. Study of massive mortality of wild population of mollusks including Ruditapes philippinarum, Ruditapes variegate and Gafrarium divaricatum in Matsu Islands : Farmers have complain of wiping out of mollusks in the intertidal zone in recent two years. A PCR study using primer sets derived from abalone herpesvirus variant has amplified a significant amplicons. Amino acid analysis revealed the 427-bp sequence has 36% (9/25) identity to ranid herpesvirus-2 and 31% (10/32) to macacine herpesvirus. No signficant histopathology and no virions were identified via the electron microscopic examination using direct negative staining of pooled organs in the study. Further study will done in next year.
2. A conservative survey of biological diversity of mollusks populations and infection of abalone herpes-like virus in wild population of Gastropoda including abalone (Haliois diversicolor) of eastern Taiwan: The project was directed by Dr. Shu-Ping Wu, Department of Natural Science, Taipei Municipal University of Education. Samples of various molluscan species were collected from two locations along the eastern coast of Taiwan, and the number of species collected was 18 and 5, respectively. The study revealed that the wild population of abalone in Yanliao area has prominently decreased this year. Initial attempt to confirm the presence of the virus gave negative results in all samples by conventional PCR procedures using primer sets derived from abalone herpes-like virus and herpes-like virus variant. Further analysis with real-time PCR protocol was unable to detect the presence of viral DNA of abalone herpes-like virus in the wild abalone samples collected from both locations.
Annual reports of OIE Reference Centres, 2011 3 Infection with abalone herpes-like virus
7. Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the pathogen and the disease concerned
Not.
8. Provision of consultant expertise to OIE or to OIE Member Countries
Not.
9. Provision of scientific and technical training to personnel from other OIE Member Countries
Ms. Yeo Jin Jung and Ms. Harim Baek, National Fisheries Research and Development Institute of South Korea, have visited my lab from August 1 to 6, 2011. The course schedule was listed as following:
Monday Tuesday Wednesday Thursday Friday
Aug. 1 Aug. 2 Aug. 3 Aug. 4 Aug. 5
Orientation PCR technique PCR technique Real time PCR technique Field trip to visit the farms
10. Provision of diagnostic testing facilities to other OIE Member Countries
Not.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
Not.
12. Participation in international scientific collaborative studies
Objectives: In this study, the characterization of the DNA polymerase gene of abalone herpes-like virus will be study. And further developed a conventional PCR targeting the virus DNA polymerase will select to design specific primers because the DNA polymerase gene seems to be highly conserved, this virus gene appears of interest for generic diagnosis purpose to detect most of abalone herpes-like virus isolates.
Project partners: Pen Heng Chang; T. Renault, Ifremer, France ; C.S. Friedman, School of Aquatic and Fishery Sciences, University of Washington, WA, USA.
Time frame: This is a conservative study from last year.
Significant outcomes: A 5781-base pair (bp) fragment of genomic DNA from Taiwanese abalone herpesvirus was obtained and showed 99% (5767/5779) homology in nucleotide sequence and 99% (1923/1926) in amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. The homology in the amino acid sequence with the DNA polymerase of ostreid herpesvirus 1 was only 30% (563/1856). In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The protocol employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed to yield 606 bp amplicons. Based on concordance of the newly developed PCR protocol and histopathology, this assay can serve as a good proxy for diagnosing herpesvirus infections in abalone suffering mortality.
13. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)
4 Annual reports of OIE Reference Centres, 2011 Infection with abalone herpes-like virus
Presentations at international conferences and meetings
Not.
Scientific publications in peer-reviewed journals
Not.
Other communications
Participate “International workshop on the emerging ostreid herpesvirus”, held in July 9-10, at Carins Quesland, Australia.
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