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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:
Infection with abalone herpes-like virus
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:
website:
[email protected]
http://www.vm.ntu.edu.tw/DVM/
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
Pen Heng Chang
Name(including Title and
Position) of OIE Reference
Expert:
Pen Heng Chang
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.
2.
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
Production and distribution of diagnostic reagents
Type of reagent
Positive control DNA
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
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.
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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 herpeslike 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
Annual reports of OIE Reference Centres, 2011
3
Infection with abalone herpes-like virus
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.
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
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Annual reports of OIE Reference Centres, 2011
Infection with abalone herpes-like virus
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)

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.
_______________
Annual reports of OIE Reference Centres, 2011
5