program - 국립수산물품질관리원 1st international... · lecture 2 the status of the oie...
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PROGRAM
Schedule of the 1st International Workshop on VHS in KoreaOpening 10:00-10:20 Introduction of participants & purpose of the event Lee Jeongseon (NFQS)Opening address Sin Hyunseok (Director General of the NFQS)Special Lecture 10:20-11:00Lecture 1. The status of the OIE reference lab of MAFRA <10:20-10:40>
Oh Soonmin (CVO) Q&ALecture 2. OIE Twinning project of the NFQS <10:40-11:00>
Dr. Kim Hyoungjun (NFQS) Q&ASession 1. Overseas research on VHS diseases 11:00-11:50 Lecture 3. VHS: The current situation or “The
disease that never stop surprising us!”Lecture 4. Molecular tracing of VHS outbreaks
<11:00-11:50>
Prof. Olesen (Denmark Technical University) Q&A
Luncheon (11:50-12:50)Session 2. VHS outbreak & research on diagnostics 13:00-14:20Lecture 5. Diagnosis of VHS in Japan, and typing
of VHSV by monoclonal antibodies<13:00-13:20>
Dr. Takafumi Ito (NRIA FRA) Q&ALecture 6. Development of diagnostic kit and
vaccine for the control of VHS in Korea<13:20-13:40>
Dr. Hwang Jeeyoun (NIFS) Q&ALecture 7. The outbreaks and cases of VHS in Jeju island <13:40-14:00>
Dr. Kang Bongjo (Jeju KOFI) Q&ALecture 8. Improvement of conventional RT-PCR
method for diagnosis of VHS<14:00-14:20>
Dr. Kim Hyoungjun (NFQS) Q&ACoffee Break (14:20-14:40)
Session 3. Research on Korean VHS characteristics & change of host genes infected by VHSV 14:40-15:20Lecture 9. Character of Korean VHSV <14:40-15:00>
Prof. Kim Wisik (Chonnam National Univ.)Q&ALecture 10. Effect of VHSV on the microRNAs expression profile of olive flounder
<15:00-15:20>
Prof. Kim Kihong (Pukyong National Univ.) Q&ASession 4. Verification research on anti-VHS vaccine 15:20-16:00Lecture 11. VHS vaccine emulsified with squalen and aluminum hydroxide adjuvant
<15:20-15:40>
Prof. Jung Sungju (Chonnam National Univ.)Q&ALecture 12. Comparative research on immunogenicity between formalin-killed and live vaccine of VHSV
<15:40-16:00>
Prof. Kwon Seryun (Sunmoon Univ.)Q&ACoffee Break (16:00-16:20)
Session 5. General Discussion 16:20-18:20
제1회 바이러스성출혈성패혈증 연구 국제 워크숍 일정표
* 기념촬영 후 동 호텔에서 만찬
개회식 4.21.(목) 10:00～10:20○ 내빈소개 및 워크숍 목적 설명 사회(수품원 이정선 연구관)○ 개 회 사 국립수산물품질관리원장 신현석특강 : 농림부 OIE 표준실험실 현황 및 수품원 OIE Twinning 프로젝트 현황 10:20～11:00(주제 1) 농림부 OIE 표준실험실 인정 현황 및 추진실적 <10:20～10:40>
․허문 연구관 (농림축산검역본부)질의․응답(주제 2) 수품원 OIE Twinning 프로젝트 추진 현황 <10:40～11:00>
․김형준 박사 (수품원 운영지원과)질의․응답세션1 : 해외 VHS 질병 연구 현황 11:00～11:50(주제 3) VHS : 현재 상황 또는 “놀라움의 연속”(주제 4) VHS 발생의 분자적 추적
<11:00～11:50>
․Olesen 교수 (덴마크 수의연구소)질의․응답오찬(11:50~12:50)
세션2 : 일본, 국내의 VHS 발병현황 및 진단법 연구 현황 13:00～14:20(주제 5) 일본의 VHS 진단 및 단일클론항체에 의한 VHS 유전형 <13:00～13:20>
․Dr. Takafumi Ito (일본 증양식연구소)질의․응답(주제 6) VHS 제어를 위한 진단 키트 및 백신 개발 연구 <13:20～13:40>
․황지연 박사 (국립수산과학원)질의․응답(주제 6) 제주지역 중심의 넙치 VHS 발생특성과 사례 <13:40～14:00>
․강봉조 박사 (제주 해양수산연구원)질의․응답(주제 7) VHS 진단을 위한 conventional PCR의 민감도 평가 <14:00～14:20>
․김형준 박사 (수품원 운영지원과)질의․응답Coffee Break (20min) <14:20～14:40>
세션3 : 우리나라 VHS 특성 및 VHSV 감염에 의한 숙주 유전자 변화 연구 14:40～15:20(주제 8) 우리나라에서 분리되는 VHSV 특성 <14:40～15:00>
․김위식 교수 (전남대학교)질의․응답(주제 9) VHSV 감염에 의한 넙치 microRNA Profile 변화 <15:00～15:20>
․김기홍 교수 (부경대학교)질의․응답세션4 : VHS에 대한 예방 백신 효과 검증 연구 15:20～16:00(주제 10) 면역보조제가 첨가된 VHSV 백신 효과 연구 <15:20～15:40>
․정성주 교수 (전남대학교)질의․응답(주제 12) VHSV 불활화백신과 생백신의 면역원성 비교 <15:40～16:00>
․권세련 교수 (선문대학교)질의․응답Coffee Break (20min) <16:00～16:20>
세션5 : 종합 토론 16:20～17:30
Lecture 1
Current situation of the designated OIE Reference Laboratory and their activities in
QIA, MAFRA
Moon Her
Animal and Plant Quarantine Agency (QIA), MAFRA, Korea
e-mail: [email protected]
The QIA has pursued major tasks, including quarantine on animals, animal products and plants;
control and prevention of animal and plant diseases; research on and development of veterinary
science and plant quarantine technology, etc.
After the QIA first achieved the recognition of the OIE Reference Laboratory for Brucellosis in 2009,
the OIE Reference Laboratory for Newcastle Disease (ND) was recognized in 2010, Chronic Wasting
Disease (CWD) and Rabies in 2012, and Japanese Encephalitis (JE) in 2013. Our agency is currently
operating 5 OIE Reference Laboratories in total and has plan to achieve additional recognition of the
OIE Reference laboratories for Foot and Mouth Disease (FMD) and Bovine Tuberculosis. These OIE
Reference Laboratories annually report 12 mandatory performances (2 general and 10 specific
activities) to the OIE as follows.
Two general activities
1. Tests in use or available for the specified diseases
2. Production & distribution of diagnostic reagents
Ten specific activities
1. International harmonization/ standardization of methods
2. Preparation and supply of international reference standards QIA
3. Research and development of new procedures
4. Collection, analysis and dissemination epizootiological data
5. Provision of consultant expertise
6. Provision of scientific and technical training
7. Provision of diagnostic testing facilities for OIE Members
8. Organization of international scientific meetings
9. Participation in international scientific studies and collaboration
10. Presentations and publications
As leading veterinary science institution with the 5 OIE Reference Laboratories, our agency will
contribute itself to the improvement of animal health all around the world.
Lecture 2
The status of the OIE Laboratory Twinning Project for viral hemorrhatic septicemia
(VHS) with Korea and Denmark
Hyoung Jun Kim1*
, Niels Jørgen Olesen2
1National Fishery Products Quality Management Service, Busan, Korea
2National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark
e-mail: [email protected]
The national fishery products quality management service (NFQS) is the competent authority for
quarantine on aquatic organisms in trade, contributing itself to quality improvement of fishery products
originated from Korea. While meaningful proportion of olive flounders exported to the U.S., Japan and
the EU is produced in aquaculture farms in Korea, outbreaks of VHS, which occurs every year in
Korea, causes considerable damages to the aquaculture industry. In October 2014, the NFQS invited
the OIE reference laboratory experts of aquatic animal diseases for the third global conference of the
OIE reference in Songdo, Incheon. The NFQS introduced the laboratory of Incheon regional office
and a VHSV research work. Also cooperative research and development of diagnostic capacity were
discussed between the NFQS and Prof. Olesen (OIE reference laboratory for VHS) for the OIE
Twinning Project. This work brought us into submission of the application letter for the OIE Twinning
Project to the OIE, resulting in final approval from the aquatic animals commission. All the
administrative works for this project have been completed and signed off by the OIE (21 April 2015).
In 2015, the NFQS secured the exclusive budget ($250,000/yr) for this project and research on VHSV.
We have constantly discussed the improvement of diagnostic methods in a kick-off meeting, EAFP
conference, TAIEX meeting and during cooperative research in Copenhagen. We plan this year to
submit an annual report to the OIE, presentation of our research result in the EURL meeting and
finally revise the OIE diagnostic manual based on our research results.
Lecture 3
VHS: The current situation or “The disease that never stop surprising us!”
Niels Jørgen Olesen
National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark
e-mail: [email protected]
VHS has been known since the 1960ties as a disease causing serious mortalities in rainbow trout
farming in continental Europe. Since the 1980ties the causative virus has been recognized in all over
the Northern hemisphere affecting more than 90 fish species in both salt and fresh water. Today VHS
is thus both affecting farmed as well as wild fish. VHSV can be divided into 4 genotypes and at least 8
subtypes and there is a close linkage between genotypes, geographic range and affected fish species.
While countries like Denmark, Norway and England have freed themselves for VHS several countries
are still struggling with the disease. An update on the control and eradication of VHS in Denmark is
given. Followed by the present VHS status in wild fish in Northern European waters. Then the recent
VHS outbreaks in a number of fish species in the great lakes in USA and Canada, in rainbow trout in
Iran, in turbot in Turkey, in wrasse in Scotland, in rainbow trout in Norway and the very recent
outbreak in lumpfish in Iceland as well as a general overview of the worldwide distribution of the
disease will be given.
Virus evolution:
Recent studies indicate that only a few amino acid changes in the structural proteins of VHSV can
change the virulence patterns significantly, thereby coming closer to assessing the risk of none to low
virulent viruses becoming high virulent. Virulence factors both depend on the ability of VHSV to enter
a cell and on the speed and efficiency of virus replication in the cells. Apparently the viral
nucleocapsid protein plays a very important role for the later- and seem to be the target for
determination of a virulence marker.
Lecture 4
Molecular tracing of viral haemorrhagic septicaemia outbreaks in Denmark
S. S. Mikkelsen
*1, V. Panzarin
2, H. Schütze
3, A. Fusaro
2, H. F. Skall
1,5, H. Korsholm
4, N.J. Olesen
1
1National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark
2IZSVe, Venice, Italy
3Friedrich-Loeffler Institut, Insel Riems, Germany
4Danish Veterinary and Food Administration, Vejle, Denmark
5Aarhus University, Aarhus, Denmark
e-mail: [email protected]
Viral haemorrhagic septicaemia Virus (VHSV) is one of the most important fish diseases and is
widely spread over the Northern hemisphere, creating significant losses every year for fish farmers.
VHSV has been endemic in Denmark since first discovered in the 1950’s but after an effective control
and eradication programme the virus was finally eradicated from Denmark in 2009 and the country
was officially declared free of VHSV in 2013 (EU 2013). Almost 300 Danish VHSV isolates from both
marine and freshwater rainbow trout farms, spanning from 1978-2009, including all known isolates
from 1993 onward, were selected for analysis in the largest study of Danish VHSV isolates to date.
The full-length G-gene was sequenced for all new isolates. Genetic data and epidemiological
information have been used to infer phylogenetic trees and phylogeographic models for viral spread
to analyse the relationship between VHS outbreaks in Denmark and to understand the diffusive
dynamics of the disease over a historical period, as well as the effectiveness of the containment and
eradication programme. Phylogenetic analysis shows that all the Danish isolates from rainbow trout
cluster into genotype I, with the majority of isolates in subgenotype Ia. Of the 295 isolates, 158 unique
sequences and clusters of 100% identical sequences were observed, some being persistent and
showing up after more than two years between outbreaks. Some of these span over several water
catchments, and years but most isolates within a cluster originated from the same water system
representing downstream infection or cross infection between farms within short distances. The
identical and very closely related isolates indicated a number of unexpected trade patterns and
underline the risk associated with inland rainbow trout processing plants and ownership of multiple
fish farms. Molecular tracing shows that VHS outbreaks in Danish marine fish farms were most likely
due to stocking these with clinically healthy VHSV infected rainbow trout carriers from inland fish
farms and not to infection with VHSV from the marine environment. From the analysis it appears that
evolution of low virulent VHSV from marine fish species is a very rare event and is most likely related
to feeding with fresh fish which is now prohibited in rainbow trout farming.
Lecture 5
Diagnosis of viral haemorrhagic septicaemia (VHS) in Japan, and typing of VHSV by
monoclonal antibodies
Takafumi Ito
*1 and Niels Jørgen Olesen
2
1 National Research Institute of Aquaculture (NRIA), Fisheries Research Agency, Mie, Japan
2 National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark
e-mail: [email protected]
In Japan, VHSV was first detected in farmed Paralichthys olivaceus in 1996. Since 2000, VHSV has
been isolated from farmed P. olivaceus at several prefectures. All VHSV isolates from Japan were
identified as genotype IVa, except for 1 isolate from 1996 which belongs to genotype Ib. It was
suggested that the genotype Ib isolate was accidentally introduced into Japan by importation of fish or
fish products from overseas. VHS of salmonid fish is designated as “Specific Disease” in Japanese
law. For “Specific Disease”, protective guidelines have been established and laboratory diagnosis of
the disease should be conducted in accordance with these guidelines. According to the procedure,
the Prefectural Fisheries Experimental Stations, which belong to local government, first conduct
epizootic and routine clinical examination on diseased fish. When the RT-PCR or IFAT test was
positive for VHSV, the sample is sent to the ‘Diagnosis and Training Center for Fish Diseases’, NRIA,
for further examination by virus isolation, IFAT, RT-PCR and sequencing for confirmation. VHSV
isolates can be divided into four major genotypes and a number of subtypes with almost-distinct
geographical distributions. The host range and the pathogenicity appear, at least to some extent, to
be linked to the genotype. Since VHS of salmonid fish is caused by VHSV genotype I, Ia and Ic, a
quick and simple detection method for discriminating between the genotypes of VHSV is strongly
desired. Therefore, monoclonal antibodies (mAbs) against VHSV were produced aiming at
establishing a complete immunoassay for typing of VHSV according to genotype. BALb-c mice were
immunized with purified preparations of 7 different genotypes (I, Ia, Ib, II, III, IVa and IVb). Established
mAbs were selected by IFAT and ELISA test against a large panel of 79 VHSV isolates. As the results,
we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing
isolates in immunoassay using a panel of nine mAbs.
Lecture 6
Development of diagnostic kit and vaccine for the control of viral hemorrhagic
septicemia virus in Korea
Jee youn Hwang*, Mi Young Cho, Seong Don Hwang,
Mun-Kyoung Kwon, Sung-Hee Jung, and Myoung Ae Park
National Institute of Fisheries Science
e-mail: [email protected]
We report research into the commercialization of diagnostics and vaccines to control viral
hemorrhagic septicemia (VHS), the major viral disease affecting flounder.
We developed two molecular biological kits. The first is the VHSV OIE Melting array kit, including a
PNA probe in the PCR product, by using the VHSV OIE PCR primer set. This kit has the advantage of
lowering the time required to improve the accuracy of the OIE VHS diagnostic PCR method. Second,
we developed a region specific VHSV PNA kit, which can simultaneously diagnose and determine
variation in VHSV. For further diagnosis, we developed the additional primer set in the G gene for
simultaneous subtyping by using a PNA probe based on the OIE guidelines (real-time PCR primer set
in N gene). Third, we developed a serological monoclonal antibody-based diagnostic kit for rapid VHS
detection. The accuracy of the kit was tested using VHSV (at various concentrations), koi herpesvirus,
and red seabream iridovirus. Viruses other than VHSV led to negative results. We assessed the
sensitivity of the kit by detecting the amount of VHSV in positive and negative samples by using real-
time PCR. The result showed that this rapid diagnosis kit has good sensitivity comparable to that of
conventional real-time PCR. The efficacy of this kit has also been confirmed for field applications, and
it is expected to be used for diagnosis in the field.
Further, we isolated a VHSV strain (FP-VHS2010-1) associated with high mortality rates in the olive
flounder (Paralichthys olivaceus) cultured in South Korea and characterized this strain to develop an
inactivated vaccine. The vaccination efficacy of intraperitoneal injection of heat-inactivated VHSV (at
60°C) was evaluated and compared between olive flounder and Epithelioma papulosum cyprini cell
lines. The relative percent survival of fish administered the heat-inactivated VHSV vaccines at high
temperature (20°C) was significantly higher than that of fish administered the vaccine at low
temperature (12°C). Thus, temperature during the vaccine response period was shown to be a critical
factor contributing to potency of the heat-inactivated VHSV vaccine.
Lecture 7
Report of characterization and case study on viral hemorrhagic septicemia (VHS)
outbreak in olive flounder on Jeju island
Bong Jo Kang and Yeoung Hwan Jang
Jeju Special Self Governing Province, Ocean and Fisheries Research Institute
e-mail: [email protected]
In olive flounder farms on Jeju island, suspected case of VHS was observed in April 2006, but it
was not officially confirmed until March 2007. The number of VHS confirmation was 197 in total in
from 2007 to 2015 (56 in 2007; 69 in 2008; 15 in 2009; 9 in 2010; 5 in 2011; 9 2012; 9 2013; 3 2014
and 2 in 2015). All viruses from those cases were found genotype IVa by the phylogenetic analysis of
glycoprotein (G) and nucleocapsidprotain (N) genes. The outbreaks occurred disproportionally in
January and July while few cases came about in October to December. However, August and
September, when the water temperature was high, showed no VHS outbreak. This could be
demonstrated by the result of VHSV challenge test: mortality was observed only at 16℃while other
two groups, 21 and 25℃, all survived. In conclusion, VHS outbreak on Jeju island occurred at low
temperature, under 20℃. By size of the fish, less than 20 cm was targeted for VHS as 163 cases fell
on the group out of 177. The result of this study provides necessary development of VHS prevention
methods using heat culture system with water disinfection.
Lecture 8
Development of a novel one-step RT-PCR for detection of viral heamorrhagic
septicemia
Hyoung Jun Kim1*
, Susie Sommer Mikkelsen2 and Niels Jørgen Olesen
2
1National Fishery Products Quality Management Service, Busan, Korea
2National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark
e-mail: [email protected]
Viral haemorrhagic septicaemia virus (VHSV) is the most serious viral disease in salmonids
and olive flounder farms. Various diagnostic methods for detection of VHS virus have been
described in the OIE Diagnostic Manual. Among them, the conventional RT-PCR method is
typically used for detecting VHSV and sometimes also genotyping into I to IV. However, we found
a low sensitivity for detection of Korean VHSV Iva isolates using these OIE primers. In addition,
the RT-PCR often showed non-specific reaction with fish cell lines. Thus, we needed to improve
the specificity and sensitivity of the conventional RT-PCR given in the OIE Diagnostic Manual in
order to detect all VHSV genotypes and to remove the non-specific reactions due to fish cell lines.
In this study, we selected the candidate primers from 5 regions in VHSV N gene, and a highly
sensitive primer set was selected among these. The reaction conditions of the selected primer set
were established and no non-specific reactions in fish, fish cell lines or with heterologous viruses
were observed. The sensitivity of new RT-PCR was tested in parallel with cell cultivation, RT-
qPCR, and conventional OIE VN RT-PCR. It was concluded that the sensitivity for all genotypes
were at the same level when using cell culture, qPCR and the conventional new RT-PCR. While it
was lower for the OIE VN RT-PCR. The novel RT-PCR was following tested on 80 VHSV isolated
representing a worldwide collection of all known genotype and subtypes. In this study clear and
unique amplicons were observed for all 80 isolates. In conclusion a new conventional RT-PCR
have been developed and validated according to all steps given in the guidelines of the OIE
diagnostic Manual, except for a reproducibility study that will be conducted between 5-6
laboratories in an inter-laboratory proficiency test which is under preparation and we thereby
recommend that this new 3F2R RT-PCR shall replace the current method given in the OIE
Manual for detection of VHSV.
Lecture 9
Character of Korean viral hemorrhagic septicemia virus (VHSV)
Wi-Sik Kim and Myung-Joo Oh
Department of Aqualife Medicine, Chonnam National University
e-mail: [email protected]
Viral haemorrhagic septicaemia virus (VHSV) is an epidemic virus in olive flounder Paralichthys
olivaceus farms in Korea, since the virus have first isolated in 2001. In the present study, partial
glycoprotein (G) gene nucleotide sequences of seven Korean VHSV from cultured olive flounder
and wild marine fishes in coastal areas of Korea were analyzed to evaluate their genetic
relatedness to worldwide isolates. Phylogenetically, all Korean VHSV formed only one minor
cluster including Japanese isolates, in genotype IVa, while the North America isolates formed a
different minor cluster in genotype IVa. These results suggest that Korean VHSV could be an
indigenous virus in Korean and Japanese coastal areas, but have not been introduced from North
America. The pathogenicities of Korean and Japanese isolates of VHSV from olive flounder were
investigated with rainbow trout Oncorhynchus mykiss fry. The cumulative mortalities of fish
challenged with FYeosu05 (Korean) and Obama25 (Japanese) isolates at 106.5
TCID50/fish were
64% and 48%, respectively. No mortality was observed in fish challenged with either of the
isolates at 105.5
TCID50/fish, or in mock-challenged fish. The affected fish showed darkening of
the body, expanded abdomen, pale gills, enlarged spleen, and diffuse necrosis in splenic and
interstitial hematopoietic tissues. From all the dead fish, VHSV was re-isolated by cell culture and
detected by reverse transcription loop-mediated isothermal amplification (RT-LAMP). It was thus
confirmed that Asian VHSV isolates from olive flounder are pathogenic to rainbow trout fry,
although with low virulence. Olive flounder are highly protected from a VHSV challenge following
Polyinosinic–polycytidylic acid [Poly(I:C)] administration. We investigated the change of VHSV
titer in olive flounder following Poly(I:C) administration to understand virus dynamics in the fish.
Fish challenged with VHSV that were not administered Poly(I:C) showed 63.8% cumulative
mortality. VHSV was detectable the next day after VHSV-challenge and multiplied very quickly to
around 107.5
TCID50/g in 3 days. About 107
TCID50/g titer was maintained until 7 days and then
subsequently decreased and almost disappeared after 21 days. In contrast, 1.7% cumulative
mortality was observed in fish administered Poly(I:C), and no VHSV titer was detected in almost
all fish for 28 days. These results confirm that multiplication of VHSV is strongly down-regulated
in olive flounder following Poly(I:C) administration.
Lecture 10
Effect of viral hemorrhagic septicemia virus (VHSV) on the microRNAs expression
profile of olive flounder (Paralichthys olivaceus)
Abdellaoui Najib1, Min Sun Kim
1, Seung Hyuk Choi
1, Yue Jai Kang
2, Ki Hong Kim
1
1Department of Aquatic Life Medicine, Pukyong National University,
2Department of Aquatic Life and Medical Sciences, Sun Moon University
e-mail: [email protected]
The aim of the present study was to analyze the effect of viral hemorrhagic septicemia virus
(VHSV) infection on the cellular microRNA expression profile in olive flounder (Paralichthys
olivaceus). Fingerlings were intra-muscularly infected with VHSV, and microRNAs expression in
the head kidney was analyzed at 0 (control), 6, 12, 24, 48 and 72 h post-infection (h.p.i.) by the
high-throughput sequencing. A total of 372 mature miRNAs were identified, and, among them, 63
miRNAs were differentially expressed during VHSV infection. The differentially expressed
microRNAs number was greatly increased from 24 h.p.i. compared to the number at 6 and 12
h.p.i., suggesting that the alteration of microRNAs expression by VHSV infection may be related
to the progression of VHSV disease. The target prediction analysis, the GO enrichment analysis,
and the KEGG pathway analysis of the predicted target genes showed that various biological
pathways could be affected by VHSV infection through the down-regulation or up-regulation of
host miRNAs. The present results provide a basic information on the microRNAs related to VHSV
infection in olive flounder. Considering broad effects of microRNAs on various biological
pathways, data in this study can be used to interpret the mechanism of VHSV pathogenesis,
which, vice versa, can be used to develop control measures against VHSV. Now, we are
conducting experiments on sponge microRNAs using Epithelioma papulosum cyprini cells instead
of fish to know the role of specific microRNAs in VHSV replication.
Lecture 11
Viral hemorrhagic septicemia (VHS) vaccine emulsified with
squalene and aluminum hydroxide adjuvant
Sung-Ju Jung, Vinay Tharabenahalli Nagaraju, Ye-Ji Kim, Myung-Hwa Jung,
Wi-Sik Kim, Do-Hyung Kim
Department of Aqualife Medicine, Chonnam National University, Republic of Korea
e-mail: [email protected]
Efficacy, duration of protection and safety of an inactivated VHSV vaccine emulsified with squalene
emulsion (5%) and aluminum hydroxide (0.5%) were evaluated. The inactivated VHS vaccine
provided a moderate protection of 37% and 47% relative percent survival (RPS) at 4 and 10 weeks
post vaccination (wpv). Addition of squalene and aluminum hydroxide into inactivated VHS vaccine
clearly enhanced the level of protection showing 58% and 83% RPS at 4 and 10 wpv respectively,
indicating the need for adjuvants to enhance the efficacy. The vaccinated fish showed significant
protection at 3, 6, 12, 18, 24, and 40 wpv (except week 57) than non-vaccinated fish to an
intraperitoneal challenge of 107.1
TCID50/fish at 15°C, with RPS of 60%, 64%, 71%, 55%, 52% and 50%
(45% at 57 week) respectively, covering the duration of natural outbreak. The study of immune gene
expression suggests that the vaccinated fish are able to control the virus replication compared to non-
vaccinated fish with the early expression of pro inflammatory cytokines (IL-1β), antigen presenting
cells (MHC-II), specific cellular immune response related genes (CD8), apoptosis related genes
(Caspase 3, Granzyme, Perforin) and provide a significant protection. However, the result also
suggests that the genes responsible for specific humoral immune response (IgM, IgD) are not
significantly up-regulated.
Lecture 12
Comparison of the efficacy of Poly(I:C) immunization with live vaccine
and formalin-killed vaccine against viral hemorrhagic septicemia virus
(VHSV) in olive flounder (Paralichthys olivaceus)
Hyoung Jun Kim1*
, Jeong Su Park2, Min Chul Choi
3, and Se Ryun Kwon
2
1National Fishery Products Quality Management Service
2Department of Aquatic Life Medical Sciences, Sunmoon University
3Incheon Fisheries Research Institute, Incheon
e-mail: [email protected]
Viral hemorrhagic septicemia (VHS) in olive flounder, Paralichthys olivaceus, causes significant
economic loss for the flounder aquaculture industry in Korea. In this study, the immunogenicity of
Poly(I:C) immunization with a live vaccine against the VHS virus (VHSV) was compared with that of a
formalin treated vaccine in the olive flounder. In vaccine trial I, fish pre-injected with Poly(I:C) were
highly protected from VHSV infection 2 d later (survival rate: 96%) and the surviving fish (Poly(I:C)-
VHSV group) showed a 100% survival rate against VHSV re-challenge. Mortality in fish pre-injected
with diethylpyrocarbonate-treated water followed by injection with formalin-treated VHSV was only 2%
(1 of 50 fish), whereas survivors (DEPC-FT VHSV group) showed an 80% survival rate. In vaccine
trial II, 100% survival was observed in all Poly(I:C) vaccination groups-Poly(I:C)-VHSV 6, Poly(I:C)-
VHSV 5, and Poly(I:C)-VHSV 4. In contrast, the survival rates of the groups administered the
formalin-treated VHSV at a dose of 106, 10
5, and 10
4 TCID50 100 mL
-1 fish
-1 (DEPC-FT VHSV 6,
DEPC-FT VHSV 5, and DEPC-FT VHSV 4) were only 8%, 12%, and 12%, respectively. The
differences in the survival rates of the formalin-treated vaccine groups in trial I and trial II were
attributed to the difference in the formalin-treatment period: the formalin-treated VHSV administered in
trial I was not completely inactivated and worked as a live vaccine, which explains the 80% survival
rate against VHSV challenge. Specific antibodies against VHSV were detected in sera from all
vaccinated survivors, except the DEPC-VHSV 4 group. Furthermore, the specific antibody titers of fish
vaccinated with the live and dead VHSV vaccines were similar, but the protective effects of the live
and dead vaccines varied considerably. Our findings show that Poly(I:C) immunization with the live
vaccine offers better protection than the formalin-treated vaccine against VHS in olive flounder and
revealed that antibody levels are not a reliable indicator of the protective effect of the vaccine against
the pathogen. In the future, elements of T cell immunity may be used as a means of evaluating the
protective efficacy of a vaccine against VHSV instead of ELISA.
The 1st International Workshop on Viral Hemorrhagic Septicemia Research in Korea
1. Director General of the NFQS
Mr. Sin Hyunseok
2. Director of General Services of the NFQS
Mrs. Kwon Hyunwook
3. Director of Quarantine & Inspection Division of NFQS
Dr. Jee Junghoon
4. Ocean and Fisheries Research Institute in Jeju
Dr. Kang Bong-jo
5. National Fishery Products Quality Management Service
Dr. Kim Hyoungjun
6. Pukyong National University
Prof. Kim Ki-hong
7. Sunmoon University
Prof. Kwon Se-ryun
8. Chonnam National University
Prof. Jung Sung-ju
9. Chonnam National University
Prof. Kim Wi-sik
10. National Fisheries Research and Development Institute
Dr. Hwang Jee-youn
11. National Veterinary Institute in Denmark
Prof. Niels Jorgen Olesen
12. Fisheries Research Agency in Japan
Dr. Takafumi Ito
13. Senior Researcher of the NFQS
Mr. Lee Jeong-seon
14. Interpreter
Mr. Park Eunwook
15. National Fisheries Research and Development Institute
Dr. Kwon Mun-kyoung
16. Animal and Plant Quarantine Agency
Dr. Her Moon
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