2nd... · viral heamorrhagic septicaemia (vhs) with korea and denmark 3 topic 2) viral haemorrhagic...

개회식 5.10.(수) 10:00～10:40○ 내빈소개 및 워크숍 목적 설명 사회(수품원 이정선 연구관)○ 개 회 사 국립수산물품질관리원장○ 발표자 소개 및 기념 촬영 사회(수품원 이정선 연구관)특강 1 10:40～11:00

(주제 1) 한·덴마크 OIE Twinning 프로젝트 소개 및 진행 현황 <발표 10:40～10:55>․김형준 박사 (수품원 운영지원과)질의․응답

특강 2 11:00～11:40

(주제 2) 무지개송어에서 VHSV의 독성 결정 요소 연구 <11:00～11:35>

․Olesen 교수 (덴마크 수의연구소)질의․응답

오찬(11:40~13:30) - 모든 참석자세션 1 : VHS 바이러스 특성 연구 및 질병 현황 13:30～15:20

(주제 3) VHSV 증식, 독성과 인터페론 반응에 있어 NV유전자의 역할 <13:40～13:55>

․김민선 교수 (세종대학교)질의․응답

(주제 4) 제주 해양수산연구원 질병 연구 현황 소개 <14:00～14:15>․원승환 연구관 (제주 해양수산연구원)질의․응답

(주제 5) 우리나라 넙치 양식장의 VHSV 조사 <14:20～14:35>․김위식 교수 (전남대학교)질의․응답

(주제 6) 우리나라 바다 송어 질병 모니터링 <14:40～14:55>․김위식 교수 (전남대학교)질의․응답

(주제 7) 국내 무지개송어 IHN 발생에 따른 대량폐사 사례 <15:00～15:15>․김형준 박사 (수품원 운영지원과)질의․응답

Coffee Break (40min) <15:20～16:00>

세션 2 : VHS 진단법 연구 16:00～17:00

(주제 8) OIE 진단법 유효성 검증 매뉴얼에 따른 VHS Kim3F2R conventional PCR법 검증 <16:00～16:15>

․김형준 박사 (수품원 운영지원과)질의․응답

(주제 9) 여러 어류 바이러스들의 감염 titer에 있어 FBS농도의 영향 <16:20～16:35>

․권세련 교수 (선문대학교)질의․응답

(주제 10) VHSV 검출을 위한 신속 면역크로마토그래피test strip의 개발

<16:40～16:55>․김위식 교수 (전남대학교)

질의․응답세션 3 : VHS 백신 연구 17:00～17:20

(주제 11) 역유전학 기술을 이용한 재조합 VHSV 백신 개발 <17:00～17:15>․김민선 교수 (세종대학교)질의․응답

워크숍 1일차 세부계획(안)

※워크숍발표자및개최관계자만찬 (18:00~20:00)

특강 3 5.11.(목) 10:00～10:20

(주제 12) OIE 구제역 표준실험실의 운영 현황 <발표 10:00～10:15>․박종현 연구관 (농림축산검역본부)

질의․응답

특강 4 10:20～11:00(주제 13) 캐나다의 VHSV와 IHNV : 자연산과 양식산

어류간 상호작용 <발표 10:20～10:55>․Garver 박사(캐나다 수산해양연구소)

질의․응답

특강 5 11:00～11:40

(주제 14) VHSV 관리 대책 : 분자 역학이 어떻게 기여할 것인가? <11:00～11:35>

․Panzarin 박사(이탈리아 동물건강연구소)질의․응답

오찬(11:40~13:00) - 모든 참석자

세션 4 : VHSV에 따른 숙주 면역반응 13:00～13:40

(주제 15) NGS를 이용한 生 VHSV 및 死 VHSV를통한 넙치의 유전자 전사 분석 <13:00～13:15>

․권세련 교수 (선문대학교)질의․응답

(주제 16) 15℃와 20℃에서 VHSV로 감염시킨 넙치의톨 유사 수용체와 세포사멸체 반응 <13:20～13:35>

․정성주 교수 (전남대학교)질의․응답

Coffee Break (20min) <13:40～14:00>

세션 5 : 세션별 토론 및 종합 토론 14:00～15:00

워크숍 2일차 세부계획(안)

※일정발표주제및내용이변경또는추가될수도있음

ContentsPROGRAMME 1

Topic 1) The status of the OIE Laboratory Twinning Project for

viral heamorrhagic septicaemia (VHS) with Korea and Denmark 3

Topic 2) Viral Haemorrhagic Septicaemia Virus (VHSV): On the search for

determinants important for virulence in rainbow trout Oncorhynchus mykiss 4

Topic 3) The role of NV gene of VHSV in viral growth, virulence, and IFN response 5

Topic 4) Introduction of Oceans and Fisheries Research Institute

In Jeju & Status of Aquatic Disease Research 6

Topic 5) Survey of viral hemorrhagic septicemia virus (VHSV) in olive flounder farms 7

Topic 6) Disease monitoring of maricultured salmonid fish 8

Topic 7) Severe mortality of farmed rainbow trout caused by infectious

haematopoietic necrosis virus in Korea 9

Topic 8) Assessment of a new conventional RT-PCR for VHSV detection

in an inter-laboratory proficiency test 10

Topic 9) Effect fetal bovine serum (FBS) concentration on the infectivity titer of several viruses 11

Topic 10) Development of rapid immunochromathographic test strip for detection

of viral hemorrhagic septicemia virus (VHSV) of olive flounder 12

Topic 11) Development of recombinant VHSV vaccines using reverse genetics 13

Topic 12) Activities for Control of Foot-and-Mouth Disease

in OIE FMD Reference Laboratory in Republic of Korea 14

Topic 13) Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis

Virus in British Columbia, Canada: interplay between wild and farmed fish 15

Topic 14) VHSV management and control: How can molecular epidemiology contribute? 16

Topic 15) Transcriptome analysis of olive flounder, Paralichthys olivaceus immunized

with Poly(I:C)-VHSV or formalin-killed VHSV 17

Topic 16) TLR and apoptotic response of viral haemorrhagic septicaemia virus

infected olive flounder at 15°C and 20°C 18

1

PROGRAMME

10 - 11 May 2017

09:30 ~ 10:00 Registration of Participants

DAY 1 (10 May 2017)

Time Theme P·TTime Speakers

10:00 ~ 10:40 Opening ceremonyIce breaking & Introduction

40min

LEE JEONG SEON(NFQS chairperson)

Opening Remark Park Shin Chul (NFQS Director)

Group photo LEE JEONG SEON(NFQS chairperson)

10:40 ~ 11:00Topic 1) The status of the OIE Laboratory Twinning Project for viral heamorrhagic septicaemia (VHS) with Korea and Denmark

20min Kim Hyoung Jun(NFQS)

11:00 ~ 11:40Topic 2) Viral Heamorrhagic Septicaemia Virus (VHSV): On the search for determinants important for virulence in rainbow trout Oncorhynchus mykiss

40min Niels Jørgen Olesen (National Veterinary Institute in Denmark)

11:40 ~ 13:30 Lunch – All participantsSession 1 Study on character of VHS and rhabdoviral disease in Korea13:40 ~ 14:00 Topic 3) The role of NV gene of VHSV in viral

growth, virulence, and IFN response 15min Kim Min Sun(Sejong University)

14:00 ~ 14:20Topic 4) Introduction of Oceans and Fisheries Research Institute in Jeju & Status of Aquatic Disease Research

15min Won Seung Hwan(Oceans and Fisheries Research Institute)

14:20 ~ 14:40 Topic 5) Survey of viral hemorrhagic septicemia virus (VHSV) in olive flounder farms 15min Kim Wi Sik

(Chonnam National University)

14:40 ~ 15:00 Topic 6) Disease monitoring of maricultured salmonid fish 15min Kim Wi Sik

(Chonnam National University)

15:00 ~ 15:20Topic 7) Severe mortality of farmed rainbow trout caused by infectious haematopoietic necrosis virus in Korea


15:20 ~ 16:00 Coffee BreakSession 2 Study on diagnostic methods of VHS and rhabdoviral diseases

16:00 ~ 16:20Topic 8) Assessment of a new conventional RT-PCR for VHSV detection in an inter-laboratory proficiency test


16:20 ~ 16:40Topic 9) Effect fetal bovine serum (FBS) concentration on the infectivity titer of several viruses

15min Kwon Se Ryun(Sunmoon University)

16:40 ~ 17:00Topic 10) Development of rapid immunochromathographic test strip for detection of viral hemorrhagic septicemia virus (VHSV) of olive flounder

15min Kim Wi Sik(Chonnam National University)

2

DAY 2 (11 May 2017)

Time Theme P·TTime Speakers

10:00 ~ 10:20Topic 12) Activities for Control of Foot-and-Mouth Disease in OIE FMD Reference Laboratory in Republic of Korea

20min Park Jong Hyeon(Animal and Plant Quarantine Agency)

10:20 ~ 11:00Topic 13) Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus in British Columbia, Canada: interplay between wild and farmed fish

40min Kyle A. Garver(Fisheries & Oceans Canada)

11:00 ~ 11:40 Topic 14) VHSV management and control: How can molecular epidemiology contribute? 40min

Valentina Maria Panzarin(Instituto Zooprofilattico Sperimentale delle Venezie in Italy)

11:40 ~ 13:00 Lunch – all guestSession 4 Host immune response about VHSV

13:00 ~ 13:20Topic 15) Transcriptome analysis of olive flounder, Paralichthys olivaceus immunized with Poly(I:C)-VHSV or formalin-killed VHSV

15min Kwon Se Ryun(Sunmoon University)

13:20 ~ 13:40Topic 16) TLR and apoptotic response of viral haemorrhagic septicaemia virus infected olive flounder at 15°C and 20°C

15min Jung Sung Ju(Chonnam National University)

13:40 ~ 14:00 Coffee Break14:00 ~ 15:00 Discussion on each session and synthesis

Session 3 Study on vaccine of VHS17:00 ~ 17:20 Topic 11) Development of recombinant VHSV

vaccines using reverse genetics 15min Kim Min Sun(Sejong University)

18:00 ~ 20:00 Dinner – Speakers and Host persons

3

The Status of the OIE Laboratory Twinning Project for viral haemorrhagic septicaemia (VHS) with Korea and Denmark

Hyoung Jun Kim1*, Niels Jørgen Olesen2

1National Fishery Products Quality Management Service, Busan, Korea2National 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 originating 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 the final approval from the OIE aquatic animal commission. All the administrative works

for this project have been completed and signed off by the OIE (21 April 2015). And then, our Vice Minister

approved plans of OIE reference laboratory on VHS by NFQS.

In 2015, we have constantly discussed the improvement of diagnostic methods in a kick-off meeting, EAFP

conference, TAIEX meeting and during cooperative research in Copenhagen. In 2016, we had many activities such

as cooperation research, host of the first international workshop on VHS in Korea, meeting with president and

member of OIE aquatic animal commission, obtaining the ISO17025 on VHS diagnostic methods, submission of the

annual report to the OIE headquarters, and several meetings between Korea and Denmark. In this year, we also

performed several activities such as an inter-proficiency test for reproducibility using novel KIM3F2R method,

consultation with OIE Tokyo office for activation of OIE activities and discussion about KIM3F2R method with

Dr. Crane and Dr. Moody from experts of OIE reference laboratory in Australia, and meeting with General

Directors between NFQS and NVI of DTU, and optimization of the serological methods for VHSV detection. In

addition, we got the approval of plan to change the OIE manual with KIM3F2R method for VHSV detection from

our Minister of the Oceans and Fisheries. We make several further plans such as submission of manuscript about

KIM3F2R method to International Journal, submission of document for change of VHS OIE manual with KIM3F2R

method, submission of application for OIE reference laboratory to OIE headquarters. Finally, NFQS hopes to get

the status of OIE reference laboratory on VHS in 2018.

Topic 1

4

VIRAL HAEMORRHAGIC SEPTICAEMIA VIRUS (VHSV): ON THE SEARCH FOR DETERMINANTS IMPORTANT FOR VIRULENCE IN RAINBOW TROUT ONCORHYNCHUS

MYKISS

Niels Jørgen Olesen1*, Anna Luiza Farias Alencar1; Argelia Cuenca Navarro1, Hyoung Jun Kim2

and Takafumi Ito3

1National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark2National Fishery Products Quality Management Service, Busan, Korea

3National Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan

A goal for research on VHS virus in the past decade has been to understand how low virulent virus isolates

becomes highly virulent and to assess if single determinants can tell if an isolate is virulent for rainbow trout or

not. Focus was primarily on the viral envelope glycoprotein (G) as this protein alone can induce neutralising

antibodies and protective immunity. It has been shown that the G-protein is important for virus entry into the host

but for virus replication recent studies have showen that the viral nucleocapsid protein appears to be a very

important player. In recent years determinants on the polymerase protein, and the non-virion protein have also been

suggested to be involved.

VHSV genotype specific MAbs were previously produced in order to establish a fast and low cost genotyping

system for VHSV isolates. In this process, we encountered a number of unpredicted reactions with various VHSV

isolates. Some MAbs were able to discriminate between rainbow trout virulent and non-virulent isolates within the

same sub-genotype. By assessing the epitope specificity of these MAbs together with cloning and full-length

genome sequencing of viruses of various virulence putative virulence markers were identified. Variable regions of

the viral nucleocapsid (N) protein i.e. aa 118-123 for Genotype III and aa 43-46 and aa 168 for Genotype Ib and

only few amino acid substitutions hereby appear to be of high significance for virulence and replication in rainbow

trout.

In vitro studies revealed that replication of non- or low virulent virus are reduced in the rainbow trout cell line

RTG-2 cell with 3-4 log reductions when compared to virulent isolates. While no reduction of non- virulent viruses

was observed in EPC or BF-2 cell lines. In vitro studies using of gill epithelial cells also showed reduced uptake

of low virulent virus compared to high virulent VHSV isolates.

Infection trials by immersion with the virulent GIII and GIb isolates indicate that the time of exposure to

rainbow trout has a significant effect on the virulence to the fish, indicating that the innate immune system is

efficient at reducing virus replication of these medium virulent viruses during short exposure.

Reverse genetics changing low virulent VHSV into high virulent isolates would be the ultimate prove of

determinants important for virulence. Studies have been undertaken to use the results from testing the virulence of

a high number of VHSV isolates and to compare their full genome sequences to determine putative AA mutations

that can enhance virulence. By site directed mutagenesis using a low virulent virus as back bone recombinant

viruses will be assess in in-vivo trials in Rainbow trout.

Topic 2

5

The role of NV gene of VHSV in viral growth, virulence, and IFN response

Min Sun Kim1* and Ki Hong Kim2

1Graduate School of Integrated Bioindustry, Sejong University2Department of Aquatic Life Medicine, Pukyong National University

The reverse-genetics approach to generate recombinant viruses from cloned cDNAs has contributed to elucidate

the function of viral genes and to develop prophylactic vaccines. In the present study, we rescued various

recombinant VHSVs using reverse genetics method to know the role of the NV protein in viral proliferation,

virulence, and IFN response. In order to produce a recombinant VHSV that expressing higher amount of NV

protein, we inserted additional NV gene ORF between N and P genes of the genome, and named it as

rVHSV-dNV. A recombinant VHSV harboring EGFP gene ORF between N and P genes was generated

(rVHSV-A-EGFP), and used as a control for rVHSV-dNV. A recombinant VHSV that is lacking the NV gene ORF

in the genome (named rVHSV-ΔNV) was also produced. Furthermore, to know the functional role of the

N-terminal region of NV protein, we generated recombinant VHSVs containing mutated NV gene, in which the

first, or the 1st and 2nd, or the 1st, 2nd and 3rd ATG codons were mutated into stop codon, and named them as

rVHSV-NV1, rVHSV-NV2, and rVHSV–NV3, respectively. In the viral growth and virulence, rVHSV-dNV,

rVHSV-A-EGFP, and rVHSV-ΔNV showed significantly lower viral growth and virulence, but induced clearly

stronger interferon response in EPC cells compared to rVHSV-wild. When the expression level of NV transcript

between cells infected with rVHSV-wild and rVHSV-dNV was compared, although one viral particle of

rVHSV-dNV expressed NV gene higher than one particle of rVHSV-wild, the total amount of NV transcript in a

cell infected with rVHSV-dNV was significantly lower than that in a cell infected with rVHSV-wild due to the

difference in viral titer. In the NV gene mutated rVHSVs, rVHSV-NV1 showed significantly higher viral growth,

virulence, and significantly lower interferon response than rVHSV-NV2 and rVHSV-NV3 indicating the NV protein

that translated from the 2nd ATG may function like the normal NV protein. The present results suggest that the

NV protein of VHSV plays an important role in suppressing interferon response in host cells, which can provide a

favorable environment for the viruses to proliferate in host cells. And genome length is a critical factor for the

determination of rVHSVs replication efficiency.

Topic 3

6

Introduction of Korea Oceans & Fisheries Research Institute in Jeju & Status of Aquatic Disease Research

Won Seung Hwan

Director of Aquatic Disease Research CenterKorea Oceans & Fisheries Research Institute in Jeju

Korea Oceans & Fisheries Research Institute in Jeju was first established in June, 1999 and it is implementing

research on fish (halibut) and shellfish disease along with Jeju coastal ecosystem, coastal fishing ground and in the

area of fish and shellfish aquaculture. The Institute is also working on creation of fishery resources by seed

production of Jeju special fish, abalone and sea cucumber and focusing on gear fishery development and targeted

technology training for fishermen so as to promote fishing industry.

The Institute is implementing disease control inspection system to make disease inspection mandatory for carrying

seed in and out of Jeju Island for disease free aquatic seed stocking in the aquatic disease research field. Safety

inspection system is also underway for mandatory detection of antibiotics residues prior to shipment in order to

secure food safety of farmed fishery products. Moreover, its work is to prevent Kudoa-infected seed stocking in

advance to control Kudoa of olive flounder for inspecting disease control of aquatic seed.

Research has been performed on development of aquatic antibiotics substituent, developed bacteriophage additives

and started to distribute them from 2016. Further, mix vaccine was developed to prevent bacterial disease of olive

flounder and is being used onsite from 2015.

The Institute is carrying out survey on filter-sterilization system for farm influent water on the basis of

fundamental research on farm’s disease control to confirm effectiveness of disease reduction in water treatment

system and its on-site application.

The Institute is closely monitoring emaciation symptoms recently found in Jeju olive flounder farm and

investigating their causes. For Kudoa which has effects on the export of Jeju halibut and its quality safety, it is

developing disinfectants.

Topic 4

7

Survey of viral hemorrhagic septicemia virus (VHSV) in olive flounder farms

Wi-Sik Kim, Min-Seok Jang, Seok-Ryel Kim1, Myung-Joo Oh

Department of Aqualife Medicine, Chonnam National University1National Fisheries Research and Development Institute

Viral hemorrhagic septicemia (VHS) generally occurs after juvenile olive flounders (Paralichthys olivaceus) are

moved from the hatchery to on-growing system in Korea during spring. Whether fish are infected by VHS virus

(VHSV) in the hatchery or the on-growing system remains unclear. In the present study, a survey was conducted

to investigate VHSV infections in 34 olive flounder hatcheries from 2014 to 2016. Samples were tested for the

presence of VHSV by inoculating sample to FHM and CHSE-214 cells to observe cytopathic effect, RT-PCR,

and antibody detection ELISA. VHSV was not detected in any of the 557 samples tested, although 34 (20.7%)

of 164 samples was found to be positive for marine birnavirus (MABV). Antibody detection ELISA results

revealed that all 212 fish sera showed OD values below of 0.1, suggesting that these fish had no VHSV-specific

antibodies. VHSV was not detected in any of 40 pooled samples (172 fish) collected after shifting rearing water

temperatures from 17-21°C to 10-15°C. In conclusion, VHSV-infection was not found in olive flounder hatcheries

in Korea.

Topic 5

8

Disease monitoring of maricultured salmonid fish

Wi-Sik Kim, Kyoung-hui Kong, Myung-Joo Oh

Department of Aqualife Medicine, Chonnam National University

Recently, mariculture of salmonid fish has been initiated in the coast areas of Korea. Mariculture of salmonid

fish has several advantages including a (i) high expected demand because of the popularity of salmonid fishes in

Korea, (ii) it grows quickly in seawater and can be easily cultured in olive flounder farms because farmers can

control the water temperature throughout the year using seawater that is underground, and (iii) it can be cultured in

empty farms on the southern coast areas during the winter season. In this study, disease monitoring was conducted

using maricultured salmonid fish at farms in Goheung and Jeju during 2014-2016.

About 70% mortality occurred in cultured coho salmon Oncorhynchus kisutch at a marine farm in Jeju in 2014.

Diseased fish showed greyish or pale patches on the gills, with no internal signs of disease. No bacteria or viruses

were isolated from diseased fish, but numerous amoebae were found on the gills. Histopathological examinations

revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between

gill filaments. The amoebae had a 630 bp partial 18S rRNA gene fragment specific to Neoparamoeba perurans.

Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that this Korean amoeba

belonged to the N. perurans group. This is the first report of N. perurans infection in Korea.

About 10% mortality occurred in cultured rainbow trout Oncorhynchus mykiss at a marine farm in Jeju in 2014.

Diseased fish showed markedly abdominal distension and distended stomach. Although parasites, bacteria or viruses

were not isolated from diseased fish, numerous Candida sp. were isolated from distended stomach. In experimental

infection with Candida sp., mortality was not observed in most fish and clinical sign of distended stomach was not

observed in the fish. These results suggest that Candida sp. may not be the etiologic agent of stomach distension

syndrome. This is the first report of stomach distension syndrome in seawater farmed rainbow trout in Korea.

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm

in Goheung during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat

with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates

produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo-214 cells. The cell culture

supernatant showing CPE contained specific 1527-bp fragment for the infectious hematopoietic necrosis virus

(IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities

with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean

IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results

suggest that this IHNV isolate might have been introduced to rainbow trout farm (land-based culture system) in

Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.

Topic 6

9

Severe mortality of farmed rainbow trout caused by infectious

haematopoietic necrosis virus in Korea

Hyoung Jun Kim1, Jung Jo Han2, Torunn Taksdal3, Ole Bendik Dale3, Niels Jorgen Olesen4,

Jeong Su Park5, Se Ryun Kwon5

1National Fishery Products Quality Management Service2Gyeonggi Province Freshwater Fisheries Research Institute

3Norwegian Veterinary Institute4National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark

5Sunmoon University


In May 2015, mass mortality of rainbow trout occurred in Jeollabuk-do province. Mortality was first observed in

April and all died in early May. The moribund fish were concentrated around airation and rainbow trout weakly

swam and died. Moreover, the visible symptoms included darkening body, exophthalmos and anatomic symptoms

included severe anemia of gills, clearing kidney. For investigating the cause, we used several methods such as

parasitological observation, bacterial culture and viral examination. In the result, any pathogen was not detected by

parasitological and bacterial examination, but cytopathic effects (CPE) appeared on RTG-2 cell line by inoculation

with kidney and spleen tissues. After 7 days, we used specific primer sets for detection of several viral diseases.

In the result, we detected the IHNV gene by RT-PCR method, and the amplified PCR product (glycoprotein gene

of full ORF) was sequenced. In phylogenetical analysis, the IHNV was grouped in IHNV JRt Nagano group. It

was reported IHNV JRT Nagano group has lower pathogenicity than IHNV JRT Shizuoka group, but the mortality

case demonstrated that IHN occurrence can lead to mass mortality even in group with low virulence group.

Topic 7

10

Assessment of a new conventional RT-PCR for VHSV detection

in an inter-laboratory proficiency test

Hyoung Jun Kim1*, Niels Jørgen Olesen2

1National Fishery Products Quality Management Service, Busan, Korea2National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark


Conventional PCR is typically used for detecting and genotyping of pathogens. However, we found a low

sensitivity for detection of VHSV IVa isolates using conventional RT-PCR on OIE aquatic manual (VN primer set).

Further, non-specific bands with fish cell lines were often observed when using the OIE RT-PCR of VN primer

set. Thus, a novel conventional RT-PCR (KIM3F2R) have been developed and validated for detection of all

genotypes of VHSV. The KIM3F2R method showed the same sensitivity as cell culture and real-time PCR.

Non-specific responses were not observed in heterologous viruses, tissue of several fish species (rainbow trout,

atlantic salmon, olive flounder) and normal fish cell lines. The novel RT-PCR was following tested on 80 VHSV

isolates representing a worldwide collection of all known genotype and subtypes, where it produced clear and

unique amplicons for all 80 isolates. It was to assess the reproducibility of a novel conventional RT-PCR for

detection of VHSV by an inter-laboratory proficiency test among 7 selected laboratories using KIM3F2R primer set.

10 samples were prepared including 6 VHSV, 3 heterologous virus and 1 normal medium. The viral supernatants

were dropped on FTA cards. First of all, the FTA cards were cut by scissors, and cut into small pieces and mixed

with lysis buffer. After then, the RNA was extracted from FTA cards of small pieces. And, we performed real-time

PCR and conventional RT-PCR using Kim3F2R primer. In the result, the PCR results from 7 institutes were

successful, and the reproducibility of KIM3F2R was confirmed by several institutes and kits. The reproducibility of

KIM3R2R was confirmed by several institutes. Finally, we suggest that the KIM 3F2R primer set shall replace the

current primer set recommended in the OIE manual for detection of VHSV.

Topic 8

11

Effect of fetal bovine serum (FBS) concentration on the infectivity titer of several viruses

Se Ryun Kwon

Department of Aquatic Life Medical Sciences, Sunmoon University

Fetal bovine serum (FBS) is the most widely used for the culture of animal cells, because it has a very low

level of antibodies and contains more growth factor. Cell culture is usually conducted using culture media

supplemented with 10% (v/v) of FBS. However, the percentage of FBS may vary with the individual in virus

quantification using endpoint dilution assay, and there is no report about them. In this study, the infectivity titer

of VHSV was compared in fish cell line using Eagle’s minimum essential medium (MEM) supplemented with

different concentration of FBS. Epithelioma papulosum cyprinid (EPC) cells grown using MEM supplemented

with 10% FBS, 100IU/ml penicillin G, and 100ug/ml streptomycin sulfate were removed from cell culture flask

by treatment of trypsin-EDTA solution. Cells were washed twice with MEM, divided into four and resuspended

with MEM supplemented with 0, 2, 5, or 10% of FBS (MEM-0, MEM-2, MEM-5 or MEM-10). Each suspended

cells was seed in 96-well tissue culture plates, and the plates were then incubated at 20°C. The next day,

VHSV, IHNV and IPNV stock were 10-fold serially diluted and seeded in each 96-well tissue culture plates.

After 14 d of culturing the appearance of CPE was evaluated to determine the 50% tissue culture infectious dose

(TCID50). As a result, infectivity titer of all virus samples using MEM-2 and MEM-5 was higher than using

MEM-0 and MEM-10. FBS concentration did not affect the cell viability and growth during the experiment.

Topic 9

12

Development of rapid immunochromathographic test strip for detection of viral hemorrhagic

septicemia virus (VHSV) of olive flounder

Wi-Sik Kim, Min-Seok Jang, Ha-Na Jeong, Myung-Joo Oh

Department of Aqualife Medicine, Chonnam National University

Viral hemorrhagic septicemia virus (VHSV) is a problematic pathogen in olive flounder (Paralichthys olivaceus)

aquaculture farms in Korea. Thus, it is necessary to develop a rapid and accurate diagnostic method to detect this

virus in the field. We produced monoclonal antibody (MAb) against VHSV and developed rapid

immunochromatographic test strip for VHSV detection. Balb/c mice were injected with purified VHSV-FYeosu05

(genotype IVa) from diseased olive flounder. Nine hybridoma clones secreting MAbs against VHSV were

established. The MAbs were recognized glycoprotein (G)-protein (4 MAbs), nucleoprotein (N)-protein (2 MAbs),

phosphoprotein (P)-protein (1 MAb), or matrix protein (M)-protein (2 MAbs). A simple strip-test kit for VHSV

detection was developed using a MAb (against the N-protein) conjugated with colloidal gold as the detector

antibody. The MAb was also used as the capture complex at the test line (T), and goat anti-mouse IgG antibody

(GAM) was used as the capture antibody at the control line (C). In test samples containing VHSV, the virus

bound to the MAb conjugated with colloidal gold and the resulting complex was captured by the antibodies at T

to give a reddishpurple band. Any unbound MAb conjugated with colloidal gold moved across T to be captured by

the GAM and formed a band at C. In VHSV specificity test, positive reaction was observed in only VHSV, but

not in infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HIRRV), marine birnavirus (MABV),

and nervous necrosis virus (NNV). This method was able to detect about 105TCID50/100ulofVHSV. Infield samples

test, it was completely distinguished between normal fish (negative reaction) and diseased fish (positive reaction),

suggesting that this tool will be useful for rapid detection of VHSV in olive flounder farms.

Topic 10

13

Development of recombinant VHSV vaccines using reverse genetics

Min Sun Kim1* and Ki Hong Kim2

1Graduate School of Integrated Bioindustry, Sejong University2Department of Aquatic Life Medicine, Pukyong National University

Viral haemorrhagic septicemia caused by viral hemorrhagic septicemia virus (VHSV) is one of the most

important viral diseases not only threatening the aquaculture industry but also affecting the wild fish fishery.

Reverse genetics is one of the most effective approaches for the development of live viral vaccines. In the first

study, the potential of the NV-gene knockout recombinant VHSV (rVHSV-ΔNV-EGFP) as a live attenuated

vaccine was assessed. In the vaccination experiment, the cumulative mortality rates of olive flounder i.m.

immunized and orally administrated with rVHSV-ΔNV-EGFP were 0-10% and 10-30%, respectively. The serum

and mucus neutralization titers were also proportional to the survival rates of vaccine groups. These results

suggest that the rVHSV-ΔNV-EGFP has a high potential to be used as a live vaccine in olive flounder farms. In

the next study, to produce a safety-enhanced live viral vaccine, we generated recombinant VHSV lacking an

essential gene for the production of infective viral particles. The rhabdoviral envelope-spiked glycoprotein (G)

plays critical roles in the attachment of viruses on the cell surface receptor and in the transfer of endocytosed

viruses to the cytoplasm by fusion to the endosomal membrane. Thus, we produced a G gene-lacking

recombinant VHSV (rVHSV-ΔG) that has no ability to form plaques in the cells without a trans-supply of the G

protein, which suggests that rVHSV-ΔG is a single-cycle virus and progenies of rVHSV-ΔG are not infectious.

One of the major advantages of attenuated vaccines is the maintenance of replication ability in the host, which

enforces the adaptive immune responses. However, in spite of lacking an ability to produce infective viral

particles, immunization with the present rVHSV-ΔG induced significantly higher serum neutralization titers and

survival rates against virulent VHSV challenge in olive flounder fingerlings, indicating that the present rVHSV-Δ

G has a high potential as a prophylactic vaccine.

Topic 11

14

Activities for Control of Foot-and-Mouth Disease in OIE FMD Reference Laboratory in

Republic of Korea

Jong-Hyeon Park

OIE FMD Reference Laboratory

Animal and Plant Quarantine Agency, 177 Hyeoksin 8-ro, Gimcheon City, Gyeongsangbuk-do, 39660,

Republic of Korea

During 2016, outbreaks in South Korea have occurred (due to the O/SEA/Mya-98 lineage). Up to March 2016,

21 farms in the west of the country were affected by FMD and sequence data indicates that the causative virus

strain is a unique sub-clade of O/SEA/Mya-98. Additional farms (147 pig holdings and 11 cattle farms) were

NSP positive in the same area of the country indicating that FMD virus may be circulating more widely.

International vaccine available are O1 Manisa+ O 3039. Since November 2016, additional vaccines (O Campos,

O Primosky) have been deployed for emergency purposes because of limited supply of other vaccines.

Post-vaccination monitoring in cattle and fattening pigs indicates good results with structural protein O ELISAs

showing a 95% and 65% protection level. In 2017 FMD outbreaks (O/ME-SA/Ind2001d, and A/Sea-97 lineage),

the implementation of national vaccination were protected the susceptible animals from the viruses in the fields.

The laboratory currently organises two national PTS twice a year (n=6). There is a collaborative network with

RRLSEA and other regional partners.

Topic 12

15

Viral Hemorrhagic Septicemia Virus and Infectious Hematopoietic Necrosis Virus in British

Columbia, Canada: interplay between wild and farmed fish

K. A. Garver

Fisheries and Oceans Canada, Pacific Biological Station, Nanaimo, British Columbia, Canada.

In the coastal waters of British Columbia, Canada, viral hemorrhagic septicemia virus and infectious

hematopoietic necrosis virus occur naturally in multiple species of wild fish. Yet upon introduction of exotic

Atlantic salmon into open net-pen marine aquaculture sites, these viruses have sporadically crossed species barrier

causing disease and losses to the Atlantic salmon aquaculture industry. Molecular epidemiological investigations of

these incursions revealed close genetic linkage between virus isolates from wild pelagic finfish species and

farmed salmonids, providing evidence for virus transmission from wild to farmed fish. For VHSV, an

inter-species laboratory challenge model was established to experimentally investigate virus transmission between

farmed Atlantic salmon and Pacific herring. Disease transmission was quantified by recording mortality, clinical

signs, histopathological changes, cellular sites of viral replication, expression of interferon-related genes, and viral

tissue titers. For IHNV, empirical studies were conducted to quantify infectious dose, shedding capacity, and

destruction rates associated with IHNV in Atlantic salmon aquaculture. These laboratory studies will be discussed

in the context of viral transmission and trafficking risk among populations of farmed salmonids and wild marine

reservoirs.

Topic 13

16

VHSV MANAGEMENT AND CONTROL: HOW CAN MOLECULAR EPIDEMIOLOGY

CONTRIBUTE?

Valentina Panzarin

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padova), Italy

Viral haemorrhagic septicaemia virus (VHSV) represents a severe threat to the industry of economically

relevant fish species such as rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus) and olive

flounder (Paralichthys olivaceus). Elucidating the patterns of viral spread is an essential requirement to understand

the mechanisms shaping disease transmission and, eventually, to develop appropriate control measures in order to

diminish the risk and the impact of VHSV in the aquatic environment. Tracing fish viruses is facilitated by the

growth of public databases and by the advent of NGS, which has considerably increased the amount of genetic

information available. Notably, collating metadata and sequence information is of utmost importance to address

key questions regarding the epidemiology of infectious diseases, i.e. identification of the origin of an outbreak,

reconstruction of transmission routes, identification of molecular markers associated with virulence, as well as

study of viral evolution and emergence. The analyses of these data have implications at all levels of clinical

practice and disease management, from the development of policies for surveillance and the implementation of

the diagnostic capacity to the application of prevention and treatment measures at a farm level.

Some recent studies addressing molecular epidemiology investigations of VHSV will be presented. A special

focus will be given to the VHSV situation in Europe. Possible scenarios for viral spread between different fish

farms, host populations, and countries will be suggested, and the potential of molecular epidemiology to

contribute to the management and control of infectious diseases in the aquatic environment will be discussed.

Topic 14

17

Transcriptome analysis of olive flounder, Paralichthys olivaceus

immunized with Poly(I:C)-VHSV or formalin-killed VHSV

Se Ryun Kwon1, Jeong Su Park1, Hyoung Jun Kim2

1Department of Aquatic Life Medical Science, Sunmoon University 2National Fishery Products Quality Management Service, Busan, Korea

Our previous study showed that Poly(I:C) immunization with the live vaccine offers better protection than the

formalin-treated vaccine against VHS in olive flounder. The purpose of this study is to clarify which genes are

involved in the protective response of olive flounder immunized with Poly(I:C)-VHSV comparing with that of

olive flounder immunized with formalin-treated vaccine. To analyze the vaccination-induced changes in the

expression profiles of genes, kidneys were collected from naive and each vaccinated fish at days 3 after

vaccination. Transcriptome analysis from kidney tissue using the next generation sequencing (NGS) technology,

Illumina Hi-seq 2000 system. Adapters and low quality reads (Q<20) from raw sequences were removed by

FastQC toolkit. Only high-quality reads were de novo assembled by platform Trinity(r20140717). The number of

reads mapped to each transcript was counted for each group and then normalized as value of fragments per

kilobases of transcripts per million fragments mapped(FPKM). The sequence of identified DEGs was annotated

with BLAST, GO analysis. Clean reads of Naïve, Poly(I:C)-VHSV and FK-VHSV samples were de novo

assembled into a transcriptome consisting of 176,732 contigs with N50 of 1,866. In addition, Poly(I:C) and

formalin-treated group were up-regulation of 1,220 and 1,615 contigs , down-regulation of 1,607 and 1,754

contigs. Top up-regulation differentially expressed genes (DEGs) of Poly(I:C)-VHSV versus FK-VHSV include

complement C-1q-like protein, interleukin-1 receptor type II, interleukin-20 receptor subunit alpha-like, caspase-3,

transmembrane 4L6 family member 5-like, cadherin-related family member 5-like isoform X3, NFK1-type

zincfinger-containing protein, lghMV VH4 region, CD9 antigen like isoform X1 and other immune related

proteins.

Topic 15

18

TLR and apoptotic response of viral haemorrhagic septicaemia virus

infected olive flounder at 15°C and 20°C

Sung-Ju Jung

Chonnam National University, Korea

The olive flounder (Paralichthys olivaceus) shows a high rate of mortality to viral haemorrhagic septicaemia

virus (VHSV) in the winter and spring but has zero mortality over 20°C. In this experiment, we studied the

effect of rearing temperature on viral replication, viral transcription, toll-like receptors (TLRs) and antiviral

apoptotic immune response in VHSV-infected olive flounder by real-time polymerase chain reaction. Olive

flounder were given intra-peritoneal injections of VHSV (107.8TCID50/ml) and were reared at 15°C or 20°C.

Five fish were randomly sampled for headkidney at 3, 6 and 12 hours post-infection (hpi) and 1, 2, 4 and 7

days post-infection (dpi). In the 15°C group, the number of viral gRNA copies peaked after 2 dpi and remained

high through 7 dpi, while in the 20°C group, the copy number was at the highest at 1 dpi but drastically

declined at later stages. Viral mRNA levels in the 15°C group gradually increased starting at 3 hpi to reach the

irmaximum value at 12 hpi and remained high until 2 dpi, where as the other group showed much lower copy

numbers that were undetectably low at 4 and 7 dpi. At 3 hpi, fish showed are lative fold increase of TLR 2,

TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7 and ISG 15. When viral copy numbers

were high, a higher expression of immune genes IL 1β, IRF 3 ,IRF 7, IFN Type I, ISG 15 and Mx was

observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immuneresponse was

considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A

high degree of correlation was observed between immune genes and viral copy number in each of the sampled

fish at 12 hpi. A fish with as cites sampled at 7 dpi displayed high viral copy but under expressed immune

genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were

down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression

of the immune genes indicates their crucial role in survival of host against VHSV.

Type II IFN expression increased as the viral copies increased and the 20°C group showed quicker and

stronger expression than the 15°C group. The MHC class I and CD8 expression was high in both the groups at

early stage of infection (3-6 hpi) but at later stages (2-7 dpi) in 15°C group expression reduced below control

levels, while they expressed higher to control in 20°C group. The expression of granzyme in 15°C fish showed a

single peak at 2 dpi, but was consistently expressing in 20°C fish. Individuals expressed very high levels of

perforin expressed very high levels of caspase 3. In 15°C fish, TNFα, FasL and p53 expressed significantly

higher than 20°C only at initial stages of infection (3-6 hpi). Caspase 3 expression found to be low in 15°C fish

whereas it was significantly elevated in 20°C group. Interestingly individual fish with high caspase 3 expression

contained very low viral RNA. Thus, from our experiment, we can conclude that an effective apoptotic immune

response in VHSV-infected olive flounder plays a crucial role in the survival of the host at higher temperatures.

Topic 16

An entry of The 2nd International Workshop on VHS and Rhbdoviral diseases in Korea

10 and 11th May 2017

Num. Name Organization E-mail

1 Niels Jørgen Olesen National Veterinary Institute in Denmark [email protected]

2 Kyle A. Garver Fisheries & Oceans Canada [email protected]

3 Valentina Maria PanzarinInstituto Zooprofilattico Sperimentale

delle Venezie in [email protected]

4 Kim Min Sun Sejong University [email protected]

5 Kim Wi Sik Chonnam National University [email protected]

6 Jung Sung Ju Chonnam National University [email protected]

7 Kwon Se Ryun Sunmoon University [email protected]

8 Park Jong Hyeon Animal and Plant Quarantine Agency [email protected]

9 Won Seung Hwan Oceans and Fisheries Research Institute [email protected]

10 Kim Hyoung JunNational Fishery Products Quality

Management [email protected]

11 Park Shin ChulNational Fishery Products Quality


12 Kwon Hyun WookNational Fishery Products Quality


13 Lee Jeong SeonNational Fishery Products Quality


14 Eom Ji YeongNational Fishery Products Quality


15 Oh So YoungNational Fishery Products Quality


16 Jeon Jeong YongNational Fishery Products Quality


17 Kim Shin HuNational Fishery Products Quality


18 Kim Min JiNational Fishery Products Quality


19 Min Byung JooNational Fishery Products Quality


20 Choi Hae MinNational Fishery Products Quality


Num. Name Organization E-mail

21 Cho Jae BumNational Fishery Products Quality


22 Lee Jang WookNational Fishery Products Quality


23 Choi Hee JungNational Fishery Products Quality


24 Park Su JiNational Fishery Products Quality


25 Kim Ho YoulNational Fishery Products Quality


26 Kim Sang BoNational Fishery Products Quality


27 Kim Hyo JinNational Fishery Products Quality


28 Lee Nam HunNational Fishery Products Quality


29 Kwak Jun Soung Pukyong National University [email protected]

30 Abdellaoui Najib Pukyong National University [email protected]

31 Woo Soo Ji Pukyong National University [email protected]

32 Qadiri Syed Shariq Nazir Chonnam National University [email protected]

33 Park Jeong Su Sunmoon University [email protected]

34 Hwang Seong Don National Institute of Fisheries Science [email protected]

35 Hwang Jee Youn National Institute of Fisheries Science [email protected]

36 Do Jeong Wan National Institute of Fisheries Science [email protected]

37 Yoo Eun Ho Oceans and Fisheries Research Institute [email protected]

38 Han Jung JoGyeonggi Province Maritime & Fisheries

Research [email protected]

39 Cho Ki TaekGyeonggi Province Maritime & Fisheries

Research [email protected]

40 Park Jae Sin SEASUN Biomaterials [email protected]

2nd... · viral heamorrhagic septicaemia (vhs) with korea and denmark 3 topic 2) viral haemorrhagic...

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