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Enzyme-linked immunosorbent assay를 이용한 바이러스성 출혈성 패혈증 바이러스 감염 넙치(Paralichthys olivaceus)의 특이 항체반응 검사
황지연,장진현,김동준,권문경,서정수,황성돈,손맹현,Hwang, Jee Youn,Jang, Jin Hyeon,Kim, Dong Jun,Kwon, Mun Gyeong,Seo, Jung Soo,Hwang, Seong Don,Son, Maeng-Hyun 한국수산과학회 2017 한국수산과학회지 Vol.50 No.5
The viral hemorrhagic septicemia virus (VHSV) has an extensive host range, and infects farmed and wild fish inhabiting both freshwater and marine ecosystems. Enzyme-linked immunosorbent assay (ELISA) is highly useful in diagnosing viral hemorrhagic septicemia. However, ELISA shows high, non-specific background reaction with fish antibodies. In this study, we optimized the antigen and antibody concentrations used for detecting specific antibodies in VHSV-infected olive flounder to reduce non-specific binding, and improve the sensitivity of ELISA. The results suggested that OD (optical Density) values were valid when ELISA was performed with $0.1{\mu}g/well$ of virus, involving blocking with blocking buffer (Roth, Roti-Block), 1:300-1:600 dilution with flounder antisera, and 1:1000 dilution with anti-flounder IgM and HRP-conjugated goat anti-mouse IgG for detecting the VHSV antibody in flounder sera. Furthermore, 11 different VHSV strains isolated in Korea from 2012 to 2016 were used to infect the fish. The results showed no correlation between viral pathogenicity and antibody production. This research is a basic study on the application of antibody detection in the diagnosis of viral hemorrhagic septicemia in the olive flounder.
황지연(Jee-Youn HWANG),권문경(Mun-Kyoung KWON),서정수(Jung-Soo SEO),김경덕(Kyoung-Duck KIM),이예숙(Ye-Suk LEE),정승희(Sung-Hee JUNG) 한국수산해양교육학회 2015 水産海洋敎育硏究 Vol.27 No.5
In this study we investigated the effect of vaccine efficacy and immune activities by feeding additives several natural herbal remedies extracts of Sweet Wormwood (Artemisia annua), Galla Rhois, Oriental raisin tree (Havenia Dulicis) and 6-mixed herb to Oilve flounder, Paralichthys olivaceus. Each group of fish fed with the herbal remedies added feed and basic formula during the seven weeks and vaccinated with Edwardsiella tarda Formalin killed vaccine after 4 weeks feeding. Relative Percentage Survival values (RPS) in the group was assessed by the challenge with E.tarda. All groups with the herbal remedies added feed enhanced growth rate, but there were not significantly different in lysozyme activity and agglutination titer. In a challenge experiment with E.tarda, RPS in the all groups feeding additives natural herbal remedies was higher than that of the control group. These results suggest that the natural herbal remedies extracts of Sweet Wormwood (Artemisia annua), Galla Rhois, Oriental raisin tree (Havenia Dulicis) and 6-mixed herb would be effective to enhance efficacy of vaccination to Olive flounder.
In vitro 조건에 따른 molecular adjuvant의 넙치, Paralichthys olivaceus 면역유전자 자극 효과
권문경(Mun-Gyeong KWON),황지연(Jee-Youn Hwang),서정수(Jung-Soo SEO),정승희(Sung-Hee JUNG) 한국수산해양교육학회 2015 水産海洋敎育硏究 Vol.27 No.5
Adjuvant is an immune enhancer commonly used during vaccination to enhance the host immune response. In the present study, we produced the several recombinant protein from immune related gene of olive flounder (Paralichthys olivaceus). Especially, to produce the soluble type of recombinant protein, we constructed the MBP (Maltose binding protein) fusion G-CSF (Granulocyte colony stimulating factor) recombinant protein among the flounder immune related genes. To verify the immune stimulatory effect and safety of this recombinant protein (rPoGCSF), expression changes of several immune genes were tested using quantitative real-time PCR method with gene specific primer from flounder head kidney leukocytes. As a result, we confirmed that the rPoGCSF has an ability of immune stimulatory effect, also it has broad range of pH and temperature.
PCR 기법을 이용한 2009년 우리나라 서해안과 남해안 바지락, Ruditapes philippinarum의 Perkinsus olseni 감염에 관한 보고
이남실 ( Nam Sil Lee ),황지연 ( Jee Youn Hwang ),최동림 ( Dong Lim Choi ),박명애 ( Myoung Ae Park ) 한국어병학회 2010 한국어병학회지 Vol.23 No.2
Prevalence of a protozoan parasite Perkinsus olseni in Manila clam Ruditapes philippinarum was surveyed from July to December 2009 on the west and south coast of Korea. P.olseni infection was diagnosed using two primer sets, P.olseni NTS Forward/P.olseni NTS Reverse set and PolsITS-140F/PolsITS-600R set in polymerase chain reaction(PCR). The results using PolsITS-140F and PolsITS-600R primer set was retained up to 60% at all stations from July to December, except for Padori. Especially, Goheung showed 100% prevalence from October to December. The results about comparison of the 4 station`s DNA sequences which were analyzed from PCR products(457bp) using PolsITS-140F and PolsITS-600R primer set, there were only 2base differences at Sunjedo.
권문경(Mun-Gyeong KWON),황지연(Jee Youn HWANG),정성희(Sung Hee JUNG) 한국수산해양교육학회 2014 水産海洋敎育硏究 Vol.26 No.6
Edwardsiella tarda, Streptococcus iniae and S. parauberis are main bacterial pathogens in aquaculture farms of olive flounder, Paralichthys olivaceus. We have discussed the efficacy and safety of 3 type-combined vaccines (A: S. iniae 1mg + S. parauberis 1mg + E. tarda 1mg, B: S. iniae 1mg + S. parauberis 1mg + E. tarda 0.5mg, C: S. iniae 1.5mg + S. parauberis 1.5mg + E. tarda 1mg) through intraperitoneal injections in olive flounder. None of the vaccines gave rise to any signigicant side effects on histopathology and blood chemistry. The antibody titers and lysozyme activities of A type were higher than those of B, C and control. Four weeks after vaccination, RPS (relative percent survival rates) was 62.5~75% (A type), 50~66.7% (B type) and 55.6~62.5% (C type) respectively. As the results, the combined vaccines are possible to prevent edwardsiellosis and streptococcosis, and A type : S. iniae 1mg + S. parauberis 1mg + E. tarda 1mg, is the most effective out of them.
Young-Chul KIM(김영철),Jee-Youn HWANG(황지연),Hae-Ryeon JEON(전해련),Da-Won LEE(이다원),Jung-Soo SEO(서정수),Kwang-Il KIM(김광일),Mun-Gyeong KWON(권문경),Bo-Young JEE(지보영),Seong-Don HWANG(황성돈) 한국수산해양교육학회 2018 水産海洋敎育硏究 Vol.30 No.6
We identified structural and non-structural gene regions encoding major capsid protein (MCP) and DNA polymerase (DPOL) of megalocytiviruses collected from infected cultured fishes in RBIVD outbreak farms in 2013-2017 in Korea. With the two PCRs using 1-F/1-R and 4-F/4-R primer sets of the Manual of Diagnosis Tests for Aquatic Animals of the World Organization for Animal Health (OIE), amplicons were generated from the spleen and kidney tissue from approxmately ~30 fishes, including rock bream (Oplegnathus fasciatus), red sea bream (Pagrus major), and rock fish (Sebastes schlegeli), from 15 outbreak regions in the aquatic farms of the South Sea and Jeju Island. In phylogenetic analysis, complete MCP and partial DPOL genes belonged to RSIV type-subgroup2. Interestingly, these genes formed a cluster indicating closer relatedness to GSIV-K1, RIE12-1, and RBIV-C1, which were previously isolated from Japan and China, than with RBIV-KOR-TY1 isolated from Korea. However, the nucleotide sequence identities of the MCP and DPOL genes of these viruses were high, at >99.8% and >99.7%, respectively, compared with RBIV-KOR-TY1. Comparisons of nucleotide and amino acid sequences showed minimal differences between the obtained strains in the MCP gene, however, one or two nucleotide sequences substitutions of the DPOL gene were detected in nine strains, including a silent mutation detected in five strains. These findings suggest a slow rate of evolution of megalocytiviruses in this region, but the potential for mutations and new pathogenic strains warrants continuous surveillance.
바이러스성 출혈성 패혈증 바이러스(Viral Hemorrhagic Septicemia Virus) 감염 넙치(Paralichthys olivaceus)의 조직별 바이러스 정량분석
장진현,황성돈,정지민,권문경,황지연,Jang, Jin Hyeon,Hwang, Seong Don,Jung, Ji Min,Kwon, Mun-Gyoung,Hwang, Jee Youn 한국환경생물학회 2021 환경생물 : 환경생물학회지 Vol.39 No.3
국내외에서 80여 종이 넘는 담수 및 해산어류를 감염시켜 대량폐사를 발생시키는 바이러스성 출혈성 패혈증 바이러스(VHSV) 진단검사를 위해 넙치의 여러 조직의 바이러스 발현량에 대한 정량적 데이터를 시간순서에 따라 분석하였다. 무작위 선별된 넙치에 3.0E+07 TCID<sub>50</sub> per 0.1mL per fish의 VHSV를 복강 주사하여 시간순서(0시간, 6시간, 12시간, 1일, 2일, 3일, 5일, 7일)에 따라 조직(아가미, 간, 신장, 비장, 근육)을 채취하였다. Real-time PCR 법을 통해 상대 정량한 결과 5일차 아가미, 간, 신장, 비장에서 바이러스의 발현량이 가장 높게 나타났다. 이번 연구를 통해 감염 초기단계에서 비장이 VHSV 확정진단을 위한 적정조직임을 입증하였으며, 국내 법정전염병 진단에 중요한 정보를 제공할 것이다. A diagnostic test for viral hemorrhagic septicemia virus (VHSV), which infects more than 80 species of freshwater and marine fish at home and abroad, causing mass mortality, was conducted to provide quantitative data on the amount of virus expression in various tissues of flounder in chronological order. The tissues were collected in chronological order after the intraperitoneal injection of 3.0E+07 tissue culture infective dose<sub>50</sub> (TCID<sub>50</sub>) per 0.1mL per fish of VHSV to randomly selected flounder. As a result of relative quantification through real-time PCR, the highest levels of virus expression were found in the spleen, kidney, gill, and liver on day 5. This study proved that the spleen was an appropriate site for the final diagnosis of VHSV in the early stages of infection and will provide important information for the diagnosis of legal infectious diseases in Korea.
바이러스성 출혈성 패혈증 바이러스 NV 단백질에 의한 glucokinase 전사 활성의 억제
조미영(Mi Young Cho),황지연(Jee Youn Hwang),지보영(Bo Young Ji),박명애(Myoung Ae Park),성미소(Mi So Seong),김소영(So Young Kim),정예은(Ye Eun Jung),정재훈(Jae Hun Cheong),최영현(Yung Hyun Choi) 한국생명과학회 2016 생명과학회지 Vol.26 No.12
바이러스성 출혈성 패혈증 바이러스(VHSV)는 넙치를 포함한 어류 양식의 막대한 피해를 일으키는 바이러스 병원체이며, VHSV가 생성하는 6개의 바이러스 단백질들 중에서 NV 단백질이 병원성에 관여하는 것으로 알려져 있다. VHSV-감염 넙치를 이용한 전사체 마이크로 어레이의 선행 분석 결과에 의하면 VHSV 감염이 해당과정 효소들의 mRNA 발현을 억제함으로써 넙치 세포에서 ATP 생성을 감소시켰음을 알 수 있었다. 이들 결과를 토대로, 본 연구에서는 VHSV NV 단백질이 해당과정 효소인 glucokinase의 발현에 미치는 영향을 검토하였다. 본 연구의 결과에 의하면, NV 단백질은 넙치 세포에서 glucokinase의 mRNA 발현을 감소시켰으며, 새롭게 동정한 glucokinase의 유전자 프로모터의 활성 실험결과, NV 단백질이 glucokinase의 프로모터 활성을 저해함을 알 수 있었다. 이와 같은 작용 결과들로 인하여 VHSV NV 단백질의 발현이 세포 내로의 포도당 흡수 또한 감소시켰다. 이러한 결과들은 VHSV NV 단백질이 유전자 발현의 전사 수준에서 음성적으로 해당과정의 효소 발현을 조절함을 의미하며, 결국 세포 내 에너지의 결핍으로 넙치의 폐사로 이어질 가능성을 보여주는 것이다. The viral hemorrhagic septicemia virus (VHSV), which belongs to the Novirhabdovirus genus of the Rhabdoviridae family, is a viral pathogen that causes severe losses in the olive flounder farming industry. Among six encoding VHSV proteins, the non-virion (NV) protein has been shown to have an impact on virulence. In our previous studies, transcriptomics microarray analysis by using VHSV-infected olive flounder showed that VHSV infection significantly down-regulated the mRNA expression of glycolytic enzymes. In addition, VHSV NV protein variants decreased the intracellular ATP level. Based on these results, we have tried to examine the effect of VHSV NV protein on glycolytic enzyme glucokinase expression, which phosphorylates glucose to glucose 6-phosphate. Our results indicated that the NV protein significantly decreased the mRNA expression of glucokinase in olive flounder HINAE cells. Furthermore, the NV protein played a negative role in the promoter activation of glucokinase. Furthermore, glucose uptake was effectively inhibited by VHSV infection and NV protein expression in olive flounder HINAE cells. These results suggest that the VHSV NV protein negatively regulates glycolytic enzyme expression by a transcription level and eventually leads to gradual morbidity of olive flounder through cellular energy deprivation. The present results may be useful for the prevention and diagnosis of VHSV infection in olive flounder.
수온변화에 따른 해수내 VHSV(Viral Hemorrhagic Septicemia Virus)의 생존능 분석
정지민(Ji Min JEONG),지보영(Bo-Young JEE),권문경(Mun-Gyeong KWON),서정수(Jung Soo SEO),황성돈(Seong Don HWANG),이지훈(Ji-Hoon LEE),황지연(Jee Youn HWANG) 한국수산해양교육학회 2019 水産海洋敎育硏究 Vol.31 No.5
Viral hemorrhagic septicemia (VHS) is a serious viral disease that infects the olive flounder in South Korea. The Korean aquaculture industry experienced an economic loss caused by the high infectivity and mortality. Wastewater is a factor in transferring infections to wild and cultured fish in waters near infected aquaculture farms. In this study, we analyzed the stability of the VHS virus in sterilized seawater depending on the water temperature. The VHS virus maintained infectivity for 8 days and was detected using quantitative polymerase chain reaction after 10 days of inoculation to sterilized seawater at low temperatures. On the other hand, at high temperatures, the virus maintained infectivity for 4 days and was detected for 7 days. These results may contribute to basic data for epidemiological studies.
서정수(Jung-Soo SEO),정승희(Sung-Hee JUNG),지보영(Bo-Young JEE),권문경(Mun-Gyeong KWON),황지연(Jee-Youn HWANG) 한국수산해양교육학회 2018 水産海洋敎育硏究 Vol.30 No.6
Scuticociliate are one of the main parasite affecting the flounder aquaculture industry, causing significant economic losses in Korea. Aquatic formalin has been approved to treat against external parasitic infestation in flounder aquaculture pond. To investigate metabolic effect after treating the parasitic agent aquatic formalin in scuticociliate Miamiensis avidus, the transcriptome analysis was conducted. Through exposing with low concentration(10~50 ppm) of formalin for 96 hr, parasite trancriptomes were sequenced cDNA libraries in the GX Plex sequencer. A total of million read for control and formalin treated group were assembled in 187,210 and 177,959 contigs, respectively. After normalization and assembling with house keeping gene(β-actin), transcripts were assigned to biological processes and functions after annotation in Gene Ontology(GO). Specifically, 10 exclusive transcripts were up- or down-regulated for control and formalin treated group, respectively. Our results provide valuable genetic information for further analysis of the biological responses mechanism of aquatic formalin exposure in M. avidus.