Bo Young Jee

Effects of RNA interference-mediated knock-down of hypoxia-inducible factor-α on respiratory burst activity of the Pacific oyster Crassostrea gigas hemocytes

In mammals, hypoxia-inducible factor-1 α (HIF-1α) is known to play important roles not only in oxygen homeostasis but also in innate immune responses. In this study, to assess the functional role of HIF-α in respiratory burst activity of Crassostrea gigas hemocytes, oysters were injected with HIF-α- or green fluorescent protein (GFP)-targeted-long double-stranded RNAs (dsRNAs), and at 1, 3, and 7 days post-injection, knock-down of C. gigas HIF-α expression and production of reactive oxygen species (ROS) were analyzed. Expression of HIF-α in mantle, gill, and hemocytes of C. gigas was clearly down-regulated by injection of the HIF-α-targeted-long dsRNA, but was not inhibited by the GFP-targeted-long dsRNA, indicating that HIF-α expression was suppressed through sequence-specific and systemic RNA interference (RNAi). Respiratory burst activity of hemocytes was significantly increased by administration of GFP-targeted-long dsRNA. However, knock-down of HIF-α expression led to significant decrease of chemiluminescence (CL) response of C. gigas hemocytes at 3 and 7 days post-administration of HIF-α-targeted-long dsRNA, indicating the critical role of HIF-α in activation of respiratory burst activity of oyster hemocytes.

Fugu double U6 promoter–driven long double-stranded RNA inhibits proliferation of viral hemorrhagic septicemia virus (VHSV) in fish cell lines

A long double-stranded RNA (dsRNA)-producing vector driven by fugu double U6 promotors, in which the two promoters were arranged in a head-to-head fashion, was newly constructed. To determine whether the DNA-vector-based long dsRNAs can induce sequence-specific RNA interference (RNAi), Epithelioma papulosum cyprini (EPC) cells and chinook salmon embryonic (CHSE-214) cells were transfected with the long dsRNA vector targeting the G gene of VHSV, and its effect on expression of the G gene and viral proliferation was investigated. The sequence-specific inhibitory effect was further confirmed by analysis of interferon (IFN)-triggered Mx1 gene expression and cross-protection against infectious hematopoietic necrosis virus (IHNV). The fugu double U6 promoter–driven vector successfully produced long dsRNAs in EPC cells, a system that allows continuous production of long dsRNAs in transfected cells. The plasmid-based long dsRNAs targeting the VHSV G gene effectively suppressed G gene expression, but control dsRNAs targeting the EGFP gene did not. Furthermore, there was no significant difference in Mx gene expression between cells transfected with the long dsRNA-producing vector and those transfected with the control empty vector. These results suggest that G gene expression was suppressed not by type-I-IFN-mediated nonspecific inhibition but in a sequence-specific manner. Both EPC and CHSE-214 cells transfected with plasmids producing long dsRNAs targeting the VHSV G gene were protected against VHSV infection but were not protected against IHNV infection, suggesting sequence-specific RNAi-mediated inhibition of viral proliferation. In conclusion, we show, for the first time, long-dsRNA-mediated RNAi in fish cells. The DNA-vector-based long dsRNAs may provide an efficient tool for analysis of gene function in fish cells without preliminary burdensome work for selection of effective siRNA clones, and it may be applied as an antiviral measure in cultured fish.

Detection of Ostreid Herpesvirus 1 from adult Pacific Oysters Crassostrea gigas Cultured in Korea

The presence of ostreid herpesvirus 1 (OsHV-1) and the percentage of viral DNA detected in Pacific oyster Crassostrea gigas adults were investigated monthly between May and November 2012 at three locations along the southern coast of Korea. Among 210 oysters examined by polymerase chain reaction (PCR) analysis, OsHV-1 DNA was detected in only one oyster collected in August. The low detection rate of OsHV-1 DNA was consistent with the lack of reported OsHV-1-associated disease in C. gigas cultured in Korea. The sequence of the present PCR product amplified with the C2/C6 primer pair was identical to that of OsHV-1 μVar except for one nucleotide, and the sequence amplified with Del36-37F2/Del36-37R showed a 605-bp deletion as in OsHV-1 μVar. Although these sequence data are insufficient to determine genotype, the results suggest that the herpesvirus detected was similar to OsHV-1 μVar. This is the first report on the presence of OsHV-1 in adult Pacific oysters cultured in Korea.

Genetically similar VHSV isolates are differentially virulent in olive flounder Paralichthys olivaceus

Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV-KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was evaluated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortality of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortality of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, suggesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substitutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Gene expression and functional characterization of serum amyloid P component 2 in rock bream, Oplegnathus fasciatus

Mammalian serum amyloid P component (SAP) recognizes a wide range of exogenous pathogenic substances and activates a complementary pathway leading to pathogen clearance. To determine the potential roles of SAP in the fish immune system, SAP (RbSAP2) gene was cloned from ESTs analysis of rock bream (Oplegnathus fasciatus), which consisted of a signal peptide and pentraxin domain. Phylogenetic analysis revealed that the RbSAP2 gene was classified with other known fish SAPs. RbSAP2 was highly expressed in the liver of healthy rock bream. Overall, pathogen exposure led to an induction of RbSAP2 in the liver and spleen, although this effect was not observed in the spleen following infection with Edwardsiella tarda. A high concentration of recombinant RbSAP2 (rRbSAP2) showed lower growth Streptococcus iniae than control in the absence of Ca(2+), whereas E. tarda growth was decreased by high concentration of rRbSAP in the presence of the Ca(2+). These results suggest that RbSAP plays an important role in the immune response against invading pathogens.

The First Report of a Megalocytivirus Infection in Farmed Starry Flounder, Platichthys stellatus, in Korea

In 2009, a systemic megalocytivirus infection associated with high mortality was detected for the first time in cultured starry floun der Platichthys stellatus in Korea. Diseased starry flounder had pale bodies and gill coloring and enlarged spleens. Histopathologi cal examinations revealed basophilic enlarged cells in various organs of diseased starry flounder. Polymerase chain reaction (PCR) was performed on tissue samples using three published primer sets developed for the red sea bream iridovirus. PCR products were detected for all primer sets, except 1-F/1-R, which are registered by the World Organization for Animal Health (OIE). The part of the gene corresponding to the full open reading frame encoding the viral major capsid protein (MCP) was amplified by PCR. PCR products of approximately 1,581 bp were cloned, and the nucleotide sequences were analyzed phylogenetically. The MCP gene of the starry flounder iridovirus, designated SFIV0909, was identical to that of the turbot reddish body iridovirus (AB166788).

Diagnosis Case of Viral Hemorrhagic Septicemia (VHS) in Adult Olive Flounder Paralichthys olivaceus

We examined the cause of a disease outbreak in adult olive flounder Paralichthys olivaceus, which occurred at a Korean aquaculture farm in Korea in 2011. The principal signs included an expanded abdomen and congested liver, with persistent mortality (a little over two months). At the beginning of the outbreak, farm administrators misjudged the disease as bacterial in origin, because of the aforementioned signs, persistent mortality, and the detection of bacterial species, including Vibrio spp. and Streptococcus spp. Moreover, the detection of viral hemorrhagic septicemia virus (VHSV) by reverse trasnscription-PCR analysis was complicated by use of the VHS-VN primer set, which has been in general use recently, because it produced weak bands in some samples. Therefore, we recommend the use of at least two different primer sets in the diagnosis of VHSV. Our histopathological findings indicate that necrotizing myocarditis could be considered a pathogenic sign of VHSV infection.

Analysis of complete genome and pathogenicity studies of the spring viremia of carp virus isolated from common carp (Cyprinus carpio carpio) and largemouth bass (Micropterus salmoides): An indication of SVC disease threat in Korea

A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.

Disease Resistance against Bacterial Infection on Treatment of Hot-water Extract with 6 Herbal Mixtures in Olive Flounder Paralichthys olivaceus

The heat extracts of six kinds of medicinal herbs (Scutellaria baicalensis, Sophora flavescens, Citrus unshiu pericarpium, Lonicera japonica, Perilla frutescens, Benincasa hispida) were tested for non-specific immune response and disease resistance effects related with fish diseases on olive flounder, Paralichthys olivaceus. The preventive effects of 6 herbal mixtures against bacterial disease on cultured flounder were examined follow as feeding EP absorbed with the heat extract of six kinds of medicinal herbs. For feeding trial for 12th week, weight gain and serum analysis of fish fed various groups were not significant differences. Lysozyme activity of the 0.01 % treated group on 4th week showed significant increase. Histopathology of the administrated group for feeding period showed no particular signs of tissue degeneration. At 0.01% oral experimental group, relative percent survival (RPS) were only 50% to 75% for four weeks and eight weeks group by intraperitoneal injection with E. tarda. The results suggest that heat extracts of six kinds of medicinal herbs (0.01%) would be effective to enhance the nonspecific immunity and protective ability of olive flounder against fish pathogen. Key word : Herb extract, Immunostimulant, Disease resistance, Paralichthys olivaceus Corresponding author : 051-720-3041, jsseosoo@korea.kr (R2016069) .