• Journal of fish pathology
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• v.30 no.1
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• pp.1-9
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• 2017

This study was conducted to monitor the prevalence of viral hemorrhagic septicemia virus (VHSV), nervous necrosis virus (NNV) and marine birnavirus (MABV) in cultured walleye pollock Gadus chalcogrammus by RT-PCR. All of the viruses tested were not detected by one-step PCR in 62 spleen sample sets, except for NNV in one brain sample set (1/55). By two-step PCR, VHSV was detected in 51.6%(32/62) and NNV was detected in 1.6%(1/62) spleen sample set, but MABV was not detected. In the brain sample sets, the detection rate of NNV was 3.6%(2/55). VHSV and NNV were detected for the first time in cultured walleye pollock in this study. However, the titers of viruses in these sample sets are thought to be very low, because most of the positive sample sets were detected by two-step PCR and none of the fish showed any clinical symptoms of each virus. Continuous monitoring, subsequent virus isolation and validation of carrier fish will be necessary.

• The Plant Pathology Journal
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• v.33 no.4
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• pp.393-401
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• 2017

Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts of a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against plant viruses are poorly documented. Here we report isolation of a soil inhabiting bacterium, Pseudomonas oleovorans strain KBPF-004 (developmental code KNF2016) which showed antiviral activity against mechanical transmission of tobamoviruses. Antiviral activity was also evaluated in seed transmission of two tobamoviruses, Pepper mild mottle virus (PMMoV) and Cucumber green mottle mosaic virus (CGMMV), by treatment of seed collected from infected pepper and watermelon, respectively. Pepper and watermelon seeds were treated with culture supernatant of P. oleovorans strain KBPF-004 or control strain ATCC 8062 before planting. Seeds germinated after treatment with water or ATCC 8062 yielded about 60% CGMMV or PMMoV positive plants, whereas < 20% of KBPF-004-treated seeds were virus-infected, a significantly reduced seed transmission rate. Furthermore, supernatant of P. oleovorans strain KBPF-004 remodeled aggregation of PMMoV 126 kDa protein and subcellular localization of movement protein in Nicotiana benthamiana, diminishing aggregation of the 126 kDa protein and essentially abolishing association of the movement protein with the microtubule network. In leaves agroinfiltrated with constructs expressing the coat protein (CP) of either PMMoV or CGMMV, less full-size CP was detected in the presence of supernatant of P. oleovorans strain KBPF-004. These changes may contribute to the antiviral effects of P. oleovorans strain KBPF-004.

• The Plant Pathology Journal
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• v.21 no.2
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• pp.167-171
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• 2005

We isolated the Cucumber green mottle mosaic virus(CGMMV) particles from watermelon leaves and designated as CGMMV-HY1 as a watermelon isolate and attempted to characterize the pathogenic isolate responsible for such an epidemic in watermelon and also to monitor dominant viral isolates in greenhouse. The watermelon plants infected with CGMMV generally showed mottle mosaic, mosaic, growth stunting, necrosis and deformed fruit. The reactions of indicator plants to CGMMV-HY1 were the local lesions on Nicotiana tabacum cv. White Burley, Nicotiana tabacum cv. Samsun, and Chenopodium amaranticola, and the mosaic symptoms only on Cucumis sativus, but the CGMMV-HY1 did not infect Nicotiana sylvesytis, Datura stramonium, Chenopodium quinoa, and Petunia hybrida. Purified virus particles were rod-shaped and about 300 nm long. The coat protein (CP) of purified CGMMV-HY1 was single band with molecular weight of about 16.5 kDa which was confirmed by western blot analysis probed with monoclonal antibody of CGMMV-HY1. The genomic and subgenomic RNAs of 6.4 kb and 0.75 kb were revealed by the electrophoresis on 1.2% formaldehydedenatured agarose gel. Viral and complementary CGMMV-specific primer sets were designed for spanning the genome using previously reported CGMMV sequences. A 464bp of CP gene of CGMMV-HY1 was amplified by RT-PCR and cloned into PGEM-T easy vector. The nucleotide sequence of CP gene of CGMMV-HY1 shared 98%, 99%, and 100% identities with that of CGMMV strains W, KOM, and KW respectively. Based on these results, we identified CGMMV-HY1 as a CGMMV isolate of watermelon, a member of Tobamovirus.

• Korean Journal of Veterinary Research
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• v.43 no.2
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• pp.239-246
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• 2003

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.

• Korean Journal of Veterinary Research
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• v.52 no.4
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• pp.223-230
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• 2012

The worldwide distribution and continuing genetic mutation of avian influenza virus (AIV) has been posed a great threat to human and animal health. A comparison of 3 isolates of AIV H9N2, A/chicken/Korea/KBNP-0028/00 (H9N2) (KBNP-0028), A/chicken/Korea/SNU8011/08 (H9N2) (SNU 8011) and an inactivated oil vaccine strain A/chicken/Korea/01310/01 (H9N2) (01310), was performed. The former 2 AIVs were isolated from field cases before and after the application of an inactivated H9N2 vaccine in 2007, respectively. The antigenic relationship, viral shedding, tissue tropism and genetic analysis were examined. The comparison of virus shedding from the cloaca and the oropharynx revealed that both isolates were more frequently isolated from the upper respiratory tract (90~100%) 1 day post inoculation (DPI) compared with isolation 5 DPI from gastrointestinal tracts (10~60%). Moreover, the isolate KBNP-0028 were recovered from all organs including bone marrow, brain and kidneys, indicating higher ability for broad tissue dissemination than that of SNU 8011. KBNP-0028 replicated earlier than other strains and with a higher titer than SNU 8011. In full-length nucleotide sequences of the NA gene and a partial sequence of the HA gene of SNU 8011, we found that there might be significant changes in tissue tropism, virus replication and genetic mutation in AIV H9N2 isolates.

• The Plant Pathology Journal
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• v.22 no.4
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• pp.329-333
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• 2006

Two distinct and stable isolates of Barley mild mosaic virus(BaMMV) designated as Naju82-S(severe) and Naju82-M(mild) were obtained. These two isolates differed in their symptomatology, virus transmission characteristics and cultivar specificity at various temperature. Thus, these isolates were referred to as strains in this study. BaMMV Naju-S strain showed severe mosaic symptoms accompanied by necrosis on the infected leaves. Naju82-S strain is more virulent demonstrated by shorter incubation period and relatively high virus concentration than Naju82-M strain. Five Korean cultivars were tested for their pathogenicity to different strains based on the rate of infection. Results showed that infection rate of cultivars to both strains did not significantly differed from each other. However, under different temperatures, the pathogenicity on the two cultivars such as cultivars Hopumbori and Sessalbori were significantly affected. Hopumbori was moderately resistant to both strains at $10-12^{\circ}C$ and susceptible at $15-18^{\circ}C$. Similarly, Sessalbori was moderately resistant at $10-12^{\circ}C$ to both strains but distinctly differentiated at $15-18^{\circ}C$ wherein it was resistant to mild strain and highly susceptible to severe strain. Other cultivars including Baegdong, Jinyangbori and Neahanssalbori consistently showed susceptible reaction to both strains at varying temperatures tested in this study.

• Korean Journal of Veterinary Pathology
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• v.3 no.1
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• pp.35-44
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• 1999

The morphologic changes of small intestinal epithelium in pigs diagnosed as porcine epidemic diarrhea(PED} by virus isolation and immunohistochemistry were studied through light microscope and transmissible electron microscope. On semi-thin section, the histologic findings showed severe villous atrophy and fusion with hyperplasia of cuboidal epithelium in the villi, inflammatory cell infiltration in lamina propria, and increased mitotic figures in the crypt. The structural changes were mostly restricted to the cytoplasm of affected absorptive epithelium of villi. 3 types of epithelial changes were found; degenerated virus-affected cells, undifferentiated cuboidal cells, and normal columnar cells. On electron microscopy, round to spherical viral particles of 50∼l00nm in diameter were found within the dilated vesicles and endoplasmic reticulums of degenerated cells, which had decreased their cytoplasmic electron density due to dilated and missing organelles(e.g. mitochondria, ERs, etc.). Microvilli were shortened and sparse, leaving denuded terminal web of the villous epithelial cells. Fat globules were often found within slightly degenerated enterocytes. On the tip of villi, severely damaged cells were exfoliated and replaced by undifferentiated cuboidal cells We found distinct ultrastructural changes in the jejunal epithelium confirming PED virus infection is involved in malabsorptive diarrhea.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2037-2043
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• 2017

The surface protein hemagglutinin (HA) mediates the attachment of influenza virus to host cells containing sialic acid and thus facilitates viral infection. Therefore, HA is considered as a good target for the development of diagnostic tools for influenza virus. Previously, we reported the isolation of single-stranded aptamers that can distinguish influenza subtype H1 from H5. In this study, we describe a method for the selective electrical detection of H1 using the isolated aptamer as a molecular probe. After immobilization of the aptamer on Si wafer, enzyme-linked immunosorbent assay (ELISA) and field emission scanning electron microscopy (FE-SEM) showed that the immobilized aptamer bound specifically to the H1 subtype but not to the H5 subtype. Assessment by cyclic voltammetry (CV) also demonstrated that the immobilized aptamer on the indium thin oxide-coated surface was specifically bound to the H1 subtype only, which was consistent with the ELISA and FE-SEM results. Further measurement of CV using various amounts of H1 subtype provided the detection limit of the immobilized aptamer, which showed that a nanomolar scale of target protein was sufficient to produce the signal. These results indicated that the selected aptamer can be an effective probe for distinguishing the subtypes of influenza viruses by monitoring current changes.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.19-28
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• 2000

Two distinct hantaviruses have been isolated from Apodemus agrarius in 1976 and Rattus norvegicus in 1980 in Korea. Since our serosurveys conducted in 1994, a genetically distinct hantavirus from Apodemus peninsulae has been investigated. To isolate hantavirus from Apen insulae captured in Korea, the lung homogenate of seropositive Apeninsulae inoculated Vero E6 cells. Viral antigen was detected in a progressively higher percentage of cells with subsequent passage after 80 days postinoculation. The new isolate from seropositive Apodemus peninsulae was designated Suchong virus after Suchong valley located in northeastern region of South Korea. Comparing with hantaan virus 76-118 strain, Suchong virus-1, 2, 3 and 4 showed the similarity of $71.0{\sim}91.8%$ at nucleotide and $90.9{\sim}94.8%$ at amino acid sequences in 231 nucleotides region of M segment, and the similarity of $75.1{\sim}81.0%$ at nucleotide and $97.5{\sim}100%$ at amino acid sequences in 237 nucleotides of S segment.

• Korean Journal of Veterinary Research
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• v.33 no.2
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• pp.249-254
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• 1993

The etiological survey on porcine epidemic diarrhea virus(PEDV) by immunofluorescence antibody test(IFA) showed the positive rusult from the intestines of piglet died from acute diarrhea. The viral agent of PED was also isolated from intestine, which showed positive reaction by immunofluorescence test. After passage in Vero cell, the viral agent was further cloned by plaque purification and designated as KPEDV-9. The immunoblotting analysis using hyperimmune sera and porcine sera revealed the presence of several polypetide bands with molecular weight(M.W.) of 88K, 74K, 70K, 58~54, 54~46K, 44~40K and 33~32K, respectively.