• Bulletin of the Korean Chemical Society
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• v.35 no.4
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• pp.1082-1086
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• 2014

A multi-channel microchip electrophoresis (MCME) method with parallel laser-induced fluorescence (LIF) detection was developed for rapid screening of H1N1 virus. The hemagglutinin (HA) and nucleocapsid protein (NP) gene of H1N1 virus were amplified using polymerase chain reaction (PCR). The amplified PCR products of the H1N1 virus DNA (HA, 116 bp and NP, 195 bp) were simultaneously detected within 25 s in three parallel channels using an expanded laser beam and a charge-coupled device camera. The parallel separations were demonstrated using a sieving gel matrix of 0.3% poly(ethylene oxide) ($M_r$ = 8,000,000) in $1{\times}$ TBE buffer (pH 8.4) with a programmed step electric field strength (PSEFS). The method was ~20 times faster than conventional slab gel electrophoresis, without any loss of resolving power or reproducibility. The proposed MCME/PSEFS assay technique provides a simple and accurate method for fast high-throughput screening of infectious virus DNA molecules under 400 bp.

• The Plant Pathology Journal
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• v.27 no.1
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• pp.93-97
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• 2011

For quarantine purpose, we selected five plant RNA viruses including Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Potato aucuba mosaic virus (PAMV), Potato yellow dwarf virus (PYDV), and Tomato chlorosis virus (ToCV), which are not reported in Korea and cause serious economic losses to the family Cucurbitaceae or Solanaceae. To detect those viruses, we employed RT-PCR technique with specific oligonucleotide primer pairs and tested their detection efficiency for each virus. To design RT-PCR primers, coat protein was used for CVYV, CYSDV, and ToCV whereas RNA polymerase and nucleocapsid regions were used for PAMV and PYDV, respectively. The development of an RT-PCR based method proved a useful tool for rapid detection and identification of quarantine virus infections.

• Journal of fish pathology
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• v.36 no.2
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• pp.389-394
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• 2023

Infectious hematopoietic necrosis virus (IHNV) is s significant viral pathogen affecting cultured rainbow trout (Oncorhynchus mykiss) in Korea. In this study, five monoclonal antibodies (mAbs) (IHNV-1, 2, 3, 4, and 5) were produced using purified IHNV. Reactivities of these mAbs were analyzed by western blot (WB), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody test (IFAT). These mAbs recognized glycoprotein (69 kDa, IHNV-1), nucleocapsid protein (39 kDa, IHNV-3, 4, and 5), or phosphoprotein (27 kDa, IHNV-2) of IHNV by WB analysis. ELISA results indicated that these five mAbs were specific to IHNV without showing any cross-reactivity against other fish viruses (hirame rhabdovirus, infectious pancreatic necrosis virus, and viral hemorrhagic septicemia virus). IFAT demonstrated specific fluorescence signals of IHNV-infected epithelioma papulosum cyprini (EPC) cells, whereas no reactivity of normal EPC cells was observed. These mAbs can be very useful for immuno-diagnosis of IHNV infection.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2018.05a
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• pp.588-591
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• 2018

Baculovirus was originally isolated from the alfalfa looper and contains a 134-kbp genome with 154 open reading frames (ORF). The major capsid protein VP39 together with some minor proteins forms the nucleocapsid ($21nm{\times}260nm$) that encloses the DNA with p6.9 protein. They are double-stranded, circular, supercoiled DNA molecules in a rod-shaped capsid. Wild-type baculoviruses exhibit both lytic and occluded life cycles that develop independently throughout the three phases of virus replication. Recombinant baculoviruses can transfer their vectors and express their recombinant proteins in a wide range of mammalian cell types. Especially, inclusion of a dominant selectable marker in these baculoviral vectors can express diverse recombinant genes in many cells. Baculoviral vectors were reconstructed with cytomegalovirus (CMV) promoter,uroplakin II promoter, polyhedron promoter, vesicular stomatitis virus G (VSVG), enhanced green fluorescent protein (EGFP), protein transduction domain (PTD) gene and so on. These reconstructed vectors were infected into various cell and cell lines. We performed transfection and expression of these recombinant vectors comparison with other control vectors. From this study, we knew that transfection and expression of these recombinant vectors have higher efficacy than any control vector. This work was supported by a grant from Mid-Career Researcher Program(NRF-2016R1A2B4016552) through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).

• International Journal of Industrial Entomology and Biomaterials
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• v.29 no.2
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• pp.207-213
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• 2014

The structural proteins of classical swine fever virus (CSFV) consist of nucleocapsid protein C and envelope glycoprotein $E^{rns}$ (E0), E1 and E2. Among them, E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. In this study, to determine the optimal expression conditions of glycoprotein E2 using baculovirus system, we investigated the influence of insect cells and media to the expression of recombinant E2. Recombinant virus containing glycoprotein E2 coding gene was constructed with bApGOZA DNA. Expression of the glycoprotein E2 was analyzed by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies. Expression of glycoprotein E2 in Sf21 cells was first observed after 3 days and reached a maximum on the 5th day after infection. Furthermore, the highest levels of glycoprotein E2 expression were observed at multiplicity of infection (MOI) of 5. When three different insect cell lines (Sf21, High-Five and Se301) were tested, High-Five cells showed the highest production. In addition, four different serum-free and serum-supplemented media, respectively, were tested for the expression of glycoprotein E2 and the budded virus (BV) titers. As a result, serum-supplemented medium provided the best conditions for protein production and the BV yield.

• Journal of Yeungnam Medical Science
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• v.15 no.1
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• pp.55-66
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• 1998

Pathophysiological mechanism of hemorrhagic fever with renal syndrome (HFRS) is not fully understood. Major clinical findings of HFRS patients are widespread hemorrhage, acute renal failure and shock. Basic lesion is vascular injury with microvascular hemorrhage and relatively little inflammation. According to autopsy findings, renal medulla shows focal hemorrhage, tubular necrosis and interstitial mononuclear infiltrates. The predominant cell type in the renal and pulmonary interstitium is a fibroblast and it participates in the healing process at the injury site by secreting a large amount of extracellular matrix proteins. Cultured human lung fibroblasts and Mongolian gerbil fibroblasts were known to be good host cells for the hantaan virus. It is possible that not only the endothelial cell but also the fibroblast is a target of Hantaan virus and the fibroblast might be involved in the pathogenesis and the healing process in HFRS. Integrins are adhesion molecules, and act as receptors for many extracellular matrix proteins. Recently, there are many reports that cell surface integrins influence on some viral infections or reversely viruses influence on the expression of integrins. The ${\alpha}_5{\beta}_1$ integrin is a major receptor for the fibronectin which is an important extracellular matrix protein secreted by fibroblasts. In this study, the role of ${\alpha}_5{\beta}_1$ integrin in the infection of Hantaan virus was examined by using anti-${\alpha}_5{\beta}_1$, integrin, anti-${\alpha}_5$ integrin and anti-${\beta}_1$, integrin antibodies in chicken embryo fibroblasts (CEF) and Mongolian gerbil fibroblasts(MGF). The treatment of anti-${\alpha}_5{\beta}_1$, integrin antibody in CEF reduced the virion titers 26.8% and the amount of nucleocapsid N protein 32.6% when compared with control CEF. When MGF were treated with anti-${\alpha}_5$, anti-${\beta}_1$ and anti-${\alpha}_5{\beta}_1$ integrin antibodies, virion titers were reduced by 26.5%, 29.4% and 28.7% and the amount of nucleocapsid N protein were reduced by 65.2%, 59.7% and 72.6%. These results suggested that ${\alpha}_5{\beta}_1$ integrin might act as a receptor for the Hantaan virus or blocking of ${\alpha}_5{\beta}_1$ integrin influences on the viral replication in CEF and MGF. It is also possible that the blocking of only one subunit of integrin represents similar results in that of whole molecule.

• Korean Journal of Veterinary Service
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• v.37 no.3
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• pp.151-156
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• 2014

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses to the Korean pig industry. ELISA tests using recombinant nucleocapsid protein of PRRSV have been most commonly used for PRRS diagnostics. In the current study, two commercial PRRSV ELISA kits (Bionote PRRSV Antibody ELISA and IDEXX 3XR PRRS Antibody ELISA) have been compared using sera collected from 19 swine farms in various stages of PRRSV infection confirmed by professional diagnostic centers. Thus 130 sera collected from 5 different farms with active PRRSV infection, 130 sera from 6 different farms with PRRS-stabilized status, and 140 sera from 8 different farms with PRRS-free status were evaluated to determine the correlation of test results between those ELISA kits. Both ELISA kits showed a good correlation [PRRSV-positive farms ($R^2$=0.6375) and stabilized farms ($R^2$=0.8928)] in sample-to-positive (S/P) ratio va lues. Among the 140 sera from negative farms, one sample was falsely positive by either of the ELISA kits. In conclusion, both of the ELISA kits showed a good correlation when applied on field samples collected from farms at various stages of PRRSV infection. Bionote ELISA or IDEXX ELISA gave a false positive result on 1 out of 140 negative samples so their specificity was calculated as 99.3%. Therefore, Bionote ELISA would be a good complementary and alternative method for IDEXX ELISA kit, and vice versa.

• Journal of Microbiology
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• v.45 no.1
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• pp.41-47
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• 2007

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.

• The Plant Pathology Journal
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• v.28 no.1
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• pp.87-92
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• 2012

$Tomato$ $spotted$ $wilt$ $virus$ (TSWV)-infected $Capsicum$ $annuum$ plants were collected from open fields during June to July in 2010. The TSWV isolates were designated as Gneung, Ghang-Kjj, Gchang-Njc, Ghae, and Pap. The nucleotide sequence of the nucleocapsid protein (N) and movement protein (NSm) of the five isolates was determined. The pathogenicity of the five isolates was determined on 14 $C.$ $annuum$ cultivars two times by using mechanical inoculation. The five isolates induced different response: Both Gneung and Gchang-Kjj did not infect any of the cultivars in the 2nd trial, while Gchang-Njc, Ghae and Pap infected 11, 6 and 13 of 14 cultivars, respectively. The five isolates also were tested on $Solanum$ $lycopersicum$ breeding line TGC09-71 and three $Nicotiana$ species. $S.$ $lycopersicum$ showed a similar response to the five isolates as did $C.$ $annuum$. Both Gchang-Njc and Ghae infected systemically all three $Nicotiana$ species tested. While both Pap and Gneung did not infect any of the $Nicotiana$ species tested. In conclusion, five TSWV isolates induced different infection spectra in $C.$ $annuum$ cultivars, $Nicotiana$ species and an $S.$ $lycopersicum$ breeding line. Amino acid sequence analysis of the NSm gene could not support or explain the different infection spectra of the five isolates. This study indicated that various isolates must be used as virus inocula for evaluation of $C.$ $annuum$ and $S.$ $lycopersicum$ cultivars in breeding programs for TSWV resistance.

• Journal of Microbiology
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• v.40 no.3
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• pp.183-192
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• 2002

Cells infected With equine herpesvirus type-1 (EHV-1) Produced both infectious and non-infectious Virus-related particles. Compared to the whole virion, non-infectious particles termed L-particles were deter-mined to lack 150 kDa protein, commonly known as nucleocapsid protein. The potential of L-particles to induce immune responses was studied in mice and foals. Intranasal immunization with L-particles or whole virions induced poor IgG antibody responses in mice. Interestingly, despite the poor antibody response, the conferred immunity protected the host from challenge infections. This was indicated by a significant reduction in virus titers in line with recovery towards normal body weight. Subsequently, the test on the usefulness of L-particles as immunizing agents was extended to foals. Immunization of specific-pathogen-free (SPF) foals resulted in similar results. As determined by a complement-fixing-antibody test (CFT), foals seroconverted when they were immunized either with inactivated L-particles or whole virions via intramuscular (i.m.) injections. The presence of the antibody correlated with the degree of protection. Beyond day 1 post challenge infection (p.i.), there was no virus shedding in the nasal mucus of foals immunized with whole EHV-1 virions. Virus shedding was observed in foals Immunized with L-particles but limited to days 6 to 8 p.i. only. In contrast, extended vim shedding was observed in non-immunized foals and it was well beyond day 14 p.i. Viremia was not detected for more than four days except in non-immunized foals. Immunization in mice via intranasal (i.n.) conferred good protection. However, compared to the i.n. route, a greater degree of protection was obtained in foals following immunization via i.m. route. Despite variation in the degree of protection due to different routes of immunization in the two animal species, our results have established significant evidence that immunization with L-particles confers protection in the natural host. It is suggested that non-infectious L-particles should be used as immunizing agents for vaccination of horses against EHV-1 infection.