• Applied Microscopy
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• v.24 no.3
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• pp.46-54
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• 1994

In ultrathin section of the tissue of odontoglossum ringspot virus (ORSV)-infected Cymbidium goeringii Reichenbach, ORSV particles appeared as bundles of irregular aggregates of various length which were called stacked plates or rounded plates. Virus particles were found in the cytoplasm in electron clear zones and they also found between cell wall and plasma membrane. They mainly clustered in parallel aggregates and sometimes oriented randomly. The X-bodies and paramural bodies were observed near the cell membrane and these contained vacuole-like cavities. The cell wall of infected tissue expanded largely. Some chloroplast in ORSV infected cell was irregular. No virus particle was present in mitochondria, nuclei, vacuoles, vesicles or other organelles. The plasmodesmata slightly enlarged, and virus-associated granules were present around it.

• Journal of Pharmaceutical Investigation
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• v.31 no.3
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.139.2-140
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• 2003

Ornithogalum showing mosaic symptoms were collected from the isolated field of National Plant Quarantine Service in Sengrimmyon of Kyungnam province. Electron microscopic examination of negatively strained preparation was filamentous particle of 740nm in lenght. Indirect-ELISA determined that the virus was serologically related to potyvirus. A single major protein band of Mr 30,000 was observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Indicator plant test showed masaic, necrotic local lesion and sunken areas in leaves of Nicotiana clevelandii and Tetragonia expansa, while the others of indicator plants did not infect. An enzyme-aided purification protocal was used, which eliminated a highly viscous mucilage from extracts of the Omithogalum. Total RNA extracted from infected Omithogalum leaves were amplified of 411b.p fragment in reverse transcription (RT)-PCR when primers specilic for the coat protein gene. An isolate of Omithogalum mosaic virus (OrMV) of the genus Potyvirus was identified as the casual agent of the disease on the basis of electron microscopic, biological and serological reaction.

• Parasites, Hosts and Diseases
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• v.56 no.5
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• pp.429-435
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• 2018

Toxoplasma gondii is a ubiquitous protozoan parasite responsible for causing toxoplasmosis. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines are of urgent need. In this study, we generated 2 virus-like particles (VLPs) vaccines expressing T. gondii rhoptry protein 4 (ROP4) or rhoptry protein 18 (ROP18) using influenza matrix protein (M1) as a core protein. Mice were intranasally immunized with VLPs vaccines and after the last immunization, mice were challenged with ME49 cysts. Protective efficacy was assessed and compared by determining serum antibody responses, body weight changes and the reduction of cyst counts in the brain. ROP18 VLPs-immunized mice induced greater levels of IgG and IgA antibody responses than those immunized with ROP4 VLPs. ROP18 VLPs immunization significantly reduced body weight loss and the number of brain cysts in mice compared to ROP4 VLPs post-challenge. These results indicate that T. gondii ROP18 VLPs elicited better protective efficacy than ROP4 VLPs, providing important insight into vaccine design strategy.

• Korean Journal Plant Pathology
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• v.14 no.1
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• pp.68-73
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• 1998

Barley mild mosaic virus (BaMMV-Kor) was isolated from the southern part of Korea, and by mechanical inoculation onto barley cultivars, purification and production of antibody. BaMMV-Kor purified form infected plants were filamentous particle, with 13 nm in diameter and 250∼300 nm and 500∼650 nm in length. Antibody of BaMMV-Kor was made by injecting the purified virus to the muscle of a rabbit. In gel-diffusion test, antibody to BaMMV-Kor created spur with BaMMV-Kal and BaMMV-M, but did not make spur with BaMMV-Kor infected New Golden, Ishukushirazu, Joshushiro Hadaka and Misato Golden, but did not infect Kashimamugi, Chikurin Ibaraki 1 and Mokusekko 3. In Korean barley cultivars, BaMMV-Kor infected most of the covered barley cultivars, but did not infect Saeolbori. It also infected naked barley cultivars except Chalbori and Hinssalbori. And all the beer barley cultivars were infected by BaMMV-Kor. BaMMV-Kor had two RNAs, RNA 1 (7.5 Kb) and RNA 2 (3.5 Kb), and coat protein (33 KDa).

• Parasites, Hosts and Diseases
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• v.59 no.6
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• pp.565-572
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• 2021

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.

• IMMUNE NETWORK
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• v.20 no.4
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• pp.35.1-35.13
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• 2020

Antigen delivery systems play critical roles in determining the quality and quantity of Ab responses in vivo. Induction of protective antibodies by B cells is essential in the development of vaccines against infectious pathogens, whereas production of IgE antibodies is prerequisite for investigation of allergic responses, or type 1 hypersensitivity reactions. Virus-like particles (VLPs) are efficient platforms for expression of proteins of interest in highly repetitive manners, which grants strong Ab responses to target antigens. Here, we report that delivery of hen egg lysozyme (HEL), a model allergen, through VLP could provoke strong HEL specific IgE Ab responses in mice. Moreover, acute allergic responses were robustly induced in the mice sensitized with VLPs that express HEL, when challenged with recombinant HEL protein. Our data show that antigen delivery in the context of VLPs could function as a platform for sensitization of mice and for subsequent examination of allergic reactions to molecules of interest.

• Korean Journal of Microbiology
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• v.22 no.2
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• pp.85-90
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• 1984

Two viruses were isolated from kidney and spleen tissues of goldfish(Carasius auratus) and the ovarian fluid of chum salmon(Oncorhynchus keta). Both viruses replicated and produced cytopathic effect in EPC, CHSE-214, and CHH-1 cell lines at $15^{\circ}C$. The isolates were resistant to pH 3 and choloroform. Antiserum to infections pancreatic necrosis virus(IPNV) serotype VR 299 neutralized the infectivity of both of the isolates. Electron microscopy showed that the particles had typical IPNV particle morphology with average diameters of 55nm, This paper describes the first isolation of viruses infecting cultured fish in Korea.

• Research in Plant Disease
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• v.15 no.2
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• pp.68-71
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• 2009

Cucumber green mottle mosaic virus (CGMMV) was not infected in figleaf gourd by sap inoculation. However CGMMV was detected by RT-PCR from the figleaf gourd collected from a field growing cucumber grafted onto figleaf gourd in Cheonan, Chungcheongnam Province in 2008. Which field showed 100% infection rate of the virus disease. In the experiment grafted with cucumber onto figleaf gourd, transportation of CGMMV through cucumber to figleaf gourd was confirmed by RT-PCR when the virus was mechanically inoculated on the leaves of the cucumber. The amplified DNA concentration of the virus on electrophoresis gel was much higher in the cucumber than in the figleaf gourd. However, the virus particles from the figleaf gourds were not observed under electron microscopy, also sap of the figleaf gourds was not transmittable to Nicotiana benthamiana. To identify the existence of CGMMV particle, the virus was purified from figleaf gourd and cucumber growing together in the graft system. CGMMV solution extracted from the cucumber represented a typical absorption spectrum of the virus but that from the figleaf gourd did not. Only a few CGMMV particles were observed in the purified preparation from the figleaf gourd. These results confirmed that CGMMV only passed through figleaf gourd in the grafting system. This study indicated that figleaf gourd is not a host of CGMMY.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.23-36
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• 2000

Japanese encephalitis virus (JEV) is the causative agent of a mosquito-borne encephalitis and is transmitted to human via persistently infected mosquito vectors. Although the virus is known to cause only acute infection, there were reports that showed neurological sequelae, latent infection in peripheral mononuclear cells, and recurrence of the disease after acute encephalitis. Innate resistance of certain cell lines, abnormal SN1 expression of the virus, and anti-apoptotic effect of cullular bcl-2 have been suggested as probable causes of JEV persistence even in the absence of defective interfering (DI) particles. Although possible involvement of DI particles in JEV persistence was suggested, neither has a direct evidence for DI presence nor its molecular characterization been made. Two questions asked in this study are whether the DI virus plays any role in JEV persistent infection if it is associated with and what type of change(s) can be made in persistently infected cells to avoid apoptosis even with the continuous virus replication, DI-free standard stock of JEV was infected in BHK-21, Vero, and SW13 cells and serial high multiplicity passages were performed in order to generate DI particles. There different-sized DI RNA species which were defective in both structural and nonstructural protein coding genes. Rescued ORFs of the DI genome maintained in-frame and the presence of replicative intermediate or replicative form RNA of the DI particles confirmed their replication competence. On the other hand, several clones with JEV persistent infection were established from the cells survived acute infections during the passages. Timing of the DI virus generation during the passages seemed coincide to the appearance of persistently infected cells. The DI RNAs were identified in most of persistently infected cells and were observed throughout the cell maintenance. One of the cloned cell line maintained the viral persistence without DI RNA coreplication. The cells with viral persistence released the reduced but continuous infectious JEV particle for up to 9 months and were refractory to homologous virus superinfection but not to heterologous challenges. Unlike the cells with acute infection these cells were devoid of characteristic DNA fragmentation and JEV-induced apoptosis with or without homologous superinfection. Therefore, the DI RNA generated during JEV undiluted serial passage on mammalian cells was shown to be biologically active and it seemed to be responsible, at least in part, for the establishment and maintenance of the JEV persistence in mammalian cells. Viral persistence without DI RNA coreplication, as in one of the cell clones, supports that JEV persistent infection could be maintained with or without the presence of DI particles. In addition, the fact that the cells with JEV persistence were resistant against homologous virus superinfection, but not against heterologous one, suggests that different viruses have their own and independent pathway for cytopathogenesis even if viral cytopathic effect could be converged to an apoptosis after all.