• Korean Journal of Veterinary Service
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• v.46 no.3
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• pp.193-202
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• 2023

Porcine epidemic diarrhea is an infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV). Especially, when suckling piglets are infected, the mortality rate is close to 100%. PEDV is classified into G1 and G2 types based on genetic differences. The G2 type PEDV outbreak in the United States in 2013 was highly pathogenic and contagious, and it has spread worldwide and caused continuous economic losses. Most commercial vaccines used are G1 type vaccines, and existing vaccines do not fully protect piglets due to genetic differences. In this study, we evaluated the safety of the newly developed G2 type attenuated HSGP vaccine strain by inoculating it into piglets and testing whether the vaccine virus spreads to the non-vaccinated, negative pigs and whether the vaccine reverts to its virulence during serial passage experiments. Each experiment lasted for 7 days for each passage, and fecal viral titers, clinical symptoms, and weight gain were measured daily. After the experiment, necropsy was performed to measure intestinal virus titer and pathological evaluation. As a result of the first passage, no transmission of the vaccine virus to negative pigs co-housed with vaccinated pigs was observed. In addition, after four consecutive passage experiments, the clinical symptoms and small intestine lesions were gradually alleviated, and no virus was detected in the feces in the fourth passage experiment. Therefore, it was concluded that the vaccine was safe without virulence reversion in accordance with the guidelines of the current licensing authority. However, further studies are needed on the genetic changes and biological characteristics of the mutant virus that occur during successive passages of the attenuated vaccine since the replication and clinical symptoms of the virus increased until the third passage during successive passages of the vaccine virus. Based on this study, it was concluded that virulence reversion and safety evaluation of attenuated vaccines through serial passage in target animals can be useful to evaluate the safety of attenuated viruses.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.101-109
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• 2000

Duck viral hepatitis is an acute, highly infectious viral disease of young dacklings aged from two days to three weeks. The significant lesion associated with the disease was enlarged liver including necrotic foci and numerous hemorrhagic spots. We have isolated five strains of duck hepatitis virus (DHV) from field cases showing about 20% mortality with a sign of opisthotonos. When a-day-old ducklings were intramuscularly inoculated with one of the isolates, 92% of the birds were died within 5 days. We attempted to develop an attenuated strain of duck hepatitis virus (DHV) using one of the isolates by serial chicken embryo passages. The propagation of DHV in chicken embryos was carried 140 passages. The virus titer increased gradually from the $21^{st}$ through the $50^{th}$ passage, but there was no significant increase of virus titer in subsequent passages after then. Through the serial passages, the virulence of the virus for chicken embryos was gradually increased but decreased for ducklings. The pathogenicity of the virus for ducklings was preserved up to the $21^{st}$ passage but disappeared at the $50^{th}$passage. An attenuated Korean isolate which was passaged 140 times in chicken embryos gave good protection in ducklings against both challenge infection to a Korean virulent strain and to a DHV-DRL strain, a type 1 reference strain of DHV, which indicated that the Korean isolates could be classified as DHV type 1. And the above results suggest that an attenuated Korean isolate can be used for developing a live DHV vaccine.

• 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.

• Korean Journal of Poultry Science
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• v.19 no.2
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• pp.107-112
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• 1992

Sporozoite of Eimeria tenella inoculated into the allantoic cavities of embryonating eggs completed their life-cycle in the chorioallantoic membranes (CAM ) and produced viable oocysts. And the strain continued to adapt to the CAM through the period of the passages. In embryos, the reproduction of the strain, judged by oocyst production increased, but the pathogenicity, judged by mortality of embryo decreased, with increasing numbers of passage in eggs.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.710-715
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• 2005

A novel recombinant baculovirus, Bactrus, was constructed by the insertion of the Bacillus thuringiensis cry1Ac gene between two polyhedrin genes of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of the polyhedrin gene promoter. Polyhedra produced by Bactrus in insect cells were incorporated with 130 kDa of polyhedrin-Cry1Ac-polyhedrin fusion protein, and 30 kDa of intact polyhedrin, resulting from a homologous recombination between two polyhedrin genes, was also expressed. The insecticidal activity of Bactrus against Spodoptera exigua larvae was similar to that of AcNPV, but it showed significantly higher toxicity towards Plutella xylostella larvae in comparison with that of AcNPV. The expression level of fusion protein and the insecticidal activity of recombinant polyhedra produced by the Bactrus against P. xylostella larvae were decreased after serial passages. In conclusion, the Bactrus had improved insecticidal activity and returned to wild-type AcNPV after several passages.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.4
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• pp.588-595
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• 2013

Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

• Korean Journal of Veterinary Service
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• v.14 no.1
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• pp.27-40
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• 1991

In order to investigate the biological properties, pathogenicity and immune responses in artficially infected SPF chickens with Avian infectious bronchitis virus that was isolated from chickens showing IB like signs in southern region of Chung buk. Results obtained throuth the experiments are summarized as follows. 1. From 15 IB suspected cases, two strains of IB virus were isolated, one each from the tracheas and lungs. 2. Infectious bronchitis specific embryo lesions were observed after four serial passages of the isolates in chicken embryos. 3. The field isolates and M-41 strain of IB virus interfered with the replication of Newcastle disease virus in chicken embryos. 4. When specific pathogen free chickens, two week old, were inoculated with the IB virus isolates, clinical respiratory signs as dyspnea, coughing were observed. Airsacculitis was observed by necropsy. 5. AGP antibody positive rates of inoculated SPF chickens were highest on day 14 and lowest on day 36, while HI antibody responses were detected on day 14 in all Groups, the reinoculated Group was shown highest titers. 6. By Indirect immunofluorescence antibody assay of artificially infected SPF chickens, the viral antigens were detected in tissues of larynx, trachea and lung on the 4 th to 7 th days post inoculation.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.24.1-24.10
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• 2022

Background: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.110-116
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• 2000

Duck viral hepatitis is an acutic, highly infectious viral disease of young ducklings. The most practical means for controlling duck viral hepatitis is the vaccination of ducklings or of a breeding stock. We attempted to develop a vaccine strain of duck hepatitis virus (DHV) using a Korean isolate by serial chicken embryo passages. The propagation of DHV in chicken embryos was carried 140 passages. After the $50^{th}$ passage, of which the virus was non-pathogenic for ducklings, approximately every $20^{th}$ passage of the virus was tested for vaccinal efficacy. Both the $70^{th}$ and $90^{th}$ passage of the virus gave good protection against challenge infection to a DHV-DRL reference strain(type 1) and a virulent Korean isolate. The $110^{th}$, $125^{th}$ and $140^{th}$ passage of the virus were less protective than the $70^{th}$ and $90^{th}$ passage, which means that more than $110^{th}$ passage may lead to over-attenuation of the virus. Ducklings vaccinated with the chicken-embryo-adapted virus by oral, intramuscular or eye drop administration showed earlier resistance to challenge infection from 3 to 7 days postvaccination. Of the above methods, ducklings vaccinated intramuscularly presented the most rapid resistance against challenge. The minimum immune dose of the chicken-embryo-adapted virus in ducklings was also studied. Ducklings inoculated with a dose of $10^{2.0}\;ELD_{50}$ and below were not fully protected against challenge with a virulent DHV, showing a protection rate of 67% to 73%, but ducklings inoculated with a dose of $10^{3.0}\;ELD_{50}$ and over were completely protected. The virus yield of the chicken-embryo-adapted DHV was examined at 24hrs and 48hrs of the incubation time in the allantoic fluid, embryo head and embryo minus head of the embryonating egg. In all three components, the titer of the virus was higher at 48 hours than that at 24 hours after incubation. And the titer of the virus was higher in the embryo minus head, embryo head and the allantoic fluid, in order. Field trials for evaluating the efficacy of the attenuated DHV as a live vaccine were done in duck farms with about 25% mortality of flocks resulting from duck viral hepatitis. After the use of the experimental vaccine, the mortality due to duck viral hepatitis was dramatically reduced in the farms. These results indicated that the attenuated DHV using a Korean isolate could be a good candidate as a live vaccine strain of DHV in Korea.

• Journal of Life Science
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• v.29 no.8
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• pp.888-894
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• 2019

Cartilage diseases, such as rheumatoid arthritis (RA) and osteoarthritis (OA), are associated with the loss of chondrocytes and degradation of articular cartilage. Recent studies revealed that inflammatory reactive oxygen species (ROS) and age-related oxidative stress can affect chondrocyte activity and cartilage homeostasis. We investigated changes in the levels of intracellular antioxidants during cellular senescence of primary chondrocytes from rat articular cartilages. Cellular senescence was induced by serial subculture (passages 0, 2, 4, and 8) of chondrocytes and measured using specific senescence-associated ${\beta}$-galactosidase ($SA-{\beta}-gal$) staining. ROS production increased significantly in the senescent chondrocytes. In addition, total glutathione (GSSG/GSH) and superoxide dismutase (SOD) levels and heme oxygenase-1 (HO-1) expression increased in senescent chondrocytes induced by serial subculture. Analysis of changes in intracellular antioxidant levels in articular cartilage from rats of different ages (5, 25, 40, and 72 wk) revealed that total glutathione levels were highest after 40 wk and slightly decreased after 72 wk as compared with those after 25 wk. SOD and HO-1 expression levels increased in accordance with age. Based on these results, we conclude that intracellular antioxidants may be associated with cartilage protection against excessive oxidative stress in the process of chondrocyte senescence and age-related cartilage degeneration in an animal model.