• Korean Journal of Veterinary Research
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• v.27 no.2
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• pp.307-316
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• 1987

This study was conducted to identify Babesia sp. strain isolated from the imported beef cattle, Aberdeen-angus heifers, in J farm of Jangsu Prefecture, Cheon-buk Province of Korea, morphologically, etiologically and serologically. Babesia sp. strain was purely isolated through the serial blood passages of three generations against splenectomized calves and one generation of blood passage against non-splenectomized calf(intact calf) by inhibiting the appearance of Theileria sergenti in the blood stream by means of injection of 20% oil pamaquine intramuscularly. In the splenectomized calves, the parasite multiplied markedly in blood stream soon after inoculation and parasitaemia was much severe. An elevated body temperature, anorexia, severe anaemia and icterus were observed clinically. Of three splenectomized calves, two were affected with haemoglobinuria and died. But in the non-splenectomized calf the clinical signs and parasitaemia were very mild. The means of the incubation period, the highest parasitaemia, the highest body temperature and the lowest PCV were 3.5 days, 41.1%, ${42^{\circ}C}$ and 9%, respectively, in the splenectomized calves. In calf erythrocytes Babesia sp. protozoa were polymorphic showing the round, oval, ameboid, piriform and paired piriform etc. The sizes of protozoa were $2.51{\sim}3.91{\times}1.32{\sim}2.19{{\mu}m}$ ($3.20{\times}1.60{\mu}m$). Serologically the isolated Babesia sp. were compared with other parasites, B. ovata, B. bigemina, B. bovis, T. sergenti, A. marginale and A. centrale by using the complement fixation(CF) test. As a result, the antibody titer between the homologous species were high. It was two tubes or more in the CF test. From the results obtained above the pure isolated Babesia species was identical with Babesia ovata Minami and Ishihara, 1980.

• Journal of Environmental Impact Assessment
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• v.22 no.2
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• pp.125-134
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• 2013

Plant virus coat (CP) gene-mediated protection is one of the best known approaches to protect against virus resistant transgenic plants. Transgenic N. benthamiana plants containing the CP gene of Zucchini green mottle mosaic virus (ZGMMV) were used for the environmental risk assessment of the living modified (LM) plants with plant virus resistance. The most optimal co-infection method of both ZGMMV and CMV (Cucumber mosaic virus) on Non-LM and CP-expressing LM tobacco plants was established and co-infection of CMV and ZGMMV was confirmed by polymerase chain reaction (PCR). To address the effects of LM tobacco plants on the mutation of the virus, in-vitro transcripts of CP and Replicase (Rep) derived from CMV and/or ZGMMV were inoculated onto Non-LM or LM tobacco plants. Mutation frequency of CP and Rep from CMV and ZGMMV was examined through six serial passages in Non-LM and LM tobacco plants. Little actual frequency of mutation was estimated, probably due to the limited number of transgenic plants tested in this study. However, it does not suggest environmental safety of these CP-mediated LM plants. Further study at a larger scale is needed to evaluate the environmental risk associated with the CP-expressing LM plants.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.751-759
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• 1999

Plaque characteristics of porcine reproductive and respiratory syndrome (PRRS) virus isolates were examined using MARC-145 line cells. The plaque morphology of PRRS virus isolates was variable in size and heterogenic in population. Upon serial passages of the PRRS virus isolates on MARC-145 tells, heterogeneity was maintained but numbers of the large plaque size virus were increased with certain isolates. A PRRS virus isolate with variable plaque sizes was subcloned into 2 populations : small plaque ($H_S$) and large plaque ($H_L$) viruses. Growth kinetics of the subclones were then determined in MARC-145 cells, and production of the structural polypeptides was analyzed by SDS-PAGE. In a comparison of the growth kinetics, the $H_S$ virus showed higher infectivity titers during the first 48 hours but slower to reach the peak titier than $H_L$ virus did. In a nucleotide sequence comparison, differences of 4 nucleotides in open reading frames 5-6 gene were found between $H_S$ and $H_L$ viruses. Both the $H_S$ and $H_L$ clones produced 5 polypeptide bands with molecular weights of 15, 19, 26, 36 and 42 kD. The 5 bands were detected at 48 hours postinoculation (PI) with antisera to $H_L$ and another large plaque virus ($W_L$) and at 72 hours PI with $H_S$ virus antiserum. The present results demonstrate differences of biologic and molecular characteristics between the two PRRS virus plaque clones.

• Korean Journal of Veterinary Research
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• v.34 no.2
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• pp.321-326
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• 1994

Korean isolate, porcine epidemic diarrhea virus (KPEDV-9) was adapted through serial passages in vero cell. The viral yield reached up to $10^{5-6}$ $TCID_{50}/ml$ at the passage level of 90th. The cell adapted virus was characterized through genetic and morphological examinations. The RNA extracted from virus infected cell revealed the presence of RNA band with molecular size of >20Kb. The electron microscopic examination on purified virus showed the pleomorphic appearance of enveloped particles with 5-10nm surface projections, which fit with the shape of coronavirus. The etiological survey on swine diarrhea by immunofluorescence test(FA) indicated 17.5% positive rate on the PEDV infection. In addition, the incidence were detected both in piglets within two weeks old as well as fattening pigs. Serological survey by ELISA revealed the overall 45% positive result, thus, indicating the PEDV infection are widespread throughout this country.

• Molecules and Cells
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• v.21 no.1
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• pp.112-120
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• 2006

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese encephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homologous DI virus intimately associated with JEV persistence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a persistent JEV infection in which the DI RNA coreplicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated during its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identification of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules retained their open reading frames despite a large deletion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these observations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.

• Korean Journal of Veterinary Research
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• v.63 no.1
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• pp.5.1-5.9
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• 2023

Feline panleukopenia virus (FPV), feline calicivirus (FCV), and feline herpesvirus type-1 (FHV-1) are major infectious pathogens in cats. We evaluated the immunogenicity of a new vaccine containing inactivated FPV, two FCVs, and FHV-1 in animals. An FPV, two FCVs, and an FHV-1 isolate were continuously passaged 70, 50, 80, and 100 times in CRFK cells. FP70, FC50, FC80, and FH100 were propagated and used as vaccine antigens. Two inactivated feline virus vaccines, feline rehydragel-adjuvanted vaccine (FRAV) and feline cabopol-adjuvanted vaccine (FCAV) were prepared and inoculated into mice and guinea pigs. Humoral immune responses were measured using hemagglutination inhibition (HI) for FPV and virus-neutralizing antibody (VNA) for two FCVs and FHV-1 tests. Serial passages in CRFK cells resulted in increase in titers of FPV and two FCVs but not FHV-1 The FCAV induced higher mean HI and VNA titers than the FRAV in guinea pigs; therefore, the FCAV was selected. Cats inoculated with FCAV developed a mean HI titer of 259.9 against FPV, and VNA titers of 64, 256, and 3.2 against FCV17D03, FCV17D283, and FHV191071, respectively. Therefore, cats inoculated with the FCAV showed a considerable immune response after receiving a booster vaccination.

• Korean Journal of Poultry Science
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• v.39 no.2
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• pp.113-120
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• 2012

Newcastle disease virus (NDV) Kr005/V strain was generated through 55 serial passages of NDV Kr005 strain in Vero cells. The Kr005/V virus yielded high infective titers of $10^{7.8}$ $TCID_{50}/mL$ in Vero cells and the infected cells showed cytopathic effects such as marked cell rounding, though less frequent syncytia. The Kr005/V virus was heat-stable and classified into the lentogenic type with a Mean Death Time (MDT) of 120h or greater while the Kr005 strain was heat-labile and velogenic (MDT of 49.6 h). Only the single amino acid substitution (T to S) was observed at position 433 of the HN protein of the Kr005/V strain, whereas no amino acid change was found in the F protein. The Kr005/V input virus correlated well (correlation coefficient $r^2$=0.97) with the Kr005 virus when ten field sera were tested by virus neutralization test. The biological properties and usefulness of Vero cell-adapted Kr005/V virus were discussed.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.997-1006
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• 2018

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over $10^8PFU/ml$. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells ($PB1_{D153N}$, $M1_{A137T}$, and $NS1_{N176S}$). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than $10^7PFU/ml$) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.129-138
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• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.313-320
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• 2010

Genetic instability of the rice blast fungus Magnaporthe oryzae has been suggested as a major factor underlying the rapid breakdown of host resistance in the field. However, little information is available on the mechanism of genetic instability. In this study, we assessed the stability of repetitive DNA elements and several key phenotypic traits important for pathogenesis after serially transferring two isolates though rice plants and an artificial medium. Using isolate 70-15, we obtained a total of 176 single-spore isolates from 10 successive rounds of culturing on artificial medium. Another 20 isolates were obtained from germ tubes formed at the basal and apical cells of 10 three-celled conidia. Additionally, 60 isolates were obtained from isolate KJ201 after serial transfers through rice plants and an artificial medium. No apparent differences in phenotypes, including mycelial growth, conidial morphologies, conidiation, conidial germination, appressorium formation, and virulence, or in DNA fingerprints using MGR586, MAGGY, Pot2, LINE, MG-SINE and PWL2 as probes were observed among isolates from the same parent isolate. Southern hybridization and sequence analysis of two avirulence genes, AVR-Pita1 and AVR-Pikm, showed that both genes were also maintained stably during 10 successive generations on medium and plants. However, one reversible loss of restriction fragments was found in the telomere-linked helicase gene (TLH1) family, suggesting some telomere regions may be more unstable than the rest of the genome. Taken together, our results suggest that phenotype and genotype of M. oryzae isolates do not noticeably change, at least up to 10 successive generations on a cultural medium and in host plants.