• The Plant Pathology Journal
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• 제22권1호
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• pp.21-27
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• 2006

We immunized BALB/c mice with purified Cucumber green mottle mosaic virus isolate HY1 (CGMMV-HY1). Through the selection of positive clones that were grown on the HAT medium, four sensitive monoclonal clones (CG99-01, CG99-02, CG99-03, and CG99-04) were selected from 500 Hypoxanthine-guanine phosphoribosyltransferase positive hybridoma cells. Four sensitive clones of CGMMV-HYI were determined as IgM type of the subclass of mouse immunoglobulins Ig group. The titer of monoclonal antiserum against CGMMVHY1 was estimated 1:12,800 by the indirect ELISA. Although monoclonal antibodies (MAbs) from CG99-01 and from CG99-04 cross-reacted with Zucchini green mottle mosaic virus and Kyuri green mottle mosaic virus, MAb from the cell line CG99-03 was highly specific to CGMMV. No MAbs cross-reacted with Cucumber mosaic virus-Fny. Only CG99-04 reacted with Pepper mild mottle virus weakly and CG99-02 reacted with both CGMMV and KGMMV. CGMMV was detected from the rind of watermelon fruit by DAS-ELISA of CGMMV-HY1, but not from the flesh of watermelon. Average seed transmission rate of CGMMV in watermelon was $24\%$ from symptomatic watermelon collected from 5 regions of Gyeongnam province. CGMMV was detected by DAS-ELISA with specific MAb of CGMMVHY1 periodically from root stock, during the sequential process for nursery seedling in Haman. Necrotic spots on cotyledons of root stock seedling progressed to reveal the typical symptomatology on the primary leaves of scion upon grafting. Here, we have established MAb based ELISA system, which could accurately detect CGMMV from watermelon seeds, nursery seedlings, transplants and field samples from greenhouse or open out door field as well.

• Journal of Microbiology and Biotechnology
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• 제26권10호
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• pp.1800-1807
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• 2016

To understand how human cytomegalovirus (HCMV) might change and evolve after reactivation, it is very important to understand how the nucleotide sequence of cultured HCMV changes after in vitro passaging in cell culture, and how these changes affect the genome of HCMV and the consequent variation in amino acid sequence. Strain JHC of HCMV was propagated in vitro for more than 40 passages and its biological and genetic changes were monitored. For each passage, real-time PCR was performed in order to determine the genome copy number, and a plaque assay was employed to get virus infection titers. The infectious virus titers gradually increased with passaging in cell culture, whereas the number of virus genome copies remained relatively unchanged. A linear correlation was observed between the passage number and the log₁₀ infectious virus titer per virus genome copy number. To understand the genetic basis underlying the increase in HCMV infectivity with increasing passage, the whole-genome DNA sequence of the high-passage strain was determined and compared with the genome sequence of the low-passage strain. Out of 100 mutations found in the high-passage strain, only two were located in an open reading frame. A G-T substitution in the RL13 gene resulted in a nonsense mutation and caused an early stop. A G-A substitution in the UL122 gene generated an S-F nonsynonymous mutation. The mutations in the RL13 and UL122 genes might be related to the increase in virus infectivity, although the role of the mutations found in noncoding regions could not be excluded.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권6호
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• pp.340-346
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• 2002

The purpose of the present study was to examine the efficacy and mechanism of the PAB (para-amino benzamidine) affinity column chromatography, Viresolve NFP virus filtration, pasteurization (60$\^{C}$ heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine as regards the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of urokinase Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID$\_$50/), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing HAV with a log reduction factor of 3.24. HAV infectivity was rarely detected in the urokinase fraction, while most of the HAV infectivity was recovered in the unbound and wash fractions. HAV was completely removed during the Viresolve NFP filtration with a log reduction factor of $\geq$ 4.60. Pasteurization was also found to be an effective step in inactivating HAV where the titers were reduced from an initial titer of 7.18 log$\_$10/ TCID$\_$50/ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was $\geq$ 4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was $\geq$ 14.08. Accordingly, these results indicate that the production process for urokinase exhibited a sufficient HAV reducing capacity to achieve a high margin of virus safety.

• KSBB Journal
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• 제16권6호
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• Fisheries and Aquatic Sciences
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• 제25권6호
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• pp.320-326
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• 2022

Most of the emerging diseases that threaten humans are caused by RNA viruses which are extremely mutable during evolution. The fish RNA virus, viral hemorrhagic septicemia virus (VHSV) can infect a broad range of aquatic animal hosts, but the transmissibility of VHSV to mammals has not been thoroughly investigated. Therefore, our study aimed to investigate the potential adverse effects of VHSV in mammals. Briefly, the survival of VHSV was determined using only minimum essential media (MEM-2) and mammalian SNU-1411 and hepa-1c1c7s cells at 15℃ and 37℃. Mice (Mus musculus, 27.3 ± 1.9 g) were intravenously injected with VHSV (2.37E+05 TCID₅₀·mice^-1) in triplicate. Clinical signs and survival rates were examined at 14 days post-challenge, and infection was confirmed in the surviving mice. The 50% tissue culture infective dose (TCID₅₀) and polymerase chain reaction analysis were used to determine viral titers and the infection rate, respectively. The titer of VHSV suspended in MEM-2 at 15℃ was reduced by only one log after 8 days, whereas the virus maintained at 37℃ was inactivated 8 days post-inoculation (dpi). There were no recognizable cytopathic effects in either SNU-1411 or hepa-1c1c7s cells inoculated with VHSV at 15℃ and 37℃. VHSV in those cell lines at 37℃ was rapidly decreased and eventually inactivated at 12 dpi, whereas virus at 15℃ remained at low concentrations without replication. In vivo experiment showed that there were no signs of disease, mortality, or infection in VHSV-infected mice. The results of this study indicate that it is highly unlikely that VHSV can infect mammals including humans.

• Journal of Veterinary Science
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• 제23권4호
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• pp.25.1-25.14
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• 2022

Background: The commercially available Newcastle disease (ND) vaccines were developed based on Newcastle disease virus (NDV) isolates genetically divergent from field strains that can only prevent clinical disease, not shedding of virulent heterologous virus, highlighting the need to develop genotype-matched vaccines Objectives: This study examined the efficacy of the NDV genotype-matched vaccine, mIBS025 strain formulated in standard vaccine stabilizer, and in carboxymethyl sago starch-acid hydrogel (CMSS-AH) following vaccination via an eye drop (ED) and drinking water (DW). Methods: A challenge virus was prepared from a recent NDV isolated from ND vaccinated flock. Groups of specific-pathogen-free chickens were vaccinated with mIBS025 vaccine strain prepared in a standard vaccine stabilizer and CMSS-AH via ED and DW and then challenged with the UPM/NDV/IBS362/2016 strain. Results: Chickens vaccinated with CMSS-AH mIBS025 ED (group 2) developed the earliest and highest Hemagglutination Inhibition (HI) NDV antibody titer (8log₂) followed by standard mIBS025 ED (group 3) (7log₂) both conferred complete protection and drastically reduced virus shedding. By contrast, chickens vaccinated with standard mIBS025 DW (group 5) and CMSS-AH mIBS025 DW (group 4) developed low HI NDV antibody titers of 4log₂ and 3log₂, respectively, which correspondingly conferred only 50% and 60% protection and continuously shed the virulent virus via the oropharyngeal and cloacal routes until the end of the study at 14 dpc. Conclusions: The efficacy of mIBS025 vaccines prepared in a standard vaccine stabilizer or CMSS-AH was affected by the vaccination routes. The groups vaccinated via ED had better protective immunity than those vaccinated via DW.

• 대한수의학회지
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• 제49권3호
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• pp.207-213
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• 2009

Rabbit hemorrhagic disease (RHD) is caused by RHD virus (RHDV) and is one of the most fatal diseases of rabbits. Acute death of rabbits occurred in a farm located in the Gyeonggi province of South Korea. The virus was isolated and confirmed as RHDV based on reverse transcription polymerase chain reaction and hemagglutination assay (HA), and the isolate was designated as KV0801. The nucleotide sequence of the complete VP60 gene of KV0801 was determined and the corresponding amino acid sequence was deduced. Molecular analysis showed that the KV0801 isolate can be classified as a pandemic antigenic variant strain, RHDVa. The VP60 nucleotide sequence and deduced amino acid homology between KV0801 and other Korean isolate, RHF89, which was isolated in 1988, were 92.1 and 94.3%, respectively. The pathogenicity of the KV0801 isolate at an HA titer ranging from 16,384 to 0.16 HA units was evaluated in five-month-old SFP rabbits. The rabbits inoculated with KV0801 isolate containing more than 1.63 HA units died within six days of inoculation. These results suggest that a highly pathogenic RHDVa is circulating in the rabbit populations of Korea.

• Journal of Microbiology and Biotechnology
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• 제25권11호
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• pp.1960-1965
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• 2015

Rabbit hemorrhagic disease virus (RHDV) is highly contagious and often causes fatal disease that affects both wild and domestic rabbits of the species Oryctolagus cuniculus. A highly pathogenic RHDV variant (RHDVa) has been circulation in the Korean rabbit population since 2007 and has a devastating effect on the rabbit industry in Korea. A highly pathogenic RHDVa was isolated from naturally infected rabbits, and the gene encoding the VP60 protein was cloned into a baculovirus transfer vector and expressed in insect cells. The hemagglutination titer of the Sf-9 cell lysate infected with recombinant VP60 baculovirus was 131,072 units/50 μl and of the supernatant 4,096 units/50 μl. Guinea pigs immunized twice intramuscularly with a trial inactivated RHDVa vaccine containing recombinant VP60 contained 2,152 hemagglutination inhibition (HI) geometric mean titers. The 8-week-old white rabbits inoculated with one vaccine dose were challenged with a lethal RHDVa 21 days later and showed 100% survival rates. The recombinant VP60 protein expressed in a baculovirus system induced high HI titers in guinea pigs and rendered complete protection, which led to the development of a novel inactivated RHDVa vaccine.

• 대한바이러스학회지
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• 제27권1호
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• pp.9-17
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• 1997

Hepatitis C virus (HCV) E2 protein is known to be one of putative envelope proteins. To develop a sensitive detection method for HCV infected tissues and cells, monoclonal antibodys (MAbs) to the E2 protein of HCV were prepared from mice immunized with recombinant baculovirus-expressing E2 protein (Bac-E2). Several hybridoma clones secreting various levels of MAb were isolated and isotypes of these MAb were determined. One clone (L.2.3.3) was used for ascites production and the E2-MAb was purified and characterized. The L.2.3.3 reacted well with both Bac-E2 and E. coli expressed glutathione-S-transferase-E2 (GST-E2) fusion proteins. Using HCV patient sera, E2 envelope protein was found to be localized in the cell membrane boundary both in CHO cells and insect cells which express HCV E2 protein. Similar result was obtained when same cells were treated with the MAb L.2.3.3. These results demonstrated that Bac-E2 protein is capable of eliciting high titer antibody production in mice.

• 한국작물학회지
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• 제7권1호
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• pp.31-63
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• 1969

A series of experiment was carried out to study on the production of disease-free seed potatoes at the Alpine Experiment Station from 1960 to 1968, which initiated a study of comparison on degeneration of plain warm region and high altitude products and the effect of latent potato virus X (PVX) and potato leaf roll virus(PLRV) on degeneration. Particular observations were made on some aspect of the nature of potato virus disease and its control such as concentrations of PVX, range of host plants, physical properties such as concentrations of PYX, range of host plants, physical properties and carrying effect of insects, by investigating 9 different areas of the main potato producing regions (Kimhae, Taegu, Choongju, Taejoen, Suwon, Kwangju, Chonju, Cheju and Chinju). Highly purified anti-serum was separated and tested for control of the virus disease and also various method of prevention and control of PLRV were observed, using cultivation of sprouted seed tubers, early harvesting method, and systemic chemicals. The results obtained are summarized as follows; 1. Potato yield in the plain region decreased by 32.8~66.3% in the first year cultivation of seed potatoes from colder region, and the rate of virus infection was 92.9 to 95.4%. 2. Plants of three families including, 20 species were susceptible to the PVX, and among the plants Salvia officinalis of a habits only was the carrier while the symptom of Digitalis purpurea of Screphulariaceae was masked. Necrosis and ring spot was occurred in most pJants of the Solanaceae and ring spot symptom also was observed in Nicotiana tabacum L. var. White Burley and in N. glutinosa. 3. The 8$C_2$ strain of virus had the following physical properties; thermal inactivation point, 68-$72^{\circ}C$ : dilution inactivation point, above 1, 000, 000 dilution: ageing in vitro, 240-360 days: and ageing in dry plant tissue, 30 days. 4. Myzus persicae and Oxya spp. did not transmit the 8$C_2$ strain of potato virus. 5. Virus was purified through the ammonium sulphate isolating method, and higher titer value, 1/2048 was obtained through anti-serum test. 6. Inhibition Chenopodiacae on the virus infection of potato was remarkable, and inhibition of local lesion host also was observed. 7, By earlier planting of sprouted seed tubers, growth period could be prolonged by 10 to 12 days. 8. Earlier harvest decreased much the rate of virus infection of seed potatoes. 9. According to the results of aphid control trial using systemic soil insecticides at Kangnung and Taekwanlyung, PSP 204, Disyston and Thimet was effective to aphid control. In particular, control effect of twice treatments of PSP 204 was great. 10. Treatmental effect of those chemicals lasted about 60-70 days. However, single foliar application of emulsified chemicals was not effective to potato virus control. 11. The effect of PSP 204, Disyston, and Thimet on the control of potato leaf roll virus was great, particularly in the case of two treatments of PSP 204, at Kangnung as well as at Taekwanlyung. Higher negative correlationship between the control effect of potato leaf roll virus and potato yield was observed showing the value r=-0.85 at Kangnung, and r=-0.87 at Taekwanlyung. 12. Differences in the control effects among PSP 204, Disyston, and Thimet was not noticed.