• Korean Journal of Plant Tissue Culture
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• v.25 no.6
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• pp.519-524
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• 1998

A simple and reliable method to diagnose cucumber green mottle mosaic virus of watermelon in Korea (CGMMV-WK) was determined by RT-PCR, and coat protein gene for CGMMV-WK was cloned. Comparing to a method reported by Lee et al. (1996), the method developed here showed a better RT-PCR reaction. RT-PCR was possible by one step in the PCR reaction mixture that contains 20 pmol of primer, reverse transcriptase (30 unit), RNasin (5 unit) using the crude RNA solution. RT-PCR condition for specifically diagnosing CGMMV-WK was that cDNA was synthesized at 42$^{\circ}C$ for 45 min followed by pre-denaturation at 95$^{\circ}C$ for 2 min, and then PCR reaction was carried out with a programmed condition that consisted of 36 sequential cycles at 96$^{\circ}C$ for 30 sec, 6$0^{\circ}C$ for 30 sec, and 72$^{\circ}C$ for 1 min. A gene encoding the coat protein of CGMMV-WK was cloned and characterized. Nucleotide sequence of coat protein gene of CGMMV-WK shared 98.77% and 99.38% of sequence identity with those of CGMMV-W and CGMMV-SH, respecitvely, however, all of amino acid sequences were same.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.8
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• pp.1089-1095
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• 2012

Neonatal Fc receptor (FcRn) gene encodes a receptor that binds the Fc region of monomeric immunoglobulin G (IgG) and is responsible for IgG transport and stabilization. In this report, the 8,900 bp porcine FcRn genomic DNA structure was identified and putative FcRn protein included 356 amino acids. Alignment and phylogenetic analysis of the porcine FcRn amino acid sequences with their homologies of other species showed high identity. Tissues expression of FcRn mRNA was detected by real time quantitative polymerase chain reaction (Q-PCR), the results revealed FcRn expressed widely in ten analyzed tissues. One single nucleotide polymorphism (SNP) (HQ026019:g.8526 C>T) in exon6 region of porcine FcRn gene was demonstrated by DNA sequencing analysis. A further analysis of SNP genotypes associated with serum Classical Swine Fever Virus antibody (anti-CSFV) concentration was performed in three pig populations including Large White, Landrace and Songliao Black pig (a Chinese indigenous breed). Our results of statistical analysis showed that the SNP had a highly significant association with the level of anti-CSFV antibody (At d 20; At d 35) in serum (p = 0.008; p = 0.0001). Investigation of expression and polymorphisms of the porcine FcRn gene will help us in further understanding the molecular basis of the antibody regulation pathway in the porcine immune response. All these results indicate that FcRn gene might be regarded as a molecular marker for genetic selection of anti-CSFV antibody level in pig disease resistance breeding programmes.

• Journal of Yeungnam Medical Science
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• v.25 no.1
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• pp.41-49
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• 2008

Background : The central physiological derangement of hemorrhagic fever with renal syndrome (HFRS) caused by hantaan virus (HTNV) is a vascular dysfunction, manifested by hemorrhage, impaired vascular tone and increased vascular permeability. Platelet activating factor (PAF), whose actions are mediated through a specific receptor, is a potent bioactive lipid. PAF has diverse biological functions in the vascular system, such as increasing vascular permeability, adhesion of leukocytes to the endothelium and reduction of cardiac output, which result in hypotension and shock. The goal of the present study was to investigate whether PAF is involved in the pathogenesis of HFRS. For this purpose, we evaluated the effect of HTNV on the expression of PAF receptor (PAF-R) and on the activity of PAF-acetylhydrolase (PAF-AH) instead of PAF because PAF is rapidly degraded by PAF-AH in vivo. Materials and methods : To evaluate the expression of PAF-R, we performed reverse-transcription PCR, western blot and FACS analyses using HTNV-infected human umbilical vein endothelial cells (HUVECs) and non-infected (control) HUVECs. In addition, we measured the activity of plasma PAF-AH in HFRS patients and normal healthy persons. Results : The mRNA and protein expression of PAF-R was increased in HTNV-infected HUVECs compared with control HUVECs at 2 and 3 days post-infection (d.p.i.). FACS analysis showed that HTNV induced the surface expression of PAF-R in HUVECs from 2 d.p.i. The activity of plasma PAF-AH was 2.5-fold lower in HFRS patients than in normal healthy persons. Conclusion : Increased PAF-R expression by HTNV might increase the responsiveness to PAF in endothelial cells. Reduced PAF-AH activity in the blood of HFRS patients might delay PAF degradation. These results suggest that changes in PAF-R and PAF-AH by HTNV might influence to PAF activity and might be involved in the vascular dysfunction of HFRS.

• Molecules and Cells
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• v.40 no.3
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• pp.202-210
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• 2017

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus (HCV) RNA genome is a multifunctional phosphoprotein. To analyse the influence of NS5A on apoptosis, we established an Hep-NS5A cell line (HepG2 cells that stably express NS5A) and induced apoptosis using tumour necrosis factor $(TNF)-{\alpha}$. We utilised the MTT assay to detect cell viability, real-time quantitative polymerase chain reaction and Western blot to analyse gene and protein expression, and a luciferase reporter gene experiment to investigate the targeted regulatory relationship. Chromatin immunoprecipitation was used to identify the combination of $NF-{\kappa}B$ and miR-503. We found that overexpression of NS5A inhibited $TNF-{\alpha}$-induced hepatocellular apoptosis via regulating miR-503 expression. The cell viability of the $TNF-{\alpha}$ induced Hep-mock cells was significantly less than the viability of the $TNF-{\alpha}$ induced Hep-NS5A cells, which demonstrates that NS5A inhibited $TNF-{\alpha}$-induced HepG2 cell apoptosis. Under $TNF-{\alpha}$ treatment, miR-503 expression was decreased and cell viability and B-cell lymphoma 2 (bcl-2) expression were increased in the Hep-NS5A cells. Moreover, the luciferase reporter gene experiment verified that bcl-2 was a direct target of miR-503, NS5A inhibited $TNF{\alpha}$-induced $NF-{\kappa}B$ activation and $NF-{\kappa}B$ regulated miR-503 transcription by combining with the miR-503 promoter. After the Hep-NS5A cells were transfected with miR-503 mimics, the data indicated that the mimics could reverse $TNF-{\alpha}$-induced cell apoptosis and blc-2 expression. Collectively, our findings suggest a possible molecular mechanism that may contribute to HCV treatment in which NS5A inhibits $NF-{\kappa}B$ activation to decrease miR-503 expression and increase bcl-2 expression, which leads to a decrease in hepatocellular apoptosis.

• Journal of Life Science
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• v.13 no.4
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• pp.541-550
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• 2003

E2 glycoprotein of hepatitis C virus (HCV) comprises a surface of viral particle together with E1 glycoprotein, and is thought to be involved in the attachment of HCV viral particle to receptor (s) on the permissible cells including hepatocytes, B cells, T cells, and monocytes. We constructed a phage library expressing cellular proteins of hepatocytes on the phage surface, which turned out to be 8.8${\times}$$10^5$ cfu of diversity and carried inserts in 95% of library. We screened both cDNA phage library and 12-mer peptide library to identify the cellular proteins binding to E2 protein. Some intracellular proteins including tensin and membrane band 4.1 which are involved in signal transduction of survival and cytoskeleton organization, were selected from cDNA phage library through several rounds of panning and screening. On the contrary, membrane proteins such as CCR7, CKR-L2, and insulin-like growth factor-1 receptor were identified through screening of peptide library. Phages expressing peptides corresponding to those membrane proteins were bound to E2 protein specifically as determined by neutralization of binding assay. Since it is well known that HCV can infect T cells as well as hepatocytes, we examined to see if E2 protein can bind to CCR7, a member of C-protein coupled receptor family expressed on T cells, using CCR7 transfected tells. Human CCR7 cDNA was cloned into pcDNA3.1(-) vector and transfected into human embryonic kidney cell, 293T, and expressed on the surface of the cell as shown by flow cytometer. Binding assay of E2 protein using CCR7 transfected cells indicated that E2 protein bound to CCR7 by dose-dependent mode, giving rise to the possibility that CCR7 might be a putative cellular receptor for HCV.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2133-2140
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• 2018

Norovirus is the most common cause of acute gastroenteritis. Its pathogenesis is poorly understood owing to the difficulty of establishing viral infection in animal models. Here, post-weaning gnotobiotic pigs were infected with human norovirus genogroup II genotype 4 (HuNoV GII.4) to investigate the pathogenesis and replication of the virus. Three groups of four pigs were infected with $1{\times}10^5$, $1{\times}10^6$, or $1{\times}10^7$ genomic equivalent (GE) copies of HuNoV GII.4. Four pigs were used as negative controls. Blood and rectal swab samples were collected after viral infection, and gross legions were examined after necropsy. Diarrhea was induced in 25% and 75% of pigs infected with $1{\times}10^6$ and $1{\times}10^7$ GE copies, respectively. Viral shedding was detected in 50%, 75%, and 50% of pigs infected with $1{\times}10^5$, $1{\times}10^6$, and $1{\times}10^7$ GE copies, respectively. Viremia was detected in 25% of pigs infected with either $1{\times}10^6$ or $1{\times}10^7$ GE copies. When gross lesions of gastroenteritis were investigated, the ileum walls of the infected pigs were thinner than those of the controls. Villi atrophy and inflammatory cell infiltration were identified in the ileum of each infected pig. Viral capsid was identified in the jejunum, ileum, colon, spleen, and mesenteric lymph node. Virus replication was newly verified in the spleen and mesenteric lymph nodes by detection of negative-sense viral RNA. In conclusion, HuNoV GII.4 could induce acute gastroenteritis and replicate in the extra-intestinal lymphoid tissues in post-weaning gnotobiotic pigs. Therefore, such pigs would be a suitable animal model for studying the pathogenesis and replication of HuNoV.

• Journal of Korean Society of Health-System Pharmacists
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• v.35 no.4
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• pp.453-462
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• 2018

Background : The prevalence of chronic hepatitis C virus (HCV) tends to be higher in the elderly. Pegylated interferon and ribavirin therapy (Peg-IFN/RBV) was recommended as the first-line treatment in the past decades, but this regimen showed unsatisfactory results in terms of safety and efficacy especially in elderly patients. Recently, it was demonstrated that dual therapy with daclatasvir and asunaprevir was well tolerated and led to high sustained virological response (SVR) rates, irrespective of age. We conducted a study to evaluate the efficacy and safety of daclatasvir plus asunaprevir by involving elderly patients aged above 65 years. Methods : We retrospectively analyzed clinical data from chronic hepatitis C virus (HCV) genotype 1b patients treated with daclatasvir plus asunaprevir from September 2015 to December 2016 at Seoul St. Mary's hospital. The patients were divided into two groups as elderly patients (older than 65 years) and non-elderly patients (younger than 65 years) and compared the efficacy and safety. Results : A total of 112 patients were treated with daclatasvir plus asunaprevir for chronic hepatitis C. Among them, 101 patients completed the whole treatment, and in 88 patients the amount of HCV RNA was measured after 12 weeks of treatment. There was no significant difference in SVR at 12 weeks between both the groups (p=0.68). Typically, 91.4%(32/35) of elderly patients and 94.3%(50/53) of non-elderly patients achieved SVR12. Common adverse events included elevation in transaminase level, headache, and gastrointestinal disorders. There was no statistical difference in the symptoms between the two groups. Conclusions : The combination therapy with daclatasvir plus asunaprevir exhibited similar rates of SVR12 in HCV elderly patients without leading to further adverse events compared to non-elderly patients. Therefore, it is proposed that daclatasvir plus asunaprevir therapy could be considered as an effective and safe treatment, even in patients aged over 65 years.

• Journal of Veterinary Science
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• v.23 no.3
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• pp.33.1-33.10
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• 2022

Background: Endemic circulation of human-specific hepatitis E virus (HEV) genotypes 1 and 2 may occult the importance of sporadic zoonotic HEV transmissions in Africa. Increasing numbers of studies reporting anti-HEV antibodies in cattle and the discovery of infectious HEV in cow milk has raised public health concern, but cattle exposure has seldom been investigated in Africa. Objectives: This study aimed at investigating the role of cows in the epidemiology of HEV in Burkina Faso and farmers habits in terms of dairy product consumption as a prerequisite to estimate the risk of transmission to humans. Methods: Sera from 475 cattle and 192 pigs were screened for the presence of anti-HEV antibodies while HEV RNA in swine stools was detected by reverse transcription polymerase chain reaction. Data on mixed farming, dairy product consumption and selling habits were gathered through questionnaires. Results: The overall seroprevalence in cattle was 5.1% and herd seroprevalence reached 32.4% (11/34). Herd seropositivity was not associated with husbandry practice or presence of rabbits on the farms. However, herd seropositivity was associated with on-site presence of pigs, 80.7% of which had anti-HEV antibodies. The majority of farmers reported to preferentially consume raw milk based dairy products. Conclusions: Concomitant presence of pigs on cattle farms constitutes a risk factor for HEV exposure of cattle. However, the risk of HEV infections associated with raw cow dairy product consumption is currently considered as low.

• IMMUNE NETWORK
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• v.23 no.4
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• pp.33.1-33.13
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• 2023

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been acknowledged as an effective mean of preventing infection and hospitalization. However, the emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) has led to substantial increase in infections among children and adolescents. Vaccine-induced immunity and longevity have not been well defined in this population. Therefore, we aimed to analyze humoral and cellular immune responses against ancestral and SARS-CoV-2 variants after two shots of the BNT162b2 vaccine in healthy adolescents. Although vaccination induced a robust increase of spike-specific binding Abs and neutralizing Abs against the ancestral and SARS-CoV-2 variants, the neutralizing activity against the Omicron variant was significantly low. On the contrary, vaccine-induced memory CD4⁺ T cells exhibited substantial responses against both ancestral and Omicron spike proteins. Notably, CD4⁺ T cell responses against both ancestral and Omicron strains were preserved at 3 months after two shots of the BNT162b2 vaccine without waning. Polyfunctionality of vaccine-induced memory T cells was also preserved in response to Omicron spike protein. The present findings characterize the protective immunity of vaccination for adolescents in the era of continuous emergence of variants/subvariants.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.2
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• pp.129-134
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• 2001

Since NNK is one of the most abundant tobacco-specific alkaloids and a strong carcinogenic nitrosamine, it has been used for evaluating a potential of carcinogenicity in the animal models. The present study has attempted to examine the potential of carcinogenicity of NNK in human epithelial cells, from which the cell type the most of cancers including oral cancer and nasal cavity cancer are originated. The cellular model used for the study is a human keratinocyte cell system immortalized by Ad12-SV40 hybrid virus. The cellular system has successfully been used for the carcinogenicity studies because of its limitless life span, epithelial morphology and nontumorigenicity. When cells were treated with a variety of NNK concentrations, levels of saturation density and soft agar colony formation were increased in a dose-dependent fashion. Colonies of large cell aggregates were above 5 at the higher doses. The results indicate that exposure of human cells with NNK induced loss of contact inhibition and increases of anchorage independence and cellular adhesion, which are typical characteristics of the neoplatically transformed cells. When cells were exposed with 100uM NNK for 2hr, mRNA levels of IL-1 and PAI-2 were increased in a dose-dependent manner, but expression of TGF- 1 was not affected. While expression of growth regulatory factors were altered with a short-term exposure, there was no alteration of these factors in the NNK-transformed cells. However, mRNA levels of fibronectin were increased both in the short-term treatment and in the transformation. The results suggest that altered expression of extracellular matrix such as fibronectin following short-term exposure might be fixed in the genome and these altered properties be continuously transfered throughout the cell division. Western blot analysis showed a translocation of PKC- from cytosolic fraction to the particulate fraction, indicating a possible role of NNK in the signal transduction pathway. The present study provided an evidence that NNK in the smoking may be associated with epithelial origin cancer such as oral and nasal cavity cancers. In addition, this study suggested that altered expression of extracellular matrix and PKC may play an important role in the carcinogenic mechanism of NNK.