• Molecules and Cells
• /
• v.40 no.3
• /
• pp.202-210
• /
• 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
• /
• v.13 no.4
• /
• pp.541-550
• /
• 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
• /
• v.28 no.12
• /
• pp.2133-2140
• /
• 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
• /
• v.35 no.4
• /
• pp.453-462
• /
• 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
• /
• v.23 no.3
• /
• pp.33.1-33.10
• /
• 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
• /
• v.23 no.4
• /
• pp.33.1-33.13
• /
• 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
• /
• v.27 no.2
• /
• pp.129-134
• /
• 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.

• Journal of Plant Biotechnology
• /
• v.34 no.1
• /
• pp.11-17
• /
• 2007

Previously developed transgenic watermelon rootstocks (gongdae) inserted by CGMMV-CP were examined to test the virus tolerance levels. In the restricted plastic house, the $T_{3}$ watermelon rootstock showed tolerance to CGMMV until 70 days after inoculation on the leaves while the non-transformed watermelon rootstock became susceptible at 20 days after inoculation. In the field, tolerance efficiency of transgenic rootstocks maintained up to 40% at 71 days after contamination with CGMMV in the soil while all of the non-transformed rootstocks became susceptible at 37 days with the same condition. In the same field, transgenic rootstocks showed more tolerance to CGMMV than the non-transformed rootstocks as those were inoculated on the leaves, but it showed only 10 days delay before being susceptible. Therefore, transgenic rootstocks have a characteristic of delay effect against CGMMV susceptibility, rather than resistance character. From $T_{3}$ rootstocks homozygous for the CGMMV-CP horticulturally favorable individuals were selected for further breeding and a transgenic line was finally obtained at the $BC_{1}T_{5}$. A material transfer experiment was conducted to find out if the DNA, RNA or expressed protein in the transgenic rootstocks could move to the grafted scion (non-transformed watermelon, Super-Kumcheon). PCR, northern, and western blot analysis were performed and no evidence of transferring of those materials from rootstock to scion was ever found.

• Pediatric Infection and Vaccine
• /
• v.6 no.1
• /
• pp.123-130
• /
• 1999

Purpose : Meningitis is an inflammation of meninges by various kinds of organisms. Almost 85% of aseptic meningitis is caused by Enterovirus. This study was done to detect the causative virus of those with aseptic meningitis through sequencing the 5'-noncoding region to compare prototype and homology. Methods : RNA was extracted from Coxsackie Bl, Echovirus 3, 7, 9, 30. DNA was synthesized by RT-PCR and we compared homology with prototype from WHO by direct sequencing. Results : 1) PCR products from these viruses showed same bands of 155 bp and 440 bp on gel electrophoresis. 2) Coxsackievirus and Echovirus 11 prototype sequences were compared, which showed 12 bp changes with 92.1%. 3) Coxsackievirus B1 from a patient showed 94.1% homology when compared with prototype. 4) Echovirus 3 showed 92.8%, echovirus 7 92.8%, echovirus 30 82.9% homology. Conclusion : 5'-NCR of enterovirus has high homology which was good for use of diagnosis and more long sequencing requires for typing of viruses.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.4
• /
• pp.595-606
• /
• 2018

Objective: The Fusarium mycotoxins of deoxynivalenol (DON) and zerolenone (ZEN) cause health hazards for both humans and farm animals. Therefore, the main intention of this study was to reveal DON and ZEN effects on the mRNA expression of pro-inflammatory cytokines and other immune related genes in the liver of piglets. Methods: In the present study, 15 six-week-old piglets were randomly assigned to the following three different dietary treatments for 4 weeks: control diet, diet containing 8 mg DON/kg feed, and diet containing 0.8 mg ZEN/kg feed. After 4 weeks, liver samples were collected and sequenced using RNA-Seq to investigate the effects of the mycotoxins on genes and gene networks associated with the immune systems of the piglets. Results: Our analysis identified a total of 249 differentially expressed genes (DEGs), which included 99 upregulated and 150 downregulated genes in both the DON and ZEN dietary treatment groups. After biological pathway analysis, the DEGs were determined to be significantly enriched in gene ontology terms associated with many biological pathways, including immune response and cellular and metabolic processes. Consistent with inflammatory stimulation due to the mycotoxin-contaminated diet, the following Kyoto encyclopedia of genes and genomes pathways, which were related to disease and immune responses, were found to be enriched in the DEGs: allograft rejection pathway, cell adhesion molecules, graft-versus-host disease, autoimmune thyroid disease (AITD), type I diabetes mellitus, human T-cell leukemia lymphoma virus infection, and viral carcinogenesis. Genome-wide expression analysis revealed that DON and ZEN treatments downregulated the expression of the majority of the DEGs that were associated with inflammatory cytokines (interleukin 10 receptor, beta, chemokine [C-X-C motif] ligand 9), proliferation (insulin-like growth factor 1, major facilitator superfamily domain containing 2A, insulin-like growth factor binding protein 2, lipase G, and salt inducible kinase 1), and other immune response networks (paired immunoglobulin-like type 2 receptor beta, Src-like-adaptor-1 [SLA1], SLA3, SLA5, SLA7, claudin 4, nicotinamide N-methyltransferase, thyrotropin-releasing hormone degrading enzyme, ubiquitin D, histone $H_2B$ type 1, and serum amyloid A). Conclusion: In summary, our results demonstrated that high concentrations DON and ZEN disrupt immune-related processes in the liver.