University of Arkansas=;Harvard Medical School=;Ewha Womans University=;Jeonbuk National University, Jeonju=;KU‐KIST Graduate School of Converging Science and Technology Korea University Seoul 02841 Republic of Korea.
Impact Factor : 1.276Journal of Microbiology 2019 Nov
Hepatitis E virus (HEV) is a causative agent of acute hepatitis and jaundice. The number of human infections is approximated to be over 20 million cases per year. The transmission is mainly via the fecal-oral route and contaminated water and food are considered to be a major source of infection. As a mouse model is not available, a recent development of a cell culture-adapted HEV strain (47832c) is considered as a very important tools for molecular analysis of HEV pathogenesis in cells. Previously, we demonstrated that HEV-encoded methyltransferase (MeT) encoded by the 47832c strain inhibits MDA5- and RIG-I-mediated activation of interferon β (IFN-β) promoter. Here, we report that MeT impairs the phosphorylation and activation of interferon regulatory factor 3 and the p65 subunit of NF-κB in a dose-dependent manner. In addition, the MeT encoded by the 47832c, but not that of HEV clinical or field isolates (SAR-55, Mex-14, KC-1, and ZJ-1), displays the inhibitory effect. A deeper understanding of MeTmediated suppression of IFN-β expression would provide basis of the cell culture adaptation of HEV.
Impact Factor : 1.276Journal of Microbiology 2019 Aug
Middle East respiratory syndrome coronavirus (MERS-CoV) is a causative agent of severe-to-fatal pneumonia especially in patients with pre-existing conditions, such as smoking and chronic obstructive pulmonary disease (COPD). MERS-CoV transmission continues to be reported in the Saudi Arabian Peninsula since its discovery in 2012. However, it has rarely been epidemic outside the area except one large outbreak in South Korea in May 2015. The genome of the epidemic MERS-CoV isolated from a Korean patient revealed its homology to previously reported strains. MERS-CoV encodes 5 accessory proteins and generally, they do not participate in the genome transcription and replication but rather are involved in viral evasion of the host innate immune responses. Here we report that ORF8b, an accessory protein of MERS-CoV, strongly inhibits both MDA5- and RIG-I-mediated activation of interferon beta promoter activity while downstream signaling molecules were left largely unaffected. Of note, MDA5 protein levels were significantly down-regulated by ORF8b and co-expression of ORF4a and ORF4b. These novel findings will facilitate elucidation of mechanisms of virus-encoded evasion strategies, thus helping design rationale antiviral countermeasures against deadly MERS-CoV infection.
Direct electrochemical (EC) monitoring in a cell culture medium without electron transporter as called mediator is attractive topic in vitro organoid based on chip with frequently and long‐time monitoring since it can avoid to its disadvantage as stability, toxicity. Here, direct monitoring with nonmediator is demonstrated based on impedance spectroscopy under the culture medium in order to overcome the limitation of mediator. The applicability of EC monitoring is shown by detecting alpha‐1‐anti trypsin (A1AT) which is known as biomarkers for cardiac damage and is widely chosen in organoid cardiac cell‐based chip. The validity of presented EC monitoring is proved by observing signal processing and transduction in medium, mediator, medium–mediator complex. After the observation of electron behavior, A1AT as target analyte is immobilized on the electrode and detected using antibody–antigen interaction. As a result, the result indicates limit of detection is 10 ng mL−1 and linearity for the 10–1000 ng mL−1 range, with a sensitivity of 3980 nF (log [g mL])−1 retaining specificity. This EC monitoring is based on label‐free and reagentless detection, will pave the way to use for continuous and simple monitoring of in vitro organoid platform.
Impact Factor : 1.769Virus Genes 2009 Aug
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a pivotal role in the regulation of gene expression at the post transcriptional level. Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus belonging to the herpesviridae family. It causes an acute respiratory disorder in chicken resulting in high mortality rates. ILTV encoding miRNAs have been identified in cell cultures infected by ILTV. Seven candidates ILTV encoding miRNAs were identified by the 454 FLX genome sequencing method. Five ILTV miRNAs identified in this study were identical to those previously reported by Waidner et al. (Virology 388:128, 2009). Two unique ILTV miRNAs, iltv-miR-I1-3p and iltv-miR-I7-3p, were identified in this study. The iltv-miR-I1-3p is the passenger strand of I1-5p, which was previously known. The iltv-miR-I7-3p showed a perfect match with the complementary passenger strand in contrast to other miRNA species showing imperfect complementarity with the passenger strand. The I7-3p was mapped in the replication origin (oriL) of the palindrome stem loop sequence of the ILTV genome. Expression of all ILTV miRNAs were confirmed by the end point PCR using small RNA libraries generated from either ILTV infected or uninfected control chicken embryo kidney (CEK) cells.
Influenza virus infections continually pose a major public health threat with seasonal epidemics and sporadic pandemics worldwide. While currently licensed influenza vaccines provide only strain-specific protection, antigenic drift and shift occasionally render the viruses resistant to the host immune responses, which highlight the need for a vaccine that provides broad protection against multiple subtypes. In this study, we suggest a vaccination strategy using cold-adapted, live attenuated influenza vaccines (CAIVs) to provide a broad, potent, and safe cross-protection covering antigenically distinct hemagglutinin (HA) groups 1 and 2 influenza viruses. Using a mouse model, we tested different prime–boost combinations of CAIVs for their ability to induce humoral and T-cell responses, and protective efficacy against H1 and H5 (HA group 1) as well as H3 and H7 (HA group 2) influenza viruses. Notably, even in the absence of antibody-mediated neutralizing activity or HA inhibitory activity in vitro, CAIVs provided a potent protection against heterologous and heterosubtypic lethal challenges in vivo. Heterologous combination of prime (H1)–boost (H5) vaccine strains showed the most potent cross-protection efficacy. In vivo depletion experiments demonstrated not only that T cells and natural killer cells contributed to the cross-protection, but also the involvement of antibody-dependent mechanisms for the cross-protection. Vaccination-induced antibodies did not enhance the infectivity of heterologous viruses, and prime vaccination did not interfere with neutralizing antibody generation by the boost vaccination, allaying vaccine safety concerns associated with heterogeneity between the vaccines and challenge strains. Our data show that CAIV-based strategy can serve as a simple but powerful option for developing a “truly” universal influenza vaccine providing pan-influenza A protection, which has not been achieved yet by other vaccine strategies. The promising results of potency, breadth, and safety demonstrated in the mouse model support further studies in higher animal models for clinical relevance.
Pattern recognition receptors (PRRs) are critical to the early detection and innate immune responses to pathogens. In particular, the TLR system and its associated adaptor proteins play essential roles in early host responses to infection. Epidemic keratoconjunctivitis, caused by the human adenovirus, is a severe ocular surface infection associated with corneal inflammation (stromal keratitis). We previously showed that adenovirus capsid was a key molecular pattern in adenovirus keratitis, with viral DNA playing a lesser role. We have now investigated the role of the adaptor molecule MyD88 in a mouse model of adenovirus keratitis in which there is no viral replication. In MyD88−/− mice infected with human adenovirus type 37, clinical keratitis was markedly reduced, along with infiltration of CD45+ cells, and expression of inflammatory cytokines. Reduction of inflammatory cytokines was also observed in infected primary human corneal fibroblasts pretreated with a MyD88 inhibitory peptide. Keratitis similar to wild type mice was observed in TLR2, TLR9, and IL-1R knockout mice, but was reduced in TLR2/9 double knockout mice, consistent with synergy of TLR2 and TLR9 in the response to adenovirus infection. MyD88 co-immunoprecipitated with Src kinase in mice corneas and in human corneal fibroblasts infected with adenovirus, and MyD88 inhibitory peptide reduced Src phosphorylation, linking MyD88 activation to inflammatory gene expression through a signaling cascade previously shown to be directed by Src. Our findings reveal a critical role for the PRRs TLR2 and 9, and their adaptor protein MyD88, in corneal inflammation upon adenovirus infection.
Impact Factor : 2.927Scientific Reports 2017 Apr
Human adenoviruses (HAdVs) shut down host cellular cap-dependent mRNA translation while initiating the translation of viral late mRNAs in a cap-independent manner. HAdV 5′ untranslated regions (5′UTRs) are crucial for cap-independent initiation, and influence mRNA localization and stability. However, HAdV translational regulation remains relatively uncharacterized. The HAdV tripartite leader (TPL), composed of three introns (TPL 1–3), is critical to the translation of HAdV late mRNA. Herein, we annotated and analyzed 72 HAdV genotypes for the HAdV TPL and another previously described leader, the i-leader. Using HAdV species D, type 37 (HAdV-D37), we show by reverse transcription PCR and Sanger sequencing that mRNAs of the HAdV-D37 E3 transcription unit are spliced to the TPL. We also identified a polycistronic mRNA for RID-α and RID-β. Analysis of the i-leader revealed a potential open reading frame within the leader sequence and the termination of this potential protein in TPL3. A potential new leader embedded within the E3 region was also detected and tentatively named the j-leader. These results suggest an underappreciated complexity of post-transcriptional regulation, and the importance of HAdV 5′UTRs for precisely coordinated viral protein expression along the path from genotype to phenotype.
Impact Factor : 3.73PLoS ONE 2017 Apr
Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in infants, young children, and the elderly. Two subtypes of RSV, A and B, circulate alternately at 1-2-year intervals during epidemics. The attachment glycoprotein (G protein) of RSV is one of the major targets for immune responses. In this study, we generated a recombinant fusion protein, GcfAB, which consists of the central regions (a.a. residues 131–230) of the G proteins of both RSV A (A2 strain) and B (B1 strain) subtypes, and investigated immunogenicity, protective efficacy, and immunopathology. We immunized mice with GcfAB plus cholera toxin as a mucosal adjuvant via intranasal (IN) or sublingual (SL) routes. The IN group showed higher levels of RSV G-specific antibody responses, including serum IgG and mucosal IgA, compared with the SL group. On the contrary, more vigorous RSV G-specific CD4+ T-cell responses were elicited in the SL group than in the IN group after RSV-A but not RSV-B viral challenge. Furthermore, the SL group showed more pulmonary eosinophil recruitment and body weight loss than did the IN group after RSV-A challenge. Both IN and SL immunization with GcfAB provided potential protection against both subtypes of infections. Together, these results suggest that vaccination with GcfAB via an IN route could be a universal vaccine regimen preventing both RSV A and B infections.
Impact Factor : 3.73PLoS ONE 2017 Jan
Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract illness in infants, young children, and the elderly. The G glycoprotein plays a role in host cell attachment and also modulates the host immune response, thereby inducing disease pathogenesis. We generated two monoclonal antibodies (mAbs; 5H6 and 3A5) against G protein core fragment (Gcf), which consisted of amino acid residues 131 to 230 from RSV A2 G protein. Epitope mapping study revealed that 5H6 specifically binds to the G/164-176 peptide that includes conserved sequences shared by both RSV A and B subtypes, and 3A5 binds to the G/190-204 peptide. Studies with mutant Gcf proteins in which cysteine residues were substituted with alanine revealed that 5H6 requires four cysteines for binding and 3A5 binds to Gcf variants with alanine substitutions better than wild-type. To determine if these mAbs reduce pulmonary viral infection, BALB/c mice were administered mAb and subsequently challenged with RSV. On day 4 post-infection, lung viral titers were reduced by up to 93% with the 5H6 injection and 90% with the 3A5 injection, indicating that prophylactic injection of these mAbs contributes to RSV clearance in vivo. Importantly, 5H6 injection reduced vaccine-enhanced diseases. Overall, our results suggest that this novel anti-G mAb could be used as a prophylactic regimen against RSV diseases.
Impact Factor : 5.372Molecular and Cellular Biology 2016 Mar
This study is designed to examine the cellular functions of human Fas-associated factor 1 (FAF1) containing multiple ubiquitin-related domains. Microarray analyses revealed that interferon-stimulated genes related to the antiviral response are significantly increased in FAF1-knockdown HeLa cells. Silencing FAF1 enhanced the poly(I·C)- and respiratory syncytial virus (RSV)-induced production of type I interferons (IFNs), the target genes of interferon regulator factor 3 (IRF3). IRF3 is a key transcription factor in IFN-β signaling responsible for the host innate immune response. This study also found that FAF1 and IRF3 physically associate with IPO5/importin-β3 and that overexpression of FAF1 reduces the interaction between IRF3 and IPO5/importin-β3. These findings suggest that FAF1 negatively regulates IRF3-mediated IFN-β production and the antiviral innate immune response by regulating nuclear translocation of IRF3. We conclude that FAF1 plays a novel role in negatively regulating virus-induced IFN-β production and the antiviral response by inhibiting the translocation of active, phosphorylated IRF3 from the cytosol to the nucleus.
Although recent studies have suggested that adherence to antihypertensive treatment reduced stroke incidence, the relationship of adherence to antihypertensives with stroke severity has not been studied. This study attempted to know whether nonadherence before stroke is associated with initial severity of acute ischemic stroke. Consecutive patients with acute ischemic stroke were identified in Soonchunhyang University Hospital from Mar 2005 to Aug 2014, excluding the cases without hypertension or information of antihypertensive adherence. We compared the mean of National Institute of Health Stroke Scale (NIHSS) score between adherence groups and insufficient medication group, and additionally in each stroke subtype. Multiple linear regression model was established for initial NIHSS score adjusting alleged factors linked to stroke severity. Initial NIHSS score were higher in insufficient medication group than adherence group (6.5 ± 7.2 VS 5.4 ± 5.7, P = .11). In large artery atherosclerosis (LAA) and small vessel occlusion (SVO), initial NIHSS score were significantly higher in insufficient medication group (6.1 ± 6.5 VS 4.4 ± 4.4, P = .004 for LAA; 3.8 ± 3.5 VS 2.7 ± 1.8, P = .014 for SVO). In multiple linear regression model, insufficient medication to antihypertensives had a significant effect on NIHSS score (t = 3.417, P = .001) after adjusting covariates. Insufficient medication of antihypertensives before stroke was independently associated with the severity of acute ischemic stroke. Further studies with prospective designs are warranted to evaluate clinical implication of adherence to antihypertensives for ischemic stroke.
Impact Factor : 3.73PloS one 2013 Oct
The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.
Impact Factor : 2.927Scientific reports 2013 May
The recent emergence of highly virulent human adenoviruses (HAdVs) with new tissue tropisms underscores the need to determine their ontogeny. Here we report complete high quality genome sequences and analyses for all the previously unsequenced HAdV serotypes (n = 20) within HAdV species D. Analysis of nucleotide sequence variability for these in conjunction with another 40 HAdV prototypes, comprising all seven HAdV species, confirmed the uniquely hypervariable regions within species. The mutation rate among HAdV-Ds was low when compared to other HAdV species. Homologous recombination was identified in at least two of five examined hypervariable regions for every virus, suggesting the evolution of HAdV-Ds has been highly dependent on homologous recombination. Patterns of alternating GC and AT rich motifs correlated well with hypervariable region recombination sites across the HAdV-D genomes, suggesting foci of DNA instability lead to formulaic patterns of homologous recombination and confer agility to adenovirus evolution.
The Smyth line (SL) of chicken is an excellent avian model for human autoimmune vitiligo. The etiology of vitiligo is complicated and far from clear. In order to better understand critical components leading to vitiligo development, cDNA microarray technology was used to compare gene expression profiles in the target tissue (the growing feather) of SL chickens at different vitiligo (SLV) states.
Compared to the reference sample, which was from Brown line chickens (the parental control), 395, 522, 524 and 526 out of the 44 k genes were differentially expressed (DE) (P ≤ 0.05) in feather samples collected from SL chickens that never developed SLV (NV), from SLV chickens prior to SLV onset (EV), during active loss of pigmentation (AV), and after complete loss of melanocytes (CV). Comparisons of gene expression levels within SL samples (NV, EV, AV and CV) revealed 206 DE genes, which could be categorized into immune system-, melanocyte-, stress-, and apoptosis-related genes based on the biological functions of their corresponding proteins. The autoimmune nature of SLV was supported by predominant presence of immune system related DE genes and their remarkably elevated expression in AV samples compared to NV, EV and/or CV samples. Melanocyte loss was confirmed by decreased expression of genes for melanocyte related proteins in AV and CV samples compared to NV and EV samples. In addition, SLV development was also accompanied by altered expression of genes associated with disturbed redox status and apoptosis. Ingenuity Pathway Analysis of DE genes provided functional interpretations involving but not limited to innate and adaptive immune response, oxidative stress and cell death.
The microarray results provided comprehensive information at the transcriptome level supporting the multifactorial etiology of vitiligo, where together with apparent inflammatory/innate immune activity and oxidative stress, the adaptive immune response plays a predominant role in melanocyte loss.
When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray.
A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems,
The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells.
Infection by infectious laryngotracheitis virus (ILTV;
Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death.
The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections.