Seok-Yong Kim

Development and Application of a Reverse Genetics System for Japanese Encephalitis Virus

ABSTRACT Japanese encephalitis virus (JEV) is a common agent of viral encephalitis that causes high mortality and morbidity among children. Molecular genetic studies of JEV are hampered by the lack of a genetically stable full-length infectious JEV cDNA clone. We describe here the development of such a clone. A JEV isolate was fully sequenced, and then its full-length cDNA was cloned into a bacterial artificial chromosome. This was then further engineered so that transcription of the cDNA in vitro would generate synthetic RNAs with authentic 5′ and 3′ ends. The synthetic RNAs thus produced were highly infectious in susceptible cells (>106 PFU/μg), and these cells rapidly generated a high titer of synthetic viruses (>5 × 106 PFU/ml). The recovered viruses were indistinguishable from the parental virus in terms of plaque morphology, growth kinetics, RNA accumulation, protein expression, and cytopathogenicity. Significantly, the structural and functional integrity of the cDNA was maintained even after 180 generations of growth in Escherichia coli. A single point mutation acting as a genetic marker was introduced into the cDNA and was found in the genome of the recovered virus, indicating that the cDNA can be manipulated. Furthermore, we showed that JEV is an attractive vector for the expression of heterologous genes in a wide variety of cell types. This novel reverse genetics system for JEV will greatly facilitate research into JEV biology. It will also be useful as a heterologous gene expression vector and will aid the development of a vaccine against JEV.

Helicobacter pylori CagA phosphorylation status determines the gp130-activated SHP2/ERK and JAK/STAT signal transduction pathways in gastric epithelial cells.

The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.

Prevalence and genetic characterization of respiratory syncytial virus (RSV) in hospitalized children in Korea

Human respiratory syncytial virus (HRSV) is the most common respiratory pathogen among infants and young children. To investigate the prevalence and genetic characteristics of HRSVs circulating in South Korea, we analyzed medical records of patients and performed molecular analysis of the G-protein gene of viruses detected from nasopharyngeal aspirates (NPA) of admitted patients at the Pediatrics Department of Chungbuk National University Hospital from April 2008 to April 2010. Epidemiological data revealed that the prevalence of HRSV infection was high during both winter seasons (October 2008 to February 2009 and November 2009 to February 2010). Of the 297 positive NPA specimens from infants or children tested, 67% were identified as HRSV-A while 33% were HRSV-B. The HRSV subgroup B was the most dominant in December 2008, but its dominance was dramatically replaced by HRSV subgroup A strains by February 2009. Phylogenetic analysis of the G protein sequences of HRSVs revealed novel genotypes within the HRSV-A (genotype CB-A) and B (genotypes BA11 and CB-B) subgroups in South Korea in addition to other strains identified in other countries. Molecular analysis also revealed genetic variability at the C-terminal end of the G proteins of the two HRSV subgroups, suggesting selection pressure in this region, which may potentially impact immune recognition. This is the first report of these HRSV variants in South Korea, indicating active genetic evolution of HRSV strains. Therefore, this study provides information on the molecular epidemiology of current HRSVs in the country and presents data for comparative analysis with other HRSV strains circulating worldwide.

c-Jun N-terminal kinase is involved in motility of endothelial cell

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (∼2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFα and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.

Isolation and Characterization of Novel H3N1 Swine Influenza Viruses from Pigs with Respiratory Diseases in Korea

ABSTRACT Pigs can play an important role in the genetic reassortment of influenza viruses and as a reservoir for another lineage of influenza viruses that have the ability to reassort and be transmitted between species. In March and April 2006, novel H3N1 influenza A viruses were isolated from pigs with respiratory diseases at two different commercial swine farms in Korea. Genetic and phylogenetic analyses of the sequences of all eight viral RNA segments showed that the novel H3N1 swine influenza viruses were reassortants that acquired the hemagglutinin gene from an H3 human-like virus and other genes from swine influenza viruses that are currently circulating in Korea. Serologic and virologic tests in the infected farms suggested that pig-to-pig and farm-to-farm transmissions occurred. Clinical signs in pigs and experimentally infected mice suggest the potential to transmit the virus between swine and other mammalian hosts. To our knowledge, this is the first report of the isolation of the swine H3N1 subtype from domestic pigs under field conditions in Korea. Further surveillance will be needed to determine whether this novel subtype will continue to circulate in the swine population.

Respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells.

Summary. Increased gelatinolytic activity was observed in respiratory syncytial virus (RSV)-infected HEp-2 cells by using zymography. The anti-matrix metalloproteinase-9 (MMP-9) antibody specifically reduced the gelatinolytic activity suggesting that the increased gelatinolytic activity was due to the MMP-9. It was also supported by the results from immunofluorescent staining, treatment of MMP inhibitors, and RSV infection of the cell clones that were transfected with plasmids to express more MMP-9 and tissue type inhibitor of metalloproteinase-1 (TIMP-1). The gelatinolytic activity of extracellular MMP-9 in RSV-infected HEp-2 cells increased 1.5 ± 0.2 fold compared with the control (p < 0.01). Cell surface MMP-9 expression was also clearly detected by immunofluorescent staining. Treatment with 1,10-phenanthroline (0.05 mM), ethylenediamine-tetraacetate (EDTA) (1.5 mM), and penta-O-galloyl-β-D-glucose (PGG) (3.3 µM) inhibited RSV multiplication as well as syncytia formation. Furthermore, the average syncytia size increased when the cells expressing more MMP-9 were infected by RSV. In contrast, syncytia formation was inhibited in the cells manipulated to express TIMP-1. Thus, this study concludes that although RSV infection induces MMP-9, which can enhance the syncytia formation leading to RSV multiplication and spread it can be inhibited by MMP inhibitors.

The transport of Staufen2‐containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen‐activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA‐binding proteins. In these RNP complexes, Staufen, a double‐stranded RNA‐binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein‐tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2‐containing RNP complexes in neurons under non‐stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2‐containing RNP complexes use kinesin as a motor protein. A mitogen‐activated protein kinase inhibitor, PD98059, inhibited the activity‐induced increase in the amount of both the Staufen2‐containing RNP complexes and Ca2+/calmodulin‐dependent protein kinase II α‐subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen‐activated protein kinase pathway.

Influenza A virus infection modulates the expression of type IV collagenase in epithelial cells

SummaryWe investigated the effect of influenza A/Beijing/353/89 (H3N2) virus infection on the expression of type IV collagenase in two different types of epithelial cell. Depending on the cell line infected, the viral infection caused changes in the expression of type IV collagenase. The expression of matrix metalloproteinase-9 (MMP-9; 92 kDa) but not of matrix metalloproteinase-2 (MMP-2; 72 kDa) was stimulated in Vero cells. In MDCK cells, the MMP-2 production increased with the virus infection. According to the enzymatic activity revealed with zymography, the MMP-9 promoter activity rose by a factor of over 1788 in influenza A virus-infected Vero cells but not in MDCK cells. The tissue inhibitor of metalloproteinase, TIMP-1, had increased slightly (2.3-fold) in Vero cells 48 hours after the infection, but in MDCK cells, influenza A virus had no effect on the TIMP-1 expression. In conclusion, the MMP-9 and -2 expression by influenza A virus infecton are modulated at transcriptional level, depending on the epithelial cell line.

Detection of Helicobacter pylori in Gastric Mucosa of Patients with Gastroduodenal Diseases by PCR-Restriction Analysis Using the RNA Polymerase Gene (rpoB)

ABSTRACT A novel PCR restriction analysis method using the RNA polymerase β-subunit- coding gene (rpoB) was employed to both detect and identify Helicobacter pylori in biopsy specimens and culture isolates. The rpoB DNAs (458 bp) were specifically amplified by PCR with the Helicobacter-specific primers (HF and HR). Based on the determined rpoB sequences of the culture isolates, an H. pylori-specific restriction site, Tru9I, was found. H. pylori can be identified by observing two discernible DNA fragments (288 and 138 bp) after Tru9I digestion and agarose gel electrophoresis. The rpoB PCR and subsequent restriction analysis (PRA) enabled the specific detection and identification of H. pylori in biopsy specimens from patients with gastroduodenal diseases. The rpoB PRA conferred a compatible or a slightly higher positive rate (53.7%) than did the Campylobacter-like organism (CLO) test (50.4%) and glmM PCR (48.8%), suggesting that it is useful for diagnosing an H. pylori infection without culture in the clinical laboratory.

Surveillance and characterization of low pathogenic H5 avian influenza viruses isolated from wild migratory birds in Korea

Migratory waterfowls are the natural reservoir of influenza A viruses. However, interspecies transmission had occasionally caused outbreaks in various hosts including humans. To characterize the genetic origins of H5 avian influenza viruses isolated from migratory birds in South Korea, phylogenetic analysis were conducted. A total of 53 H5 viruses were isolated between October 2005 and November 2008. Full genetic characterization indicated that most of these viruses belong to the Eurasian-like avian lineage. However, some segments of the AB/Korea/W235/07 and the AB/Korea/W236/07 isolates were clustered with North American lineage viruses rather than those of the Eurasian lineage, suggesting the occurrence of reassortment between these two avian virus lineages. Phylogenetic analysis further demonstrated that the H5N2 and H5N3 virus isolates were of the low pathogenicity H5 phenotype. The H5 viruses appear to be antigenically similar to each other, but could be distinguished from a recent HPAI H5N1 (EM/Korea/W149/06) virus by hemagglutinin inhibition (HI) assays. Experimental inoculation of representative viruses indicated that certain isolates, particularly AB/Korea/W163/07 (H5N2), could be detected in trachea and lungs of chickens but none could be transmitted by direct contact. Furthermore, all of the viruses could be detected in mice lung without prior adaptation which is indicative of their pathogenic potential in a mammalian host. Overall, our results emphasize the important role that migratory birds play in the perpetuation, transport, and reassortment of avian influenza viruses stressing the need for continued surveillance of influenza virus activity in these avian populations.