Ki Joon Song

Novel mutations in the VKORC1 gene of wild rats and mice – a response to 50 years of selection pressure by warfarin?

BackgroundCoumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control.ResultsIn the present study, we have sequenced the VKORC1 genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteinsConclusionOur results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.

Effects of Endocytosis Inhibitory Drugs on Rubella Virus Entry into VeroE6 Cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin‐dependent endocytosis and not by caveolae‐mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule‐disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin‐mediated endocytosis is likely to be a major route of RV infectious entry.

Characterization of tula virus from common voles (Microtus arvalis) in Poland: Evidence for geographic-specific phylogenetic clustering

Tula virus (TULV), a recently identified arvicolid rodent-borne hantavirus, is harbored by the European common vole (Microtus arvalis) in Central Russia and the Czech and Slovak Republics. We report the isolation and characterization of this hantavirus from M. arvalis captured in Poland, a country where human disease caused by hantaviruses has not been recognized. Of 34 arvicolid rodents (24 Clethrionomys glareolus, 9 M. arvalis, 1 Pitymys sp.) captured in Lodz and Tuszyn, Poland, during June to September 1995, sera from 3 M. arvalis and 3 C. glareolus contained IgG antibodies to Puumala virus (PUUV), as determined by an indirect immunofluorescent antibody assay. Alignment and comparison of the 1852-nucleotide S segment and a 1676-nucleotide region of the G2 glycoprotein-encoding M segment, amplified from lung tissues of two hantavirus-seropositive M. arvalis, revealed 83.9–85.2% and 82.3–83.5% sequence similarity, respectively, with TULV strains from Central Russia and the Czech and Slovak Republics. A > 98% sequence conservation was found at the amino acid level. Phylogenetic analysis indicated that the newly found TULV strains from Poland were closely related to, but distinct from, TULV from elsewhere in Europe.

Phylogenetic Analysis of the Small Hydrophobic (SH) Gene of Mumps Virus in Korea: Identification of a New Genotype

Viral RNAs extracted from fifteen mumps virus isolated from throat swab, saliva, blood, urine or CSF during mumps epidemics between 1997–1998 in Korea were amplified by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and compared by nucleotide sequencing of the small hydrophobic (SH) gene. The deduced amino acid sequences of the SH gene were aligned with the published sequences of mumps virus isolated in different geographic areas. A comparison of the SH gene of mumps viruses in Korea indicated 96.2–100% and 91.2–100% similarity at the nucleotide and amino acid levels, respectively. Phylogenetic analysis, using the neighbor‐joining method, showed that Korean mumps virus strains formed a genetically distinct monophyletic group from previously reported genotypes based on the 315‐bp length nucleotide and 57 deduced amino acid sequences of the SH gene, and possibly be designated as a new genotype (I).

Axin localizes to mitotic spindles and centrosomes in mitotic cells.

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3beta) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3beta in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.

Ecological surveillance of small mammals at Firing Points 10 and 60, Gyeonggi Province, Republic of Korea, 2001–2005

Abstract Throughout Korea, small mammals are hosts to a number of disease-causing agents that pose a health threat to U.S. and Korean military forces while they conduct field-training exercises. A seasonal rodent-borne disease surveillance program was established at two firing points (FP), FP-10, and FP-60, and conducted over five years from 2001 through 2005 in response to hantavirus cases among U.S. soldiers. The ecology of these sites consisted primarily of tall grasses associated with semi-permanent and temporary water sources (drainage ditches and a small stream) and dry-land agriculture farming. Eight species of rodents and one species of insectivore were collected, including Apodemus agrarius, Micromys minutus, Mus musculus, Rattus norvegicus, Tscherskia triton, Microtus fortis, Myodes regulus, and Crocidura lasiura. The striped field mouse, A. agrarius, (primary reservoir for Hantaan virus, the causative agent of Korean hemorrhagic fever), was the most frequently collected, representing 90.6% of the 1,288 small mammals captured at both sites. Reported herein are the ecological parameters, seasonal population densities, and seasonal population characteristics associated with small mammals collected at two military training sites in the Republic of Korea.

In vivo characterization of the integrin β3 as a receptor for Hantaan virus cellular entry

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the αvβ3 integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether β3 is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of β3 integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 ± 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either β3 or av prolonged survival significantly to a maximal average survival of 19.7 ± 1.5 days (P<0.01) and 18.4 ± 0.9 days (P<0.01), respectively. XT-199, a chemical blocker of the αvβ3 receptor also prolonged survival to 19.5 ± 1.3 days (P<0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine β1 antibody comparatively prolonged survival (19.0 ± 1.2 days), suggesting that HTNV infection is partly mediated through integrin β1 receptors as well as through β3 receptors in vivo. Our data demonstrate that the β3 receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond β3 for cellular entry within an organism.

The molecular characteristics of circumsporozoite protein gene subtypes from Plasmodium vivax isolates in Republic of Korea

Abstract. The circumsporozoite protein (CSP) of Plasmodium vivax has two sequence types, VK210 and VK247, each of which has a characteristic tandem amino acid repetition. Previous studies report that a resurgent Korean strain has unique amino acid sequences in tandem repeats and post-repeat areas compared to other strains of P. vivax. We have found another molecular subtype of the CSP gene from resurgent Korea isolates. New strains show a total of eighteen amino acid repeats and different amino acid repeat patterns compared with previous resurgent Korean isolates. In the post-repeat region, AGGNAANKKAEDAGGNA and three repeats of the sequence GGNA was found in all six isolates. These results of sequence comparison revealed that there are two types of isolates based on CSP in South Korea.

Hemorrhagic fever with renal syndrome in 4 US soldiers, South Korea, 2005.

Four US soldiers acquired hemorrhagic fever with renal syndrome while training near the Demilitarized Zone, South Korea, in 2005. Hantaan virus sequences were amplified by reverse transcription–PCR from patient serum samples and from lung tissues of striped field mice (Apodemus agrarius) captured at training sites. Epidemiologic investigations specified the ecology of possible sites of patient infection.

Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro

Hantaviruses are human pathogens that cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. The mechanisms accounting for the differences in virulence between pathogenic and non-pathogenic hantaviruses are not well known. We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. Pathogenic hantaviruses were found to replicate more efficiently in interferon-competent A549 cells than putative non-pathogenic hantaviruses. The former also suppressed the expression of the interferon-β and myxovirus resistance protein genes, while the transcription level of both genes increased rapidly within 24 h post-infection in the latter. In addition, the induction level of interferon correlated with the activation level of interferon regulatory factor-3. Taken together, these results suggest that the observed differences are correlated with viral pathogenesis and further indicate that pathogenic and putative non-pathogenic hantaviruses differ in terms of early interferon induction via activation of the interferon regulatory factor-3 in infected host cells.