Based on the geographic range and distribution of its rodent reservoir host, the European common vole (Microtus arvalis), Tula virus is likely to be widespread throughout Eurasia. Tula virus-infected voles have been captured in Central Russia, Austria, Czech and Slovak Republics, and the former Yugoslavia. Although serologic evidence for Hantaan (HTN) or Seoul (SEO) virus infection can be found in the vast majority of the more than 300 cases of hemorrhagic fever with renal syndrome (HFRS) occurring annually in Korea, approximately 4% of Korean patients with HFRS show a more than 4-fold higher antibody titer to Puumala (PUU) virus than to HTN or SEO virus by double-sandwich IgM ELISA, suggesting the existence of pathogenic Puumala-related hantaviruses in Korea. To further define the geographic distribution and genetic diversity of Tula virus in Eurasia and to investigate the existence of previously unrecognized Microtus-borne hantavirus in Korea, arvicolid rodents were captured in Lodz, Poland in 1995 and in Yunchon-kun, Kyungki-do during April to May, 1998. In addition, sera from 18 Korean HFRS patients who showed higher (or the same) antibody titer to Tula virus than HTN and SEO viruses were examined for hantavirus RNA by RT-PCR. Hantaviral sequences were not detected in any of the 18 patients or in 35 reed voles (Microtus fortis) in Korea. Alignment and comparison of a 208-nucleotide region of the S segment, amplified from lung tissues of two hantavirus-seropositive M. arvalis captured in Poland, revealed 80.8~83.2% sequence similarity, respectively, with Tula virus strains from Central Russia and the Czech and Slovak Republics. Phylogenetic analysis indicated that the newfound Tula virus strains from Poland were closely related to other Tula hantaviruses from Eurasia.
Arvicolinae* ; Austria ; Enzyme-Linked Immunosorbent Assay ; Genetic Variation ; Gyeonggi-do ; Hantavirus* ; Hemorrhagic Fever with Renal Syndrome ; Humans ; Immunoglobulin M ; Korea* ; Lung ; Poland* ; RNA ; Rodentia ; Russia ; Seoul ; Yugoslavia

Human cytomegalovirus (HCMV) has the ability to activate the espremission of many viral and cellular genes. Among various viral proteins, the immediate early proteins (IE1-72kDa, IE2-86kDa) have been known to be potent transactivators. The product of c-jun photo-oncogene is important in cell activation and differentiation. Here, we tried to find out if the IE could activate the c-jun promoter and also tried to identify the responsible sequence elements in the c-jun activation by IE1-72kDa. We found HCMV IE expression transactivated the c-jun promoter in human embryonal lung fibroblasts (HEL). The activation fold by IE1-72kDa, IE2-86kDa and IE2-55kBa was 23, 35, and 5, respectively. When the expression of each IE was combined, it showed synergism. Expression of (IE1-72kDa + IE2-86kBa) and (IE1-72kDa + IE2-86kDa + IE2-55kDa) resulted in 131 and 162 fold increase, respectively. The c-jun promoter region between -117 and -59 contains binding sites for the transcription factors Spl, CAAT, AP-1 like (ATF/CREB), and MEF2. Transient expression assays were performed using various reporter plasmids containing the c-jun promoter-regulatory region linked to the luciferase gene and a plasmic expressing HCMV IE1 gene. Deletional and point mutational analysis showed that the sequence between -225 to -160 and the CTF binding site were involved in the up-regulation of c-jun promoter.
Binding Sites ; Cytomegalovirus* ; Fibroblasts ; Humans* ; Immediate-Early Proteins ; Luciferases ; Lung ; Plasmids ; Promoter Regions, Genetic ; Trans-Activators ; Transcription Factor AP-1 ; Transcription Factors ; Up-Regulation ; Viral Proteins

To determine the sequence and expression of the VP7 gene of Korean isolates (CAU-9), viral RNA was purified and used for cDNA amplification by RT-PCR. The VP7 cDNA was cloned, sequenced, and expressed using baculovirus expression system. The result showed that the sequence homologies CAU-9 compared with foreign isolated strains Wa, 417, TMC-II, 95B and SA11 were ranged from 74.0% to 95.1% of nucleotide sequence and 35% to 43% of amino acid sequence, respectively High homology of CAU-9 was observed in Japanease isolates 417(nucleotide sequence homology was 95.1% and amino acid sequence homology was 43%). To express VP7 gene, the VP7 cDNA was cloned into pCR-Bac vector and inserted into the genome of baculovirus adjacent to the polyhedrin promoter by cotransfection of Spidoptera frugiperda (Sf9) insect cells with wild type baculovirus DNA. In antigenic analysis of Sf9 cells inoculated with the recombinant VP7, immunofluorescence assay revealed positive for viral antigens. In metabolic labeling of Sf9 cell lysates infected with recombinant baculoviruses, it was revealed that the protein of 34 kDa was expressed. The limited study of expressed VP7 protein inoculated with guinea pigs failed to elicit neutalizing antibody. As a results, the sequence analysis and expression of VP7 protein of rotavirus CAU-9 isolated from an infant in Korea could permit the conformation and development of virus like particles which may be useful in designing vaccine strategy.
Amino Acid Sequence ; Animals ; Antigens, Viral ; Baculoviridae ; Base Sequence ; Clone Cells ; DNA ; DNA, Complementary ; Fluorescent Antibody Technique ; Genome ; Guinea Pigs ; Humans ; Infant* ; Insects ; Korea ; RNA, Viral ; Rotavirus* ; Sequence Analysis* ; Sequence Homology ; Sequence Homology, Amino Acid ; Sf9 Cells

The gene encoding F protein of CBP-1 strain, a heat-stable Newcastle disease virus (NDV) isolated from the diseased pheasants in Korea, was characterized by reverse transcription-polymerase chain reaction (RT-PCR), nucleotide and amino acid sequences. Virus RNA was prepared from the chorioallatoic fluid infected with NDV CBP-1 virus and cDNA was amplified by RT-PCR, cloned and sequenced to analyze. The PCR was sensitive as to detect the virus titer above 25 hemagglutination unit. 1.7kb (1,707bp) size of the cDNA was amplified and cloned into BamHI site of pVL1393 Baculo transfer vector. The nucleotide sequences for F protein were determined by dye terminator cyclic sequencing using four pairs of primers, and 553 amino acid sequences were predicted. In comparison of the nucleotide sequence of F gene of CBP-1 with those of other NDV strains, the homology revealed 88.8%, 98.5% and 98.7% with Kyojungwon (KJW), Texas GB and Beaudette C strains, respectively. As the deduced 553 amino acid sequences of F protein of CBP-1 were compared with those of other NDV strains, the homology appeared 89.9%, 98.7% and 98.9% with KJW, Texas GB and Beaudette C strains, respectively. The putative protease cleavage site (112-116) was R-R-Q-K-R, indicating that CBP-1 strain is velogenic type. The amino acid sequences include 6 sites of N-asparagine-linked glycosylation and 13 cysteine residues. These data indicate that the genotype of CBP-1 strain is more closely associated with the strains of Texas GB and Beaudette C than KJW strain.
Amino Acid Sequence ; Animals ; Base Sequence* ; Clone Cells ; Cloning, Molecular* ; Cysteine ; DNA, Complementary ; Genotype ; Glycosylation ; Hemagglutination ; Korea ; Newcastle disease virus* ; Newcastle Disease* ; Polymerase Chain Reaction ; RNA ; Texas ; Viral Load

We have expressed GFP in Sf9 and Bm5 cells or Bombyx by larvae by using Ac-Bm hybrid virus capable of replicating in both Bm5 and Sf9 cells. Genomic DNA of Ac-Bm hybrid virus expressing P-galactosidase was cotransfected with baculovirus transfer vector containing GFP gene, pBacPAK-GFP in Sf9 cells. The Ac-Bm hybrid virus harboring GFP was named as Ac-Bm hybrid virus-GFP. The Ac-Bm hybrid virus-GFP-infected insect cells were easily selected by detecting the emission of GFP from each well of cell culture dish on the UV illuminator. GFP produced by Ac-Bm hybrid virus-GFP in Sf9 and Bm5 cells or B. mori larvae was confirmed by SDS-PAGE and Western blot analysis using GFP antibody. In addition, B. mori larvae infected with Ac-Bm hybrid virus-GFP was apparently appeared fluorescence from the whole body at 5 days postinoculation. The fluorescence of GFP from the hemolymph and fat body of B. mori larvae infected with Ac-Bm hybrid virus-GFP was also observed by fluorescence microscope. In conclusion, our results demonstrated that in baculovirus expression vector system, use of Ac-Bm hybrid virus have an additional advantage of expanded host range for producing recombinant proteins.
Animals ; Baculoviridae ; Blotting, Western ; Bombyx* ; Cell Culture Techniques ; DNA ; Electrophoresis, Polyacrylamide Gel ; Fat Body ; Fluorescence ; Hemolymph ; Host Specificity ; Insects ; Larva* ; Recombinant Proteins ; Sf9 Cells ; Spodoptera*

Cloning, sequencing and expressing in E. coli of the thymidine kinase (TK) gene of Herpes simplex virus type-1 (HSV-1) strain F was investigated. The TK gene, located in the BamHI 3.74 kb DNA flagment of the plasmid PHLA-12, was amplified by polymerase chain reaction (PCR). The 1,131 kb PCR product was cloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The TK gene was subcloned into the BamHI and BglII sites of pQE-30, and named pQE-TK recombinant. The nucleotide sequence of the 1,131 kb TK gene was determined, and the GC content was 65.13%. There were deduced 367 amino acid residues with a total molecular weight of 43 kDa. The weight was confirmed by the protein produced by E. coli M15/pQE-TK on the SDS-PAGE and Western blot. The production of the TK protein in the IPTG induced cells was measured over 4 h. At the end of 1, 2 and 3 h the level increased by 146,204 and 242%, respectively. The amount of the protein at the highest fraction Purified with Ni-NTA resin chromatography was 0.68 ug Per ml. The soluble state TK protein was present in the cytoplasm. In these results the F strain was different in base sequence and amino acid sequence from that of the CL101 strain, which caused difference in their strains.
Amino Acid Sequence ; Base Composition ; Base Sequence ; Blotting, Western ; Chromatography ; Clone Cells* ; Cloning, Organism* ; Cytoplasm ; DNA ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli* ; Escherichia* ; Herpesvirus 1, Human ; Isopropyl Thiogalactoside ; Molecular Weight ; Plasmids ; Polymerase Chain Reaction ; Simplexvirus ; Thymidine Kinase* ; Thymidine*

HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of P53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3Tl cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein .interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.
Animals ; Cats ; Genes, Tumor Suppressor ; HIV-1* ; Humans ; Jurkat Cells ; Phosphorylation ; Plasmids ; Protein Kinases ; RNA Interference ; Trans-Activators ; Transcriptional Activation ; Yeasts

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli cordon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.
Amino Acid Sequence ; Antibodies ; Base Sequence ; Blotting, Western ; Chromatography, Gel ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Glycoproteins ; HIV* ; HIV-1* ; Humans* ; Inclusion Bodies ; Korea ; O Antigens

Synthetic genes encoding the gag p24 and the part of the envelope protein gp41 of the human immunodeficiency virus (HIV-1) were cloned and overexpressed as fusion proteins in Escherichia coli, using an expression vector carrying 77 promoter and the poly-histidine leader sequence. The overexpressed p24 fusion protein was purified by centrifugation, Ni-affinity chromatography and CM-sepharose chromatography The overexpressed gp41 fusion protein was purified by centrifugation, C4 chromatography and DEAE-sepharose chromatography. The purified fusion proteins showed a high level of purity and immunoreactivity in SDS-polyacrylamide gel electrophoresis and western blot analysis. These results suggest that this prokaryotic expression-purification method is suitable for obtaining a large amount of the viral antigen which may be useful for screening of antibodies to HIV-1 in human blood samples.
Antibodies ; Blotting, Western ; Centrifugation ; Chromatography ; Clone Cells ; Electrophoresis ; Escherichia coli ; Genes, Synthetic ; HIV* ; HIV-1* ; Humans* ; Mass Screening

Twenty isolates of Hantavirus were isolated from patients and reserovirs from 1988 to 1994 in Korea. Isolation rate was 1.9% (10/538) in patients, 6.2% (5/81) in Apodemus sp., 2.6% (1/38) in Rattus sp. and 0.6% (4/677) in bats. Reciprocal mean IFA titers ranged from 27.5 to 1,024 at the specimen collection. According to the growth rate and reaching peak titer of infectivity, the isolates were grouped as rapid, intermediate, and slow growing groups. All isolates were confirmed as Hantaan type by the nested RT-PCR on the Gl region of the M segment. Comparison of nucleotide sequence (Nt: 2101 - Nt: 2280) of the G2 region revealed that the sequence homology between Hantaan 76/118 virus and the isolates was more than 90%. Several nucleotide positions of the isolates showed high variation. The variation rate of patientisolates was about one-half when compared with that of rodentisolates. On the basis of phylogenetic analysis Hantaan viruses isolated were divided into two genogroups. These results indicate that Hantaan virus is highly dominant serotype in Korea and the virologic property and genogroup are not correlated.
Animals ; Base Sequence ; Chiroptera ; Genotype ; Hantaan virus* ; Hantavirus ; Humans ; Korea* ; Murinae ; Rats ; Sequence Homology ; Specimen Handling