Human Cytomegalovirus IE1 Protein Enhances Herpes Simplex Virus Type 1-induced Syncytial Formation in U373MG Cells

Co-infection of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) is not uncommon in immunocompromised hosts. Importantly, organ transplant recipients concurrently infected with HSV-1 and HCMV have a worse clinical outcome than recipients infected with a single virus. However, factors regulating the pathologic response in HSV-1, HCMV co-infected tissues are unclear. We investigated the potential biologic role of HCMV gene product immediate early 1 (IE1) protein in HSV-1-induced syncytial formation in U373MG cells. We utilized a co-infection model by infecting HSV-1 to U373MG cells constitutively expressing HCMV IE1 protein, UMG1-2. Syncytial formation was assessed by enumerating nuclei number per syncytium and number of syncytia. HSV-1-induced syncytial formation was enhanced after 24 hr in UMG1-2 cells compared with U373MG controls. The amplified phenotype in UMG1-2 cells was effectively suppressed by roscovitine in addition to inhibitors of viral replication. This is the first study to provide histological evidence of the contribution of HCMV IE1 protein to enhanced cytopathogenic responses in active HSV-1 infection.

Reduced Th1 Response with Herpes Simplex Virus Type 1 in Behcet's Disease / 대한류마티스학회지

OBJECTIVE: To investigate the interaction of herpes simplex virus type 1 antigen and T cells in Behcet's disease. METHODS: Intracellular interferon-gamma response of T cells was measured before and after stimulation with herpes simplex virus type 1 in 17 patients with Behcet's disease and 20 healthy controls. The proliferation rate and the changes of CD4+/CD8+ T cell subsets were also measured. RESULTS: In the basal status, the proportions of interferon-gamma producing CD4+ or CD8+ T cells were higher in patients with Behcet's disease than healthy controls. The number of interferon-gamma producing CD4+ or CD8+ T cells in patients with Behcet's disease was significantly decreased after stimulation with herpes simplex virus (mean+/-SD, 14.6+/-6.4 % vs 10.0+/-4.7%, p=0.0052 for CD4+ T cells; 29.5+/-14.2% vs 21.2+/-11.8%, p=0.045 for CD8+ T cells), while it did not change in healthy controls (11.5+/-7.4% vs 11.2+/-6.3%, p=0.88 for CD4+ T cells; 19.8+/-15.2 vs 20.7+/-10.8, p=0.84 for CD8+ T cells). There was no difference in the proliferation rate or CD4+/CD8+ subset changes between patients with Behcet's disease and healthy controls. CONCLUSION: The stimulation of peripheral blood mononuclear cells with herpes simplex virus type 1 leads to the reduction of interferon-gamma producing T cells in patients with Behcet's disease. These findings suggest that the stimulation with herpes simplex virus type 1 antigen may reverse the interferon-gamma dominant status of the Behcet's disease.

Phase Variation of Biofilm Formation in Staphylococcus aureus by IS 256 Insertion and Its Impact on the Capacity Adhering to Polyurethane Surface

While ica gene of Staphylococcus epidermidis is known to undergo phase variation by insertion of IS256, the phenomenon in Staphylococcus aureus has not been evaluated. Six biofilm-positive strains were tested for the presence of biofilm-nega-tive phase-variant strains by Congo red agar test. For potential phase-variant strains, pulsed-field gel electrophoresis was done to exclude the possibility of contamination. To investigate the mechanism of the biofilm-negative phase variation, PCR for each ica genes were done. Changes of ica genes detected by PCR were confirmed by southern hybridization, and their nucleotides were analyzed by DNA sequencing. Influence of ica genes and biofilm formation on capacity for adherence to biomedical material was evaluated by comparing the ability of adhering to polyurethane sur-face among a biofilm-negative phase-variant strain and its parent strain. A biofilm-negative phase-variant S. aureus strain was detected from 6 strains tested. icaC gene of the phase-variant strain was found to be inactivated by insertion of additional gene segment, IS256. The biofilm-negative phase-variant strain showed lower adher-ing capacity to polyurethane than its parent strain. This study shows that phase variation of ica gene occurs in S. aureus by insertion of IS256 also, and this biofilm-neg-ative phase variation reduces adhering capacity of the bacteria.

Immune Responses to Viral Infection

Viruses are obligate intracellular parasites which cause infection by invading and replicating within cells. The immune system has mechanisms which can attack the virus in extracellular and intracellular phase of life cycle, and which involve both non-specific and specific effectors. The survival of viruses depends on the survival of their hosts, and therefore the immune system and viruses have evolved together. Immune responses to viral infection may be variable depending on the site of infection, the mechanism of cell-to-cell spread of virus, physiology of the host, host genetic variation, and environmental condition. Viral infection of cells directly stimulates the production of interferons and they induce antiviral state in the surrounding cells. Complement system is also involved in the elimination of viruses and establishes the first line of defence with other non-specific immunity. During the course of viral infection, antibody is most effective at an early stage, especially before the virus enters its target cells. The virus- specific cytotoxic T lymphocytes are the principal effector cells in clearing established viral infections. But many viruses have resistant mechanism to host immune responses in every step of viral infection to cells. Some viruses have immune evasion mechanism and establish latency or persistency indefinitely. Furthermore antibodies to some viruses can enhance the disease by the second infection. Immune responses to viral infection are very different from those to bacterial infection.

Molecular Taxonomic Survey of Mycobacteria Clinical Isolates from Patients in Jeju Island by rpoB Gene Based Molecular Biological Methods

The rpoB gene based sequencing analysis enabled not only the detection of rifampin resistant Mycobacterium tuberculosis, but also the differentiation of species in the genus Mycobacterium. In the present study, we applied the method to 68 isolates of M. tuberculosis (29 from initial treatment cases and 39 from recurrent cases) and 11 clinical isolates of nontuberculous mycobacteia (NTM) isolated from patients in Jeju island. Among rifampin resistant M. tuberculosis, two of 29 strains isolated from patients of initial cases (6.9%) and five of 39 strains isolated from patients of recurrent cases (12.8%) were confirmed to have rifampin resistant genotypes harboring mutations in rif r region of the rpoB gene. In NTM strains, M. fortuitum complex was the most frequently isolated species at the frequency of 54.5% (6/11).

Human Cytomegalovirus Immediate-Early 2Prote in Transactivates c-jun Promoter Through ATF and MEF2 Site / 감염

BACKGROUND: Human cytomegalovirus (HCMV) has the ability to activate the expression of many viral and cellular genes. The c-jun proto-oncogene has known to be induced at immediate early time of HCMV infection, however, the mechanism of up-regulation of the gene was not known. We found HCMV immediate-early (IE) 2 expression transactivate the c-jun promoter in human embryonal lung cell (HEL). METHODS: The c-jun promoter region between -117 and -59 contains binding sites for the transcription factors Sp1, CAAT, AP-1 like (ATF/CREB), and MEF2. We tried to map the sequences in the c-jun promoter responsible for activation of the promoter by HCMV IE2 expression. Transient expression assays were performed using various reporter plasmids containing the c-jun promoter-regulatory region linked to the luciferase gene and a plasmid expressing HCMV IE2 gene. RESULTS: Deletional and point mutational analysis showed that ATF, MEF2, and another down stream elements were involved in the up-regulation of c-jun promoter. Gel mobility shift assay showed that there are several factors in HEL cell nuclear extracts that specifically bind to these sites and in vitro translated IE2 could not move or supershift the specific bands. CONCLUSION: This study delineate the mechanism of c-jun up-regulation in HCMV infection and would give the clue for the possible contribution of HCMV in tumorigenesis.

Differentiation of Mycobacterium avium Complex Isolated in Korea by DT1-BT6 PCR

Recently, selective PCR method using DT1 and DT6 sequences was introduced to identify and differentiate the Mycobacterium avium complex (MAC) into M. intracellulare and M. avium. We applied this method to 49 MAC clinical isolates identified by biochemical tests. They were differentiated into 39 strains of M. intracelluare and 10 strains of M. avium. Compared to those results obtained by 16S rDNA sequencing, DT1-DT6 PCR method showed 100% specificity. While the sensitivity of DT6 PCR for M. avium was 100%, that of DT1 PCR for M. intracellulare was 84.6%. These results show heterogeneity of M intracellulare Korea clinical isolates from Korea. In conclusion, although the in-house DTl-DT6 PCR is an easy and convenient method in differentiating MAC members, other methods such as 16S rDNA sequencing analysis should be performed for the correct identification, especially of M intracellulare.

Identification of Fastidious Mycobacteria other than M. tuberculosis ( MOTT ) by Comparative Sequence Analysis of rpoB and 16S rDNA

Conventional tests for the identification of mycobacteria may frequently result in erroneous identification and underestimate the diversity within the genus Mycobacterium. However, this problem can be overcome by molecular approach like as 16S rRNA gene (rDNA) or RNA polymerase gene (rpoB) sequence analysis. In this study, a molecular approach analyzing partial sequence of 16S rDNA and rpoB gene was applied to mycobacteria other than M tuberculosis (MOTT) isolates that had not been definitely identified by conventional physical and biochemical tests. Among the eighteen isolates included in this study, twelve isolates could be identified to the species level and six were identified to the complex level. Compared with the results by 16S rDNA analysis, the rpoB analysis could di6erentiate some of the strains into the subspecies level.

Effect of Human Cytomegalovirus ( HCMV ) Replication on the Production of Alkaline Phosphatase in Osteosarcoma Cell Line ( Saos - 2 )

HCMV infection can evoke the broad spectrum of symptoms, which may be caused by the infection of responsible cell types. It is important to identify the cell types to be infected and replicated with HCMV infection for characterizing the property of HCMV infection and symptoms. Bone marrow stroma consists of heterogeneous cells, which have many cellular functions. This study was performed to verify the infectivity of HCMV to osteoblasts using the osteogenic sarcoma cell line, Saos-2, and the effect of HCMV infection to them on the cellular function. Immediate-early antigens, IE1 and IE2, were detected from 1 day postinfection (d.p.i.), and early (ppUL44) and late (gB) antigen were detected from 2 d.p.i. by the immunoperoxidase staining. All the antigens were expressed as far as observed (9 days). It was found that the virus titer in the culture supernatant and the cell pellet were 150 to 2,200 pfu/ml and 50 to 800 pfu/ml, respectively, after 4 days when the cells were infected with 2 m.o.i. Alkaline phosphatase production in Saos-2 cells infected with the different amount of HCMV was decreased to 8 to 15%, 31 to 47%, and 11 to 52% on 4, 6, and 11 d.p.i., respectively, as compared with mock-infected cells. This result suggested that HCMV could replicate in some bone marrow stromal cells and disturb the cellular function such as production of alkaline phosphatase.

DNA-mediated Immunization Methods with the HCMV gB for the Induction of Neutralizing Antibodies to HCMV in BALB/c Mice

No abstract available.

Establishment of Measurement of Human Cytomegalovirus with in situ ELISA

No Abstract Available.

Little role of anti-gB antibodies in neutralizing activity of patient's sera with human cytomegalovirus (HCMV) infection

Human cytomegalovirus (HCMV) gB is known to play important roles in cell surface attachment, virion penetration, spread of infection from cell to cell, and provocation of neutralizing antibody. This study was performed to determine the role of anti-HCMV gB antibody in overall neutralizing response in patients with HCMV infection and healthy control with past infection. HCMV gB was stably expressed in 293 cells. With the stable cell line expressing gB as a specific immunosorbent, anti-gB antibody was removed from the current and past HCMV-infected sera and the remaining neutralizing activity was measured by plaque assay. It was shown that 19-50% of the total virus-neutralizing activity of sera with past HCMV infections was derived from anti-gB antibody, but anti-gB antibody had little effect on the total serum virus-neutralizing activity in patients currently infected with HCMV. This result suggests that neutralizing antibody to HCMV gB may reflect disease status.

Gene Cloning of the Epstein-Barr Virus (EBV) Antigen Reactive with the Serum from EBV-infected Patients / 감염

BACKGROUND: Epstein-Barr virus (EBV) is causative agent of infectious mononucleosis and nasopharyngeal carcinoma and associated with Burkitt lymphoma and other tumors. The recombinant protein is needed for the rapid and sensitive serodiagnosis of EBV infection. METHODS: EBV gene encoding the protein reactive with the sera of EBV-infected patient was cloned and characterized with lambda gt11 expression library of cDNA of EBV B95-8 strain. RESULTS: The recombinant proteins from clone 12, 15 and 21 were expressed as 120, 118, 160 kDa-usion protein with beta-galactosidase, respectively, which were reactive with IgG anti-EBV antibody-positive sera, but not with anti-EBV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with EBV B95-8 sequences revealed that those were located at 61716~62087, 61898~62085, and 102128~103158, respectively. These positions correspond to BFRF3, BFRF3, and BZLF1, respectively, which were reported as immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. All the patients' sera were reactive with clone 12 protein, but only 5 out of 9 patients' sera were reactive with clone 21 protein. CONCLUSION: Clone 21 protein expressing BFRF3 fragment was immunoreactive in patient sera from natural EBV infection and was regarded as useful candidate for the serodiagnosis of EBV infection.

Development of the Enzyme Immunoassay for the Detection of Anti-HSV-2 Antibody with HSV-2 Specific Monoclonal Antibody / 감염

BACKGROUND: The serological diagnosis of herpes simplex virus type 2 (HSV-2) infection has pitfalls, in that most of the antibodies against HSV-2 cross-react with HSV-1 and the prevalence of HSV-1 infection is high, especially in Korea. In this study, we tried to establish the serological diagnostic method, which could detect and measure the specific antibodies against HSV- 2 by competitive immunofluorescent staining method as well as competitive ELISA based on the specific monoclonal antibody, MH2-7. METHODS: Immunofluorescent staining and western blot analysis were used to characterize the antigens recognized by MH2-7. Competitive immunofluorescent staining (IF), competitive enzyme immunoassay (ELISA), and western blot analysis were used to detect specific antibodies against HSV-2 in patients' sera. RESULTS: In western blot analysis, the sera from two of six patients clinically diagnosed as genital herpes showed characteristic band patterns, which have been known to be compatible with HSV-2 infection. In competitive immunofluorescent staining, only the sera from the two patients clinically diagnosed as genital herpes and with characteristic band pattern showed competition with MH2-7 monoclonal antibody. The dilution range of the serum showing specific competition was between 1:10 and 1:80. Competitive ELISA was also performed and evaluated as the diagnostic efficacy as ELISA has been known to be advantageous over IF staining in mass screening. The result showed linear dose-response relationship for the patient's sera in inhibition of the reactivity of MH2-7. CONCLUSION: We suggest that the competitive immunofluorescent staining method and competitive ELISA based on the specific monoclonal antibody MH2-7 is a simple, accurate, and precise method, which can be used in serological diagnosis of HSV-2 infection.

The Change of c-jun Promoter Activity in TPA-Induced U937 Cells Infected with Human Cytomegalovirus (HCMV)

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.

The Change of c-jun Promoter Activity in TPA-Induced U937 Cells Infected with Human Cytomegalovirus (HCMV)

Transient transfection assay has been done to evaluate whether the c-jun activation would be prerequisite to the induction of permissiveness against human cytomegalovirus using in vitro cell model in which U937 has been induced to express CD11b and CDl4 to become potential monocyte/macrophage cells by TPA treatment. U937 cells were treated with 10 microM, 50 microM or 100 microM of TPA. The cell morphology change was observed and the expression of the CD11b and CDl4 was confirmed by FACS. Differentiated cells were transfected with pJLuc reporter vector which contained the wild type murine c-jun promoter spanning the SP1, CTF, ATF/CREB and MEF-2 binding sites upstream of the firefly luciferase gene. After 48 hrs of transfection, the cells were infected with HCMV Towne strain and the luciferase activity was assessed at 1 h and 4 h pi. The transfection assay showed no activation of the c-jun promoter at 1 h pi, instead, it showed 2 times increase of the its activity at 4 h pi. There was no difference of the c-jun promoter activation between TPA treated and untreated U937 cells, implying that c-jun activation might not be prerequisite for allowing cells to be premissive to HCMV, although HCMV infection itself could activate c-jun promoter.

Eukaryotic Expression of Human Cytomegalovirus ( HCMV ) Glycoprotein H ( gH )

Human cytomegalovirus (HCMV) glycoprotein H (gH) is one of target molecules in the human immune response to HCMV infection. This study was performed to rneasure the immune responses to HCMV gH in human sera by the expression of HCMV gH in eukaryotic cells. Amplified DNA from the gene encoding gH of HCMV by polymerase chain reaction was cloned into pcDNA3 to construct eukaryotic expression vector. Immunofluorescent staining revealed that the expressed gH in mammalian cells was reactive with the specific monoclonal antibody. Antibody titer in patient's sera with HCMV infection was measured with HCMV-infected fibroblasts and HCMV gH expressed in mammalian cells. Anti-HCMV gH antibody titer was higher in patient group than in healthy control group. There was no correlation between the antibody titer to the whole HCMV and neutralizing antibody titer, and between the antibody titer to whole HCMV and whole gH. Conclusively it is highly recommendable to use the defined antigen such as HCMV gH for the detection of antibody in HCMV-infected persons in the aspect of immunological properties.

The Differential Assessment of Human Cytomegalovirus Infectivity by in Situ Polymerase Chain Reaction

No abstract available.

Mapping of Human Cytomegalovirus IE1 Responsive Elements in the c-jun Promoter

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.

Eukaryotic Kxpression of the Major Antigenic Determinants Evoking Neutralizing Antibodies in Human Cytomegalovirus ( HCMV ) Isolated in Korea

Human cytomegalovirus (HCMV) isolated from Korean patients is different in the antigenic and genomic structure of gB from the laboratory-adapted strain. To dissect the reactivity to HCMV glycoprotein B (gB) domains, each domain gene of gB of HCMV SNUCH1, Korean isolate, was amplified from the extracted DNA of the virus-infected fibroblasts with the specific primers by polymerase chain reaction (PCR). Amplified DNA was cloned into pcDNA3. Immunofluorescent staining and western blot analysis revealed that the expressed gB in mammalian cells was immunoreactive and equivalent to the naturally expressed gB in virus-infected fibroblasts. The antigenic component reactive with monoclonal antibodies, MCMVA 57, 88, and 98 appeared at the D3 domain of gB molecule, and that with MCMVA 66 and 135 at the D2b domain. Antibody titer was measured with HCMV-infected fibroblasts and the domains of gB expressed in mammalian cells. There was no correlation between the antibody titer to the whole HCMV and neutralizing antibody titer, and between the antibody titer to whole HCMV and whole gB. It was more reasonable to use whole gB than whole HCMV in the comparison with the neutralizing antibody titer. D3 was representative domain in gB molecule in the anti-gB reactivity. Conclusively it is highly recommendable to use the representing isolates in Korea and its domains for the detection of antibody or the analysis of antigen in the aspect of immunological properties and molecular structures.