OBJECTIVETo observe the effect of human cytomegalovirus infection on the host cellular DNA synthesis and expression of cyclones.

METHODSHCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres. The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell. We harvested infected cell at different time 0 h, 3 h, 6 h, 24 h, 72 h and 96 h post infection. Then the cell cycle progress was measured. Meanwhile, the DNA content and expression of proteins of cycline E, cycline A and cycline D1 were determined with FCM and Western Blot respectively.

RESULTSWe found that the amount of S stage cell infected by HCMV had increased dramatically, and that of G2/M stage cell reduced during 24 h-96 h PI, and no G2/M stage cell was detected within 96 h PI. The content of 2N DNA maintained unchangeable for 24 h after infection and the content of total DNA in infected cells began to increase within 48 h PI, and the substantial cell with 2N DNA were observed 72 h after infection. However, DNA content was not altered in control group of normal HEL and HCMV PAA group. CyclinE protein was induced 12 h PI and peak induction occurred 24 h PI in contact-inhibited cells. CyclinA protein expression was not induced in HCMV infected density-arrested cells. The abundance of cyclinD1 decreased 24 h PI.

CONCLUSIONThe expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV, which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage. HCMV can not activate cellular DNA synthesis, and increase of total DNA content in infected cells result from the viral DNA replication.

Cell Cycle ; Cells, Cultured ; Cyclins ; genetics ; metabolism ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; genetics ; metabolism ; Gene Expression ; Humans

OBJECTIVE: To determine whether U(251) cells are permissive for human cytomegalovirus (HCMV), and to investigate the characteristics of temporal expression of proteins IE1 and pp65. METHODS: U(251) cells were infected with HCMV, and then the cells were observed under the transmission electronic microscope, and the viral nucleic acid was detected by PCR, and the expression levels of IE1 and pp65 were analyzed by immunohistochemical assay with anti-IE1 monoclonal antibody and anti-pp65 monoclonal antibody at various time spost infection. RESULTS: Morphological changes of the infected cells appeared under the transmission electron microscope. The viral nucleic acid was detected successfully by PCR. The expression of IE1 was detected firstly at 4h post infection, and reached a peak within 14h, and then decreased. The incoming pp65 was detected at 1h, the low expression levels of pp65 were detected firstly at 4h, and they could remain relatively constant through 96 h, but the maximum expression occurred at 120 h. CONCLUSION Human glioma U(251) cells are permissive for HCMV, the temporal cascade of HCMV gene expression can be observed in the infected U(251) cells, but it is delayed obviously in the human fibroblast.
Cell Line, Tumor ; Cytomegalovirus ; Cytomegalovirus Infections ; Glioma ; metabolism ; virology ; Humans ; Immediate-Early Proteins ; metabolism ; Phosphoproteins ; metabolism ; Viral Matrix Proteins ; metabolism

The human cytomegalovirus (HCMV) is a widespread herpesvirus. Virus reactivation from latency can lead to stillbirth, miscarriage, fetal anomalies, and intrauterine growth retardation. During latent infection with the HCMV, the virus can be cleared by the immune response or apoptosis of host cells. However, the HCMV has developed several strategies to manipulate expression of its genes and the microenvironment of host cells. Recent studies have shown that latent infection with the HCMV is associated with viral: regulation of early expression of genes; evasion of cell death; evasion of the immune response; regulatin of non-coding RNAs. This review summarizes recent research progress on the mechanisms underpinning latent infection with the HCMV.
Animals ; Cytomegalovirus ; genetics ; physiology ; Cytomegalovirus Infections ; immunology ; virology ; Humans ; Viral Proteins ; genetics ; metabolism ; Virus Latency

Tyrosinemia type 1 is an autosomal recessive inborn error of tyrosine metabolism that caused a mutation in the gene coding for the enzyme fumarylacetoacetate hydrolase(FAH). As a result, maleylacetoacetate(MAA) and fumarylacetoacetate(FAA) are formed. The accumulated FAA is converted into succinylacetone(SA) and succinylacetoacetate(SAA) which are excreted in urine. The first report with typical clinical and biochemical findings was presented by Sakai in 1957. Clinically, the disorder is characterized by progressive liver damage with liver failure, a high risk of hepatocellular carcinoma and renal tubular dysfunction hypophosphataemic rickets. Some patients have porphyria-like episodes. Liver transplantation has been the ultimate treatment of tyrosinemia. However pharmacological therapy with 2-(2-nitro-4-trifluoromethylbenzoyl) -1,3-cyclohexanedione(NTBC) has offered a new therapeutic option in addition to dietary restriction of tyrosine and phenylalanine. We experienced a case of tyrosinemia type 1 with cytomegalovirus infection in a 4-month-old male who improved by dietary restriction of tyrosine and phenylalanine.
Carcinoma, Hepatocellular ; Clinical Coding ; Cytomegalovirus Infections* ; Cytomegalovirus* ; Humans ; Infant ; Liver ; Liver Failure ; Liver Transplantation ; Male ; Metabolism ; Phenylalanine ; Rickets ; Tyrosine ; Tyrosinemias*

The objective of this study was to investigate the effect of cytomegalovirus (CMV) infection on actin and microfilament in human embryo fibroblast cells (HF) and to explore the possible relationship with CMV replication. The cell shape was observed by microscopy after the infection of CMV, RT-PCR assay was used to detect the mRNA expression of beta-actin gene, while Westen-blot was used to measure the level of beta-actin protein. CMV immediately early antigen (IE) in HF cells was analyzed by indirect immunofluorescence assay. Microfilament alteration was determined by cytoskeleton fluorescence probe. The results showed that CMV IE was observed in more than 95% of HF cells after infection, primarily located in nucleus. HF cells infected by CMV changed from thin shuttle shape to round and thick ball shape, even detached from wall. Beta-actin got a significant and gradual decreasing of mRNA level in time-dependent manner (P < 0.05). Compared with uninfected group, the expression of beta-actin protein decreased to (74.2 +/- 13.4)% at 96 hours after infection (P < 0.05). In infected HF cells, microfilaments were ruptured, arranged turbulently, as well as cells merged and fluorescence density of microfilament obviously reduced. It is concluded that cytomegalovirus can induce alteration of actin and microfilament, which may be helpful for CMV to infect, replicate and reactivate in host cells.
Actin Cytoskeleton ; metabolism ; ultrastructure ; Actins ; metabolism ; Cell Line ; Cytomegalovirus Infections ; metabolism ; pathology ; Fibroblasts ; pathology ; ultrastructure ; virology ; Humans

BACKGROUNDCloning recombinant human Fab fragment against HCMV for the purpose of prophylaxis and control of HCMV infection.

METHODSThe authors constructed a HCMV phage display library with 2 x 10(6) clones, then used purified HCMV viral lysates to pan the library, then screened by ELISA.

RESULTSThree clones showed positive responses in ELISA, they also showed high specificity in IFA, two of them could neutralize HCMV in neutralizing assays.

CONCLUSIONThe specific binding of Fab antibodies to HCMV was demonstrated by ELISA, IFA and neutralizing activities. These results provide us the basis for further research of neutralizing recombinant human whole IgG molecule.

Antibodies, Viral ; genetics ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; immunology ; virology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library

Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10(6) IU/mL (R2 ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values <1,000 copies/mL, 100% of the samples had a variation <0.7 log10 copies/mL; >1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had <0.5 log10 copies/mL. The Real-Q assay is useful for quantifying CMV in WB with the two real-time PCR platforms.
Cytomegalovirus/*genetics/isolation & purification ; Cytomegalovirus Infections/diagnosis/virology ; DNA, Viral/*blood/metabolism ; Humans ; Limit of Detection ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction

Objective: To investigate the relationship between human cytomegalovirus (HCMV) infection and peripheral blood CD14 CD16 monocytes in the pathogenesis of coronary heart disease (CHD), and to elucidate the mechanism of pathogenesis in CHD by analyzing the correlation between infection, inflammation, and CHD, to provide a basis for the prevention, evaluation, and treatment of the disease. Methods: In total, 192 patients with CHD were divided into three groups: latent CHD, angina pectoris, and myocardial infarction. HCMV-IgM and -IgG antibodies were assessed using ELISA; CD14 CD16 monocytes were counted using a five-type automated hematology analyzer; mononuclear cells were assessed using fluorescence-activated cell sorting; and an automatic biochemical analyzer was used to measure the levels of triglyceride, cholesterol, high- and low-density lipoprotein cholesterols, lipoprotein, hs-CRp and Hcy. Results: The positive rates of HCMV-IgM and -IgG were significantly higher in the CHD groups than in the control group. HCMV infection affects lipid metabolism to promote immune and inflammatory responses. Conclusion HCMV infection has a specific correlation with the occurrence and development of CHD. The expression of CD14 CD16 mononuclear cells in the CHD group was increased accordingly and correlated with acute HCMV infection. Thus, HCMV antibody as well as peripheral blood CD14 CD16 mononuclear cells can be used to monitor the occurrence and development of CHD.
Angina Pectoris ; epidemiology ; virology ; China ; epidemiology ; Coronary Disease ; epidemiology ; virology ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; complications ; Humans ; Incidence ; Inflammation ; epidemiology ; etiology ; Leukocyte Count ; Monocytes ; metabolism ; Myocardial Infarction ; epidemiology ; virology

OBJECTIVETo study the changes of p38MAPK expressions, the frequency of apoptosis and the distribution of cell cycle of hunan Glioma U251 cells after HCMV infection.

METHODSThe expression of total p38 (both phosphorylated and nonphosphorylated p38) and phosphorylated p38 in U251 cells were detected by Western blotting at 15 min, 30 min, 1 h, 6 h, 10 h, 16 h, 24 h, 36 h and 48 h after HCMV infection. The apoptosis percentage and the cell cycle distribution of U251 cells at 2 d, 5 d and 7 d after HCMV infection were detected by flow cytometry (FCM).

RESULTSThe results of Western blotting demonstrated that a strong increase in phosphorylated p38 was detected from 6 h to 10 h after HCMV infection, with mean gray scales 186.33 +/- 7.51 (t = 5.37, P < 0.01) and 188.00 +/- 7.02 (t = 5.26, P < 0.01 for all) at 6 h and 10 h, respectively, and p38 phosphorylation decreased to the basic level at 16 h after HCMV infection. But the overall levels of p38 protein were not significantly altered during the course of infection. FCM analysis showed that HCMV could significantly increase the apoptotic rates of U251 cells compared with controls (t = 10.84, P < 0.01), and the apoptotic percentages of the cells reached to peak [(10.18 +/- 1.24)%] at 5 d after HCMV infection. The data of FCM showed that HCMV could decrease the number of U251 cells in G1 phase and arrest the cells in S and G2 phase. The numbers of G1 phase U251 cells were significantly lowered to (56.50 +/- 2.57)% (t = 26.45, P < 0.01), (62.33 +/- 2.64)% (t = 21.20, P < 0.01) and (67.45 +/- 4.44)% (t = 10.61, P < 0.01), respectively at 2 d, 5 d and 7 d after infection.

CONCLUSIONHCMV could activate p38MAPK pathway and trigger apoptosis and interfere cell cycle in U251 cells.

Apoptosis ; Blotting, Western ; Cell Cycle ; Cell Line, Tumor ; Cytomegalovirus ; isolation & purification ; Cytomegalovirus Infections ; metabolism ; physiopathology ; Flow Cytometry ; Glioma ; metabolism ; microbiology ; pathology ; Humans ; MAP Kinase Signaling System ; Phosphorylation ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.
Antiviral Agents/therapeutic use ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/drug therapy/pathology/virology ; DNA, Viral/*blood/metabolism ; Ganciclovir/therapeutic use ; Humans ; *Immunoassay ; Organ Transplantation ; Phosphoproteins/genetics/immunology/*metabolism ; *Real-Time Polymerase Chain Reaction ; Viral Matrix Proteins/genetics/immunology/*metabolism ; Virology/*methods