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

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

OBJECTIVETo investigate the effect of HCMV infection on the expression of interleukin-8 (IL-8) and regulated on activation, normal T expressed and secreted (RANTES) so as to explore the possible mechanism of the immune-pathological changes caused by HCMV infection.

METHODSExpression of both IL-8 and RANTES mRNA was detected by RT-PCR. Secretion of IL-8 and RANTES protein was quantitated by enzyme linked immune-sororbant assay (ELISA).

RESULTSAfter HCMV infection, the expression of IL-8 in HEL cells was found to be increased gradually concomitant with the prolonged infection time at both the transcriptional level and the protein level. By the time of 72 h.p.i, as compared with mock-infected cells, IL-8 mRNA expression was increased up to 12-fold and IL-8 protein up to 9-fold in HCMV-infected cells. The expression of RANTES mRNA was occurred at 8 h.p.i, with a maximum at 24 h.p.i, since then it remained relatively high level. The expression of RANTES protein peaked at 24 h.p.i, then dropped sharply and was almost undetectable by the time at 72 h.p.i.

CONCLUSIONHCMV infection of HEL cells may induce the transcription of IL-8 gene as well as the production of IL-8 orotein. HCMV may down-regulate extra-cellular production of RANTES protein.

Cells, Cultured ; Chemokines ; genetics ; immunology ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; genetics ; immunology ; virology ; Female ; Fibroblasts ; immunology ; virology ; Gene Expression ; Humans ; Pilot Projects ; Pregnancy

BACKGROUNDTo study the composition and significance of the inclusion bodies of human cytomegalovirus (HCMV).

METHODSMicrodissection of inclusion bodies, PCR and Southern blot were adopted to detect DNA, and immunohistochemistry method and catalyzed signal amplification (CSA) were used to detect the different antigens of HCMV.

RESULTSThe inclusion bodies of HCMV were separated from the tissue section of human salivary gland. The fragments amplified by PCR from these dissected inclusion bodies were confirmed to be the DNA of HCMV. With the immunohistochemical method CSA, the immediately early and early antigens of HCMV were detected with monoclonal antibodies DDG9/CCH2, while matrix protein AAC10 was negative in the inclusion bodies.

CONCLUSIONThe ingredient of inclusion bodies of HCMV included the DNA and the antigens expressed in specific stage of the virus.

Antigens, Viral ; analysis ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; diagnosis ; immunology ; virology ; DNA, Viral ; analysis ; genetics ; Humans ; Immunohistochemistry ; Inclusion Bodies ; chemistry ; immunology ; virology ; Microdissection ; Salivary Glands ; chemistry ; immunology ; virology

OBJECTIVETo further study the role of human cytomegalovirus (HCMV) infection in the pathogenesis of the type 2 diabetes mellitus.

METHODSHCMV DNA levels in sera from 29 type 2 diabetic patients and 23 controls were measured by quantitative polymerase chain reaction (PCR), and comparative analyses was made between HCMV DNA and T cell subsets, blood glucose (BG), insulin (Ins) and C peptide (C-P).

RESULTSThe levels of HCMV DNA were (1.81+/-1.67) x 10(8) copies/ml for type 2 diabetic patients, a level significantly higher than that (5.50+/-4.30) x 10(7) copies/ml of controls. The percentage of CD8 for type 2 diabetic patients with positive HCMV DNA was 29.53%+/-2.00%, being much higher than that for controls (27.13%+/-4.12%), while the ratio of CD4/CD8 1.24+/-0.05 was significantly lower than that 1.41+/-0.10 of controls. Fasting C-P of type 2 diabetic patients with positive HCMV DNA was far lower than that of those with negative HCMV DNA, but the differences of BG and Ins between the two groups were not significant.

CONCLUSIONActive HCMV infection of type 2 diabetic patients may suppress cellular immunity and its influence on the pathogenesis of the type 2 diabetes mellitus should be further studied.

Adult ; Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; complications ; immunology ; DNA, Viral ; blood ; Diabetes Mellitus, Type 2 ; immunology ; virology ; Female ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; T-Lymphocyte Subsets

OBJECTIVETo provide experimental evidence for development of human cytomegalovirus (HCMV) nucleic acid vaccine, HCMV surface protein (gB), membrane protein (pp150), and gB-pp150 fused gene eukaryotic expression vector were constructed.

METHODSgB and pp150 genes were amplified and fused into gB-pp150, then were cloned into pcDNA 3.1 (+) to obtain recombinant expression plasmids pcDNA 3.1 (+) -gB, pcDNA 3.1 (+) -pp150 and pcDNA 3.1 (+) -gB-pp150, which were encapsulated with chitosan. Mouse were vaccinated and the humoral and cell immune response were determined by ELISA, specific proliferative response of plenic lymphocytes.

RESULTSThe gB, pp150 and gB-pp150 fusion gene eukaryotic expression vector were successfully constructed. The antibodies A value induced by pcDNA3.1(+) -gB or pcDNA3.1 (+) -gB-pp150 were much higher than that of pcDNA3.1 (+) (P < 0.01). The IFN-gamma levels induced by pcDNA3.1 (+) -pp150 and pcDNA3.1 (+) -gB-pp150 were significantly higher than that of pcDNA3.1 (+). There are significant diference between the stimulating indexes of pcDNA3.1(+) -pp150 or pcDNA3.1 (+) -gB-pp150 immunized and normal mice.

CONCLUSIONThe DNA vaccine pcDNA3.1 (+) -gB can induce significant humoral immunity response, and pcDNA3.1 (+) -pp150 can induce high cellular immune response, whereas pcDNA3.1 (+) -gB-pp150 can induce both humoral and cellar immune responses in BALB/c mice.

Animals ; Cytomegalovirus ; genetics ; immunology ; Humans ; Immunity, Cellular ; immunology ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; immunology ; Vaccination ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; immunology

BACKGROUNDRheumatic diseases involve multiple organs that are affected by immunological mechanisms. Treatment with corticosteroids and immunosuppressive agents may also increase the frequency of infection. Cytomegalovirus (CMV) is a widespread herpes virus and a well-recognized pathogen, which causes an opportunistic and potentially fatal infection in immunocompromised patients. This retrospective study aimed to investigate the clinical and laboratory characteristics of CMV pneumonia in patients with rheumatic diseases after immunosuppressive therapy in a single center in Shanghai, China.

METHODSEight hundred and thirty-four patients with rheumatic diseases who had undergone CMV-DNA viral load tests were included, and the medical records of 142 patients who were positive for CMV-DNA in plasma samples were evaluated. GraphPad Prism version 5.013 (San Diego, CA, USA) was used to conduct statistical analysis. The correlation between CMV-DNA viral loads and lymphocyte counts was assessed using the Spearman rank correlation coefficient test. Significance between qualitative data was analyzed using Pearson's Chi-squared test. The cut-off thresholds for CMV-DNA viral load and lymphocyte count were determined by receiver operating characteristic (ROC) curve analysis.

RESULTSOne hundred and forty-two patients had positive CMV viral load tests. Of these 142 patients, 73 patients with CMV pneumonia were regarded as symptomatic, and the other 69 were asymptomatic. The symptomatic group received higher doses of prednisolone (PSL) and more frequently immunosuppressants than the asymptomatic group (P < 0.01). The symptomatic group had lower lymphocyte counts, especially CD4+ T-cells, than the asymptomatic group (P < 0.01). By ROC curve analysis, when CD4+ T-cell count was <0.39 × 109/L, patients with rheumatic diseases were at high risk for symptomatic CMV infection. The CMV-DNA load was significantly higher in the symptomatic patients than that in asymptomatic patients (P < 0.01; threshold viral loads: 1.75 × 104 copies/ml). Seven patients had a fatal outcome, and they had lower peripheral lymphocyte counts (P < 0.01), including CD4+ and CD8+ T-cells (P < 0.01).

CONCLUSIONSWhen CD4+ T-cell count is <0.39 × 109/L, patients are at high risk for pulmonary CMV infection. Patients are prone to be symptomatic with CMV-DNA load >1.75 × 104 copies/ml. Lymphopenia (especially CD4+ T-cells), presence of symptoms, and other infections, especially fungal infection, are significant risk factors for poor outcome, and a higher PSL dosage combined with immunosuppressants may predict CMV pneumonia.

CD4-Positive T-Lymphocytes ; metabolism ; China ; Cytomegalovirus ; pathogenicity ; Cytomegalovirus Infections ; genetics ; immunology ; therapy ; virology ; Humans ; Immunosuppression ; methods ; Pneumonia ; genetics ; immunology ; therapy ; virology ; Polymerase Chain Reaction ; Retrospective Studies ; Rheumatic Diseases ; genetics ; immunology ; therapy ; virology ; Viral Load

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.
Animals ; Antibodies, Viral ; genetics ; immunology ; Cricetinae ; Cytomegalovirus ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Humans ; Immunoglobulin M ; immunology ; Mice ; Protein Sorting Signals ; Recombinant Fusion Proteins ; immunology

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.
Adult ; Antibodies, Monoclonal ; Antibodies, Viral/immunology* ; Antibodies, Viral/blood ; Antigens, Viral/immunology ; Antigens, Viral/genetics ; Cells, Cultured ; Cytomegalovirus/immunology* ; Cytomegalovirus Infections/prevention & control ; Cytomegalovirus Infections/immunology* ; Female ; Fetus/cytology ; Fibroblasts/cytology ; Gene Expression Regulation, Viral/immunology ; Human ; Immunosorbents ; Lung/cytology ; Male ; Middle Age ; Neutralization Tests ; Recombinant Proteins/genetics ; Viral Envelope Proteins/immunology* ; Viral Vaccines

The human cytomegalovirus(HCMV) gene encoding the protein reactive with the sera of HCMV-infected patient was cloned and characterized. A reactive phage clone was screened from a lambda gt11 expression library of cDNA of HCMV AD169 strain using HCMV-infected patient sera. The recombinant protein was expressed as 138 kDa-fusion protein with beta-galactosidase, which was reactive with IgM or IgG HCMV antibody-positive sera, but not with anti-HCMV antibody-negative sera. A homology search of the DNA sequence of the cloned gene with HCMV AD169 sequences revealed that it was composed of 709 base pairs spanning between 0.174 and 0.177 map units of the UL32 region of the HCMV AD169 strain genome. This position corresponded to a part of the gene encoding 150 kDa phosphoprotein-(pp150), a major tegument protein, which was reported as an immunogenic protein which evoked strong and longstanding antibody response and had no sequence homology with the proteins of other herpesviruses. These results suggested that pp150 was an immunogenic protein in natural HCMV infection and therefore this clone was regarded as a useful candidate for developing an antigen for the serodiagnosis of HCMV.
Antibodies, Viral/*blood/immunology ; Antigens, Viral/*genetics/immunology ; Cloning, Molecular ; Cytomegalovirus/genetics/*immunology ; Cytomegalovirus Infections/blood/*immunology/virology ; DNA, Complementary/genetics ; DNA, Viral/genetics ; Gene Library ; *Genes, Structural, Viral ; Human ; Recombinant Fusion Proteins/biosynthesis/immunology ; Sequence Homology, Nucleic Acid ; Support, Non-U.S. Gov't ; Viral Matrix Proteins/*genetics/immunology