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

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

This study aimed to investigate the effect of curcumin (Cur) against human cytomegalovirus (HCMV) in vitro. Human embryonic lung fibroblasts were cultured in vitro. The tetrazolium salt (MTS) method was used to detect the effects of Cur on cell viability. The cells were divided into control group, HCMV group, HCMV + (PFA) group and HCMV + Cur group in this study. The cytopathic effect (CPE) of each group was observed by plaque test, then the copy number of HCMV DNA in each group was detected by quantitative polymerase chain reaction (qPCR), and the expression of HCMV proteins in different sequence was detected by Western blot. The results showed that when the concentration of Cur was not higher than 15 μmol/L, there was no significant change in cell growth and viability in the Cur group compared with the control group (P>0.05). After the cells were infected by HCMV for 5 d, the cells began to show CPE, and the number of plaques increased with time. Pretreatment with Cur significantly reduced CPE in a dose-dependent manner. After the cells were infected by HCMV, the DNA copy number and protein expression gradually increased in a time-dependent manner. Pretreatment with Cur significantly inhibited HCMV DNA copies and downregulate HCMV protein expression levels in a concentration-dependent manner, and the difference was statistically significant (P<0.05). In conclusion, Cur may exert anti-HCMV activity by inhibiting the replication of HCMV DNA and down-regulating the expression levels of different sequence proteins of HCMV. This study provides a new experimental basis for the development of anti-HCMV infectious drugs.
Humans ; Curcumin/therapeutic use* ; Cytomegalovirus/genetics* ; Cytomegalovirus Infections/drug therapy* ; Plaque, Atherosclerotic

The objective of this study was to observe the expression of hoxc4 and hoxc6 genes in the process of differentiation of hematopoietic stem cell (HSC) to colony forming unit-T Lymphocyte (CFU-TL) in vitro. and to explore the possible mechanism of HCMV-induced maldevelopment of human cord blood CFU-TL on genetic level through effecting the differentiation progress by human cytomegalovirus (HCMV) with and/or all-trans retinoic acid (ATRA), Normal CFU-TL culture was used as blank control. After detection with MTT, mRNA expression levels in the human cord blood CFU-TL hoxc4 and hoxc6 genes following HCMV infection and ATRA treatment were detected by fluorogenic quantitative reserve transcription polymerize chain reaction (FQ-RT-PCR) method. HCMV of 10(6) plaque formation unit (PFU)/ml was diluted to 0.1 ml 10(5) PFU/ml and added into the infected group. The results showed that the expression of hoxc4 and hoxc6 genes in the differentiation process increased slightly on day 3, and were up to the most on day 7 (p < 0.05), while became lower on day 12 respectively in normal group, HCMV group and ATRA group. Compared with the expression of hoxc6, the expression of hoxc4 was obviously higher in each group (p < 0.05). Compared with the expression of hoxc4 and hoxc6 genes in normal group, the expressions of hoxc4 and hoxc6 in ATRA group were up-regulated remarkably (p < 0.05), while the expressions of hoxc4 and hoxc6 in group HCMV were down-regulated (p < 0.05). It is concluded that the regular expression of hoxc4 and hoxc6 genes mRNA appeared in each group. A positive co-relationship exits between hoxc4/hoxc6 genes and lymphocytic progenitor hematopoiesis. Compared with the expression of hoxc6 gene, the expression of hoxc4 gene is obviously higher in each group. HCMV can down-regulate the expression of hoxc4 and hoxc6 genes and lead to suppression effect on cell morphology, which confirms that the normal hematopoietic lineage determination and maturation rely on the stable and consistent expression of homeobox gene. At the same condition, ATRA (6 x 10(-8) mol/L at 60 nmol/ml) can up-regulate hoxc4 and hoxc6 genes expression. ATRA can up-regulate the expression of hoxc4 and hoxc6 genes.
Cell Line ; Cell Proliferation ; Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; genetics ; Homeodomain Proteins ; genetics ; Humans ; Lymphoid Progenitor Cells ; cytology ; Tretinoin ; pharmacology

OBJECTIVEHuman cytomegalovirus (HCMV) displays genetic polymorphisms. Nineteen open reading frames (ORFs, UL133-UL151) found in the Toledo strain of HCMV and other low-passage clinical isolates may be essential for viral infection. This study aimed to analyze the polymorphism of HCMV UL134 gene in clinical isolates and explore the relationship between the polymorphism and HCMV infection.

METHODSPCR was performed to amplify entire UL134 region in 32 clinical isolates, which had been proven as HCMV-DNA positive by FQ-PCR. PCR products were sequenced.

RESULTSAll of the 32 isolates were amplified and sequenced successfully. HCMV UL134 gene was highly conserved in the clinical isolates. UL134 ORF and its predicted protein in the clinical strains displayed 96.4%-98.3% nucleotide identity and 92.7%-94.9% amino acid identity respectively compared to those in the Toledo strain. A new posttranslational modification site, sulfationcamp (SUL) site, was found in UL134 protein of all of the clinical isolates except 35j.

CONCLUSIONSHCMV UL134 gene in clinical isolates was highly conserved. No substantial relation was found between UL134 gene and HCMV infectious diseases.

Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; virology ; Genes, Viral ; Humans ; Open Reading Frames ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Viral Proteins ; genetics

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

OBJECTIVE: To investigate the consistency of cytomegalovirus deoxyribo nucleic acid (CMV-DNA) and immunoglobulin M (IgM) antibody detections in patients with different clinical characteristics and their guiding value for clinical practice. METHODS: From December 2014 to November 2019, a total of 507 patients who were detected with both CMV-IgM and CMV-DNA were collected in Peking University International Hospital. Their general information, such as gender, age and clinical data, including the patient's diagnosis, medication, and outcome were also collected. The groups were stratified according to whether CMV-DNA was negative or positive, CMV-IgM was negative or positive, age, gender, and whether they received immunosuppressive therapy or not. The Pearson Chi-square test or Fisher's exact test was used for comparison of the rates between the groups. P < 0.05 means the difference is statisti-cally significant. RESULTS: Of the 507 patients submitted for examination, 55 (10.85%) were positive for CMV-DNA, 74 (14.60%) were positive for CMV-IgM, and 20 (3.94%) were positive for both CMV-DNA and CMV-IgM. Of the 55 patients with CMV-DNA positive, 37 were male, accounting for 67.27%. In addition, 25 patients were older than 60 years, accounting for 45.45% and 33 patients received immunosuppressive therapy, accounting for 60%. The rates were higher than that of CMV-DNA negative group, 47.35% (P=0.005), 68.14% (P=0.043), 46.02% (P=0.050), respectively. Of the patients with both CMV-DNA and IgM positive, 45% received immunosuppressive threapy, which was lower than that of CMV-DNA positive but IgM negative patients (68.57%, P=0.086), and also lower than CMV-DNA negative but IgM positive patients (68.52%, P=0.064). In the patients with both CMV-DNA and IgM positive, 91.67% showed remission after receiving ganciclovir, whereas in the patients with CMV-DNA positive but IgM negative, the rate was only 60% (P=0.067). CONCLUSION CMV-IgM antibody detection is affected by age, gender, and immune status. It is not recommended to use CMV-IgM alone to determine CMV infection in patients with immunosuppressive status and those older than 60 years. CMV-DNA and CMV-IgM combined detection may help to predict patients' immune status and outcomes of antiviral therapy.
Antibodies, Viral ; Cytomegalovirus/genetics* ; Cytomegalovirus Infections/drug therapy* ; DNA ; Female ; Humans ; Immunoglobulin M ; Immunosuppressive Agents/therapeutic use* ; Male ; Nucleic Acids

OBJECTIVETo investigate the variability of human cytomegalovirus (HCMV) UL138 open reading frame (ORF) in clinical strains.

METHODSHCMV UL138 ORF was amplified by polymerase chain reaction (PCR) and PCR amplification products were sequenced directly, and the data were analyzed in 19 clinical strains.

RESULTSUL138 ORF in all 30 clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identities of UL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%, respectively. All of the nucleotide mutations were substitutions. The spatial structure and post-translational modification sites of UL138 encoded proteins were conserved. The result of phylogenetic tree showed that HCMV UL138 sequence variations were not definitely related with different clinical symptoms.

CONCLUSIONHCMV UL138 ORF in clinical strains is high conservation, which might be helpful for UL138 encoded protein to play a role in latent infection of HCMV.

Amino Acid Sequence ; Cytomegalovirus ; classification ; genetics ; Cytomegalovirus Infections ; congenital ; genetics ; Humans ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; Protein Structure, Secondary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics

Cytomegalovirus ; genetics ; Cytomegalovirus Infections ; virology ; DNA, Viral ; genetics ; Female ; Genotype ; Hepatitis, Viral, Human ; virology ; Humans ; Infant ; Infant, Newborn ; Male ; Polymorphism, Restriction Fragment Length ; Viral Envelope Proteins ; genetics

It has been reported that baculoviruses could serve as a new gene-transfer vehicle for mammalian cells. We have previously constructed recombinant baculovirus BacV-CMV-EGFPA and have proven that mammalian cells could be effectively infected by the recombinant baculovirus. In this report, we studied the efficiency of baculovirus to deliver exogenous gene into twenty mammalian cells, including twelve human cell lines (WI-38, Hela, HepG2, 293, PLC/PRF/5, 143B, MCF-7, BGC-223, DMS 114, CNE, Raji, LCL-cm), seven murine cell lines (BNL 1ME A.7R.1, CHO-K1, L-929, JC, PT67, NIH3T3, P815) and one monkey cell line (CV1). Results showed that most mammalian cell lines could be transduced by the recombinant baculovirus, the transduction efficiencies of the human and monkey cell lines were markedly higher than that of murine cell lines, and the transduction efficiencies in adherent culture cell lines higher than that of suspend culture cell lines, implying that the infection efficiency of the baculovirus may be correlative with the organism used and the growth properties of the cell lines. The plasmid pcDNA3. 1-EGFP, which contains the CMV promoter and EGFP reporter gene, was next transfected by LipofectAMINE into a number of mammalian cells, especially those cells that were low in the baculovirus transfection. Results showed that the CMV promoter could effectively direct the expression of the reporter gene in these mammalian cells. Therefore the gene-expression efficiencies in different mammalian cell lines by the recombinant baculovirus which contains the same CMV promoter were dictated by the ability of the baculovirus to enter the cell lines. This study suggested that the recombinant baculovirus vector is more suitable for gene expression in primate adherent culture cells than in murine cells and suspend culture cells.
Animals ; Baculoviridae ; genetics ; Cytomegalovirus ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Promoter Regions, Genetic ; Spodoptera