Summary: This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expre...Summary: This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCIDs0was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by vi- rus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in in- fected HPT-8 cells was decreased (P〈0.05), and the levels of VIP mRNA and protein, and the concen- tration were raised to the normal (P〉0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.展开更多
Objective To investigate the variability of human cytomegalovirus (HCMV) UL138 open reading frame (ORF) in clinical strains. Methods HCMV UL138 ORF was amplified by polymerase chain reaction (PCR) and PCR amplif...Objective To investigate the variability of human cytomegalovirus (HCMV) UL138 open reading frame (ORF) in clinical strains. Methods HCMV 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. Results LIL138 ORF in all 30 clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identifies of LIL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%, respectively. All of the nucleofide mutations were substitutions. The spatial structure and post-translational modification sites of HL138 encoded proteins were conserved. The result of phylogenetic tree showed that HCMV HL138 sequence variations were not definitely related with different clinical symptoms. Conclusion HCMV 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.展开更多
Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isola...Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.展开更多
Objective To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). Methods U251 cells were cultured in RPMI 1640 culture medium and ...Objective To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P〈0.05). Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.展开更多
HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infec...HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.展开更多
The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion ...The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 (CK7), vimentin (Vim) and cerbB-2 were immunocytochemically detected to identify source of cells, and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining. The EVTs were divided into two groups: control group and HCMV group, and the expression of c-erbB-2, matrix metalloproteinase-2 (MMP-2) and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting. Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV. The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV. The cell source identification showed that the cells obtained were highly-pure primary EVTs, and primary EVTs could be infected by HCMV. Primary EVTs could express c-erbB-2, MMP-2 and MMP-9 proteins, and as compared with control group, the protein expression was decreased significantly in HCMV groups (P〈0.05). Primary EVTs could secrete active MMP-2 and MMP-9 in vitro, and the activity of two MMPs was decreased sig- nificantly in HCMV groups (P〈0.05). The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased (P〈0.05). We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability, suggesting that the im- paired EVT's invasion capability might be related to the abnormal expression of c-erbB-2, MMP-2 and MMP-9 proteins.展开更多
Summary: The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus (HCMV) infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosi...Summary: The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus (HCMV) infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosis-regulating genes, bcl-2 and fas mRNA. The sequential changes of apoptotic cell rate in high and low MOI (MOI=2.5 and 0.25 respectively) of HCMV infected human embryonic lung fibroblasts (HELFs) at 1 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h post-infection were measured by flow cytometry. The expression levels of caspase-3 protein and bcl-2 and fas mRNA in HCMV infected cells (MOI=0.25) at 72 h post-infection were detected by Western blot and in situ hybridization methods, respectively. It was found that the ratio of apoptotic cells in normal controls was consistently lower, but the rates in low and high MOI infected cells were gradually increased with time prolonged, reached peak at 96 h (8.85 %) and 72 h (25.63 %), respectively. By Western blot analysis, only a narrow band of 32 kD (1 kD=0. 992 1 ku) procaspase-3 was found in normal cells, but a wider procaspase-3 band and a much wider band of 17 kD proteins (p17) ap- peared in the infected cells. Meanwhile, the expression of bcl-2 mRNA was higher and that of fas mRNA was lower in the normal HELF cells, whereas there were significantly lower bcl-2 mRNA and higher fas mRNA expression levels in HCMV infected cells. It was concluded that HCMV was a stronger inducer of apoptosis in HELF cells. Caspase-3, as the marker of undergoing apoptosis, was expressed increasingly and activated in the infected cells, indicating its action in HCMV-inducing apoptosis. Down-regulating bcl-2 mRNA expression and up-regulating fas mRNA expression were also involved in the mechanism of HCMV-induced apoptosis.展开更多
Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloni...Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.展开更多
Objective To investigate the variability of human cytomegalovirus (HCMV) UL140 open reading flame (ORF) in clinical strains, and to explore the relationship between the variability of UL140 ORF and different sympt...Objective To investigate the variability of human cytomegalovirus (HCMV) UL140 open reading flame (ORF) in clinical strains, and to explore the relationship between the variability of UL140 ORF and different symptoms of HC-MV infection. Methods HCMV UL140 ORF was amplified by polymerase chain reaction and sequenced selectedly in 30 clinical strains. Results UL140 ORF of all clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identities among all strains were 96.5% -100.0% and 95.2% -100. 0%, respectively. All of the nucleotide changes were substitutions. The post-translational modification sites were conserved. The result of phylogenetic tree showed that the strains did not cluster according to different clinical symptoms. Conclusion HCMV UL140 ORF in clinical strains is highly conserved, which may play an important role in HC-MV infection.展开更多
Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogen...Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.展开更多
The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 ...The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56. 25 % and 43. 75 % , respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19. 00 % , 40. 58 % and 46. 15 %, respectively, with a significant difference found between group 2, 3 and group 1 (P<0. 01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10. 00 %, 15. 94 % and 30. 77 %, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4. 00, P<0. 001; OR=2. 343, P<005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA( + ) or mRNA( + ) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.展开更多
As a response factor of interferon,tripartite motif(TRIM)22 was reported to exert antiviral activity against viruses.In this study,THP-1 macrophages were infected with human cytomegalovirus(HCMV)to establish the HCMV ...As a response factor of interferon,tripartite motif(TRIM)22 was reported to exert antiviral activity against viruses.In this study,THP-1 macrophages were infected with human cytomegalovirus(HCMV)to establish the HCMV lytic infection model.The mRNA levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α)and interferonbeta(IFN-β)were significantly up-regulated in THP-1 macrophages at different infection time and titers.Moreover,for the first time,upregulation of TRIM22 expression was found during HCMV infection at both mRNA and protein levels in THP-1 macrophages.Furthermore,IFN-β could induce TRIM22 expression in THP-1 macrophages or HCMV infected THP-1 macrophages.Depletion of TRIM22 increased replication activity of HCMV with increasing of HCMV titers and HCMV proteins.In conclusion,it is the first report that HCMV can induce TRIM22 activation through IFN-β signaling and TRIM22 can suppress replication of HCMV in THP-1 macrophages.展开更多
To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from murine brain tissues by human cytomegalovirus(HCMV) infection and paired murine brain tissues and to identify the d...To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from murine brain tissues by human cytomegalovirus(HCMV) infection and paired murine brain tissues and to identify the differential expression proteins. Methods: Forty Kunming mice were randomly divided into infection group (20) injected with HCMVAD169 and control group (20) injected with saline into their brain. After 30 days, the murine brain tissues by HCMV infection and paired murine brain tissues were separated by two-dimensional gel electrophoresis(2-DE), analyzed by Image Master 2D software, and identified by peptide mass fingerprint(PMF) and database searching, and make Western blotting analyses the differential expression of individual proteins. Results: Well resolved, reproducible 2-D maps of the above tissues were obtained. Some of the different proteins identified by mass spectrometry(MS) were matched in the SWISS-2D PAGE database, Western blotting analyses were further carried out to verify the differential expression of individual proteins. Conclusion: These data will be valuable for studying the diagnosis of disease at an early stage, mechanisms of pathogenic and the key to the development of anti-HCMV medicine.展开更多
The possibility of infection of the human male genital tract by human herpes virus type 2 (HSV2) or human cytomegalovirus (HCMV) is well established and their sexual transmission has been the object of many studies. M...The possibility of infection of the human male genital tract by human herpes virus type 2 (HSV2) or human cytomegalovirus (HCMV) is well established and their sexual transmission has been the object of many studies. Moreover, medically assisted procreation, which helps in numerous fertility problems, raises the question of new viral risks linked to the application of these new technologies. In this review, we shall consider current knowledge in terms of the presence of HSV 2 and HCMV in the different parts of the genital tract of immunocompetent or immunodepressed men. We shall also consider the possibility of viral transmission by the sexual act or by the various techniques used in medically assisted procreation. We shall describe studies in human beings and in animals.展开更多
Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of...Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia ceils by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.展开更多
Objective: To investigate human cytomegalovirus infec-tion and genetic variations in glycoprotein B(gB) inliver transplant recipients in south-east China.Methods:EDTA-blood samples were obtained from 21liver transplan...Objective: To investigate human cytomegalovirus infec-tion and genetic variations in glycoprotein B(gB) inliver transplant recipients in south-east China.Methods:EDTA-blood samples were obtained from 21liver transplant recipients. The semi-nested PCR wasused to amplify a region of high sequence variabilityin the gB gene of human cytomegalovirus (HCMV)followed by direct sequence analysis.Results: Out of the 21 liver transplant recipients, 5were proved HCMV positive 62 to 180 days aftertransplantation. The nucleotide and encoded aminoacid sequences were compared with published se-quences of AD169 and Towne laboratory strains.Within the region sequenced, 2 out of 5 strains pos-sessed a peptide configuration similar to that of strainAD169, while another 2 strains displayed a peptideconfiguration similar to that of strain Towne. Onestrain had amino acid substitution, which was differ-ent from those of both AD169 and Towne in thecleavage site.Conclusion: Our results provide molecular epidemio-logical data for HCMV strains circulating among trans-plant recipients in south-east China.展开更多
Human cytomegalovirus(HCMV)is a common herpesvirus that persistently infects a large portion of the world's population.Despite the robust host immune response,HCMV is able to replicate,evade host defenses,and esta...Human cytomegalovirus(HCMV)is a common herpesvirus that persistently infects a large portion of the world's population.Despite the robust host immune response,HCMV is able to replicate,evade host defenses,and establish latency throughout the lifespan by developing multiple immunomodulatory strategies,making the studies on the interaction between HCMV infection and host response particularly important.HCMV has a strict host specificity that specifically infects humans.Therefore,most of the in vivo researches of HCMV rely on clinical samples.Fortunately,the establishment of humanized mouse models allows for convenient in-lab animal experiments involving HCMV infection.Single-cell RNA sequencing enables the study of the relationship between viral and host gene expressions at the single-cell level within host cells.In this study,we assessed the gene expression alterations of PBMCs at the single-cell level within HCMV-infected humanized mice,which sheds light onto the virus-host interactions in the context of HCMV infection of humanized mice and provides a valuable dataset for the related researches.展开更多
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 path...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.展开更多
Rasmussen's encephalitis(RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses, including human cytomegalovirus(HCMV), has been speculated to be a potential trigger fo...Rasmussen's encephalitis(RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses, including human cytomegalovirus(HCMV), has been speculated to be a potential trigger for RE. However, no viral antigens have been detected in the brains of patients with RE; thus, a possible clinical linkage between viral infections and RE has not been firmly established. In this study, we evaluated the expression of HCMV pp65 antigen in brain sections from 26 patients with RE and 20 non-RE patients by immunohistochemistry and in situ hybridization, and assessed the associations between HCMV infection and clinical parameters.Elevated expression of HCMV pp65 protein and DNA was observed in 88.5%(23/26) and 69.2%(18/26) of RE cases, respectively. In the non-RE group, HCMV pp65 antigen was detected only in two cases(10%), both of which were negative for DNA staining. Additionally, the intensity of HCMV pp65 staining was correlated with a shorter duration of the prodromal stage, younger age of seizure onset, and more severe unilateral cortical atrophy. Elevated expression of HCMV pp65 was observed in RE brain tissue and was correlated with the clinical features of RE disease. In summary, our results suggested that HCMV infection may be involved in the occurrence and progression of RE disease. Thus, further studies are needed to determine whether early treatment with anti-HCMV antibodies could modulate the course of RE.展开更多
基金supported by grants from the National Natural Science Foundation of China (Nos. 39970769, 30371488,30672243, and 81200354)the Natural Science Foundation of Hubei Province (No. 2009CDD216)
文摘Summary: This paper aimed to study the ability of baicalein to block human cytomegalovirus (HCMV) infection in extravillous cytotrophoblasts (EVT) and its effect on the vasoactive intestinal peptide (VIP) expression in HCMV-infected EVT in vitro. A human trophoblast cell line (HPT-8) was chosen in this study. HCMV with 100 TCIDs0was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by vi- rus titration. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in in- fected HPT-8 cells was decreased (P〈0.05), and the levels of VIP mRNA and protein, and the concen- tration were raised to the normal (P〉0.05). Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.
基金Supported by the National Natural Science Foundation of China (30801254)
文摘Objective To investigate the variability of human cytomegalovirus (HCMV) UL138 open reading frame (ORF) in clinical strains. Methods HCMV 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. Results LIL138 ORF in all 30 clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identifies of LIL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%, respectively. All of the nucleofide mutations were substitutions. The spatial structure and post-translational modification sites of HL138 encoded proteins were conserved. The result of phylogenetic tree showed that HCMV HL138 sequence variations were not definitely related with different clinical symptoms. Conclusion HCMV 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.
基金Supported by the National Natural Science Foundation of China (30170986).
文摘Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.
基金supported by the National Natural Science Foundation of China, (No. 30770105)Mt. Tai Scholar Construction Engineering Special Foundation of Shandong Province
文摘Objective To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV). Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P〈0.05). Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.
基金National Natural Science Foundation ofChina (30770105)Qingdao Technology Project (08-1-3-30-jch) Mt. Tai Scholar Construction Engineering Special Foundation of Shandong province, China.
文摘HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.
基金supported by a grant from the National Natural Science Foundation of China(No.30672243)Hubei Provincial Natural Science Foundation(No.2009CDB216)
文摘The effect of human cytomegalovirus (HCMV) on invasive capability of early pregnant extravillous cytotrophoblasts (EVTs) was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 (CK7), vimentin (Vim) and cerbB-2 were immunocytochemically detected to identify source of cells, and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining. The EVTs were divided into two groups: control group and HCMV group, and the expression of c-erbB-2, matrix metalloproteinase-2 (MMP-2) and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting. Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV. The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV. The cell source identification showed that the cells obtained were highly-pure primary EVTs, and primary EVTs could be infected by HCMV. Primary EVTs could express c-erbB-2, MMP-2 and MMP-9 proteins, and as compared with control group, the protein expression was decreased significantly in HCMV groups (P〈0.05). Primary EVTs could secrete active MMP-2 and MMP-9 in vitro, and the activity of two MMPs was decreased sig- nificantly in HCMV groups (P〈0.05). The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased (P〈0.05). We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability, suggesting that the im- paired EVT's invasion capability might be related to the abnormal expression of c-erbB-2, MMP-2 and MMP-9 proteins.
文摘Summary: The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus (HCMV) infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosis-regulating genes, bcl-2 and fas mRNA. The sequential changes of apoptotic cell rate in high and low MOI (MOI=2.5 and 0.25 respectively) of HCMV infected human embryonic lung fibroblasts (HELFs) at 1 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h post-infection were measured by flow cytometry. The expression levels of caspase-3 protein and bcl-2 and fas mRNA in HCMV infected cells (MOI=0.25) at 72 h post-infection were detected by Western blot and in situ hybridization methods, respectively. It was found that the ratio of apoptotic cells in normal controls was consistently lower, but the rates in low and high MOI infected cells were gradually increased with time prolonged, reached peak at 96 h (8.85 %) and 72 h (25.63 %), respectively. By Western blot analysis, only a narrow band of 32 kD (1 kD=0. 992 1 ku) procaspase-3 was found in normal cells, but a wider procaspase-3 band and a much wider band of 17 kD proteins (p17) ap- peared in the infected cells. Meanwhile, the expression of bcl-2 mRNA was higher and that of fas mRNA was lower in the normal HELF cells, whereas there were significantly lower bcl-2 mRNA and higher fas mRNA expression levels in HCMV infected cells. It was concluded that HCMV was a stronger inducer of apoptosis in HELF cells. Caspase-3, as the marker of undergoing apoptosis, was expressed increasingly and activated in the infected cells, indicating its action in HCMV-inducing apoptosis. Down-regulating bcl-2 mRNA expression and up-regulating fas mRNA expression were also involved in the mechanism of HCMV-induced apoptosis.
文摘Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.
基金Supported by the National Natural Science Foundation of China(30170986)
文摘Objective To investigate the variability of human cytomegalovirus (HCMV) UL140 open reading flame (ORF) in clinical strains, and to explore the relationship between the variability of UL140 ORF and different symptoms of HC-MV infection. Methods HCMV UL140 ORF was amplified by polymerase chain reaction and sequenced selectedly in 30 clinical strains. Results UL140 ORF of all clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identities among all strains were 96.5% -100.0% and 95.2% -100. 0%, respectively. All of the nucleotide changes were substitutions. The post-translational modification sites were conserved. The result of phylogenetic tree showed that the strains did not cluster according to different clinical symptoms. Conclusion HCMV UL140 ORF in clinical strains is highly conserved, which may play an important role in HC-MV infection.
基金Supported by the National Natural Science Foundation of China(30170986,30371492).
文摘Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.
文摘The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56. 25 % and 43. 75 % , respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19. 00 % , 40. 58 % and 46. 15 %, respectively, with a significant difference found between group 2, 3 and group 1 (P<0. 01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10. 00 %, 15. 94 % and 30. 77 %, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4. 00, P<0. 001; OR=2. 343, P<005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA( + ) or mRNA( + ) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.
基金funded by the National Nature Science Foundation of China(81701535 and 81671495)the Medical Scientific Projects from Health Department of Zhejiang Province(2015KYA119)the National Nature Science Foundation of Zhejiang(LQ18H040001)and Ai You Foundation.
文摘As a response factor of interferon,tripartite motif(TRIM)22 was reported to exert antiviral activity against viruses.In this study,THP-1 macrophages were infected with human cytomegalovirus(HCMV)to establish the HCMV lytic infection model.The mRNA levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α)and interferonbeta(IFN-β)were significantly up-regulated in THP-1 macrophages at different infection time and titers.Moreover,for the first time,upregulation of TRIM22 expression was found during HCMV infection at both mRNA and protein levels in THP-1 macrophages.Furthermore,IFN-β could induce TRIM22 expression in THP-1 macrophages or HCMV infected THP-1 macrophages.Depletion of TRIM22 increased replication activity of HCMV with increasing of HCMV titers and HCMV proteins.In conclusion,it is the first report that HCMV can induce TRIM22 activation through IFN-β signaling and TRIM22 can suppress replication of HCMV in THP-1 macrophages.
基金Supported by the Program of Science and Technology from Shenzhen City, Guangdong Province (200802051)
文摘To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from murine brain tissues by human cytomegalovirus(HCMV) infection and paired murine brain tissues and to identify the differential expression proteins. Methods: Forty Kunming mice were randomly divided into infection group (20) injected with HCMVAD169 and control group (20) injected with saline into their brain. After 30 days, the murine brain tissues by HCMV infection and paired murine brain tissues were separated by two-dimensional gel electrophoresis(2-DE), analyzed by Image Master 2D software, and identified by peptide mass fingerprint(PMF) and database searching, and make Western blotting analyses the differential expression of individual proteins. Results: Well resolved, reproducible 2-D maps of the above tissues were obtained. Some of the different proteins identified by mass spectrometry(MS) were matched in the SWISS-2D PAGE database, Western blotting analyses were further carried out to verify the differential expression of individual proteins. Conclusion: These data will be valuable for studying the diagnosis of disease at an early stage, mechanisms of pathogenic and the key to the development of anti-HCMV medicine.
文摘The possibility of infection of the human male genital tract by human herpes virus type 2 (HSV2) or human cytomegalovirus (HCMV) is well established and their sexual transmission has been the object of many studies. Moreover, medically assisted procreation, which helps in numerous fertility problems, raises the question of new viral risks linked to the application of these new technologies. In this review, we shall consider current knowledge in terms of the presence of HSV 2 and HCMV in the different parts of the genital tract of immunocompetent or immunodepressed men. We shall also consider the possibility of viral transmission by the sexual act or by the various techniques used in medically assisted procreation. We shall describe studies in human beings and in animals.
基金National Natural Science Foundation of China (30770105)Mt. Tai Scholar Construction Engineering Special Foundation of Shandong province.
文摘Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia ceils by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.
文摘Objective: To investigate human cytomegalovirus infec-tion and genetic variations in glycoprotein B(gB) inliver transplant recipients in south-east China.Methods:EDTA-blood samples were obtained from 21liver transplant recipients. The semi-nested PCR wasused to amplify a region of high sequence variabilityin the gB gene of human cytomegalovirus (HCMV)followed by direct sequence analysis.Results: Out of the 21 liver transplant recipients, 5were proved HCMV positive 62 to 180 days aftertransplantation. The nucleotide and encoded aminoacid sequences were compared with published se-quences of AD169 and Towne laboratory strains.Within the region sequenced, 2 out of 5 strains pos-sessed a peptide configuration similar to that of strainAD169, while another 2 strains displayed a peptideconfiguration similar to that of strain Towne. Onestrain had amino acid substitution, which was differ-ent from those of both AD169 and Towne in thecleavage site.Conclusion: Our results provide molecular epidemio-logical data for HCMV strains circulating among trans-plant recipients in south-east China.
基金supported by the National Key R&D Program of China(2023YFC23066000 to Y.R.)the National Natural Science Foundation of China(32100106 to Y.R.,and U21A20423 and 32225004 to X.Z.)+2 种基金the CAS Youth Innovation Promotion Association(2023351 to Y.R.)Hubei Province Natural Science Funds(2023AFB582 to Y.R.and 2023AFA008 to X.Z)the Fund of the Science and Technology Bureau of Wuhan(2023020201010086 to Y.R.).
文摘Human cytomegalovirus(HCMV)is a common herpesvirus that persistently infects a large portion of the world's population.Despite the robust host immune response,HCMV is able to replicate,evade host defenses,and establish latency throughout the lifespan by developing multiple immunomodulatory strategies,making the studies on the interaction between HCMV infection and host response particularly important.HCMV has a strict host specificity that specifically infects humans.Therefore,most of the in vivo researches of HCMV rely on clinical samples.Fortunately,the establishment of humanized mouse models allows for convenient in-lab animal experiments involving HCMV infection.Single-cell RNA sequencing enables the study of the relationship between viral and host gene expressions at the single-cell level within host cells.In this study,we assessed the gene expression alterations of PBMCs at the single-cell level within HCMV-infected humanized mice,which sheds light onto the virus-host interactions in the context of HCMV infection of humanized mice and provides a valuable dataset for the related researches.
基金Funded by the National Natural Science Foundation of China[81471048]the Natural Science Foundation of Shandong Province[ZR2019MC059]Shandong Province Government-Sponsored Overseas Study Project.&These authors contributed equally to this work.
文摘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.
基金supported by the following funds: the National Natural Science Foundation of China (81571275)the Beijing Municipal Natural Science Foundation (7144217)+5 种基金the Capital Applied Clinic Research Programs of Science and Technology (Z131107002213171)the Beijing Rising-star Plan of Science and Technology (Z141107001814042)the Open Research Fund of the Beijing Key Laboratory of Epilepsy Research (No. 2014DXBL02)Capital Medical University (15JL08)Scientific Research Common Program of Beijing Municipal Commission of Education (KM201610025001)Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2014 1685)
文摘Rasmussen's encephalitis(RE) is a rare and severe progressive epileptic syndrome with unknown etiology. Infection by viruses, including human cytomegalovirus(HCMV), has been speculated to be a potential trigger for RE. However, no viral antigens have been detected in the brains of patients with RE; thus, a possible clinical linkage between viral infections and RE has not been firmly established. In this study, we evaluated the expression of HCMV pp65 antigen in brain sections from 26 patients with RE and 20 non-RE patients by immunohistochemistry and in situ hybridization, and assessed the associations between HCMV infection and clinical parameters.Elevated expression of HCMV pp65 protein and DNA was observed in 88.5%(23/26) and 69.2%(18/26) of RE cases, respectively. In the non-RE group, HCMV pp65 antigen was detected only in two cases(10%), both of which were negative for DNA staining. Additionally, the intensity of HCMV pp65 staining was correlated with a shorter duration of the prodromal stage, younger age of seizure onset, and more severe unilateral cortical atrophy. Elevated expression of HCMV pp65 was observed in RE brain tissue and was correlated with the clinical features of RE disease. In summary, our results suggested that HCMV infection may be involved in the occurrence and progression of RE disease. Thus, further studies are needed to determine whether early treatment with anti-HCMV antibodies could modulate the course of RE.