Effect of Baicalein on the Expression of VIP in Extravillous Cytotrophoblasts Infected with Human Cytomegalovirus In Vitro

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.

Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

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.

Effect of Human Cytomegalovirus on Invasive Capability of Early Pregnant Extravillous Cytotrophoblasts

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.

Effects of Human Cytomegalovirus Infection on Apoptosis and Expression of Apoptosis-Regulating Factors

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.

Recombinant Human IgG antibodies against Human Cytomegalovirus

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.

Evaluation on Clinical Application of Three Testing Methods for Human Cytomegalovirus Infection in Pregnancy

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.

Association of TRIM22 with the type 1 interferon response during primary human cytomegalovirus infection in THP-1 macrophages

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.

Human Herpes Virus Type 2 ( HSV2 ), Human Cytomegalovirus (HCMV) in the Male Genital Tract and Fertilization

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.

Expression of human cytomegalovirus components in the brain tissues of patients with Rasmussen＇s encephalitis

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.

Modulation of HLA Expression in Human Cytomegalovirus Immune Evasion

Human cytomegalovirus （hCMV） has evolved multiple mechanisms to escape the host immune recognition and innate or adaptive immune responses. Among them, hCMV has developed strategies to modulate the expression and/or function of human leukocyte antigens （HLAs）, including by encoding series of infection stage-dependent hCMV proteins to detain and destroy the expression of HLA molecules on the surface of infected cells. This disturbs the antigen presentation and processing, by encoding MHC class I homologues or selective up-regulation of particular HLA class I molecules binding to NK cell inhibitory receptors, and by encoding specific ligand antagonists to interfere with NK cell activating receptors. Here we discussed the molecular mechanisms utilized by the hCMV to alter the formation, transportation and expression of HLA antigens on the infected cell surface. The knowledge about hCMV modulating HLA expression could benefit us to further understand the pathogenesis of viral diseases and may eventually develop novel effective immunotherapies to counteract viral infections and viral associated diseases.

Sequence variability of human cytomegalovirus UL143 in low-passage clinical isolates

Background Human cytomegalovirus （HCMV） infects a number of organs and tissues in vivo. The different symptoms and tissue tropisms of HCMV infection perhaps result from genetic polymorphism. A new region of DNA containing at least 19 open reading frames （ORFs） （denoted UL133 to 151） was found in the low-passage HCMV clinical strain, Toledo, and several other low-passage clinical isolates, but not present in the HCMV laboratory strain, AD169. One of these genes, UL143, was studied to explore the sequence variability of UL143 ORF in HCMV clinical isolates and examine the possible association between gcne variability and the outcome of HCMV infection. Methods The UL143 gone of the strains obtained from suspected congenitally HCMV-infectcd infants was amplified by polymerase chain reaction （PCR） and sequenced. Results Nineteen sequences of the strains were divided into 2 major groups, G1（n=16） and G2（n=3）. All of the sequences had frame-shift mutation compared to Toledo. Nucleotide polymorphisms conferred substantial amino acid substitutions when compared with Toledo. All 16 UL143 putative proteins of the strains in G1 had a new myristylation site and loss of two PKC sites owing to missense mutations. No convincing relationships were observed between the presence of HCMV disease and the UL143 sequence group. Conclusions HCMV-UL143 existed in low passage isolates. Sequence variability caused by frame -shift mutation was found in all HCMV clinical strains. No obvious linkage was observed between UL143 polymorphisms and the outcome of suspected congenital HCMV infection.

Classical and non-classical MHC I molecule manipulation by human cytomegalovirus： so many targets--but how many arrows in the quiver？

Major mechanisms for the recognition of pathogens by immune cells have evolved to employ classical and non-classical major histocompatibility complex class I （MHC I） molecules. Classical MHC I molecules present antigenic peptide ligands on infected cells to CD8＋ T cells, whereas a key function for non-classical MHC I molecules is to mediate inhibitory or activating stimuli in natural killer （NK） cells. The structural diversity of MHC I puts immense pressure on persisting viruses, including cytomegaloviruses. The very large coding capacity of the human cytomegalovirus allows it to express a whole arsenal of immunoevasive factors assigned to individual MHC class I targets. This review summarizes achievements from more than two decades of intense research on how human cytomegalovirus manipulates MHC I molecules and escapes elimination by the immune system.

Human cytomegalovirus miR-US5-1 inhibits viral replication by targeting Geminin mRNA

Viruses commonly create favorable cellular conditions for their survival through multiple mechanisms. Micro RNAs(mi RNAs), which function as post-transcriptional regulators, are utilized by human cytomegalovirus(HCMV) in its infection and pathogenesis. In the present study, the DNA replication inhibitor Geminin(GMNN) was identified to be a direct target of hcmv-mi R-US5-1. Overexpression of hcmv-mi R-US5-1 could block the accumulation of GMNN during HCMV infection, and the decrease of GMNN expression caused by hcmv-mi R-US5-1 or GMNN specific si RNA reduced HCMV DNA copies in U373 cells. Meanwhile, ectopic expression of hcmv-mi R-US5-1 and consequent lower expression of GMNN influenced host cell cycle and proliferation. These results imply that hcmv-mi R-US5-1 may affect viral replication and host cellular environment by regulating expression kinetics of GMNN during HCMV infection.

Human Cytomegalovirus Influences Host circRNA Transcriptions during Productive Infection

Human cytomegalovirus(HCMV)is a double-strand DNA virus widely infected in human.Circular RNAs(circ RNAs)are non-coding RNAs with most functions of which keep unknown,and the effects of HCMV productive infection on host circ RNA transcriptions remain unclear.In this study,we profiled 283 host circ RNAs that significantly altered by HCMV productive infection in human embryonic lung fibroblasts(HELF)by RNA deep sequencing and bioinformatics analysis.Among these,circ SP100,circ MAP3 K1,circ PLEKHM1,and circ TRIO were validated for their transcriptions and sequences.Furthermore,characteristics of circ SP100 were investigated by RT-q PCR and northern blot.It was implied that circ SP100 was produced from the sense strand of the SP100 gene containing six exons.Kinetics of circ SP100 and SP100 m RNA were significantly different after infection:circ SP100 levels increased gradually along with infection,whereas SP100 m RNA levels increased in the beginning and dropped at 24 h post-infection(hpi).Meanwhile,a total number of 257 proteins,including 10 HCMV encoding proteins,were identified potentially binding to cytoplasmic circ SP100 by RNA antisense purification(RAP)and mass spectrometry.Enrichment analysis showed these proteins were mainly involved in the spliceosome,protein processing,ribosome,and phagosome pathways,suggesting multiple functions of circ SP100 during HCMV infection.

Discovery of N-benzyl hydroxypyridone carboxamides as a novel and potent antiviral chemotype against human cytomegalovirus (HCMV)

Current drugs for treating human cytomegalovirus(HCMV)infections are limited by resistance and treatment-associated toxicities.In developing mechanistically novel HCMV antivirals,we discovered an N-benzyl hydroxypyridone carboxamide antiviral hit(8a)inhibiting HCMV in submicromolar range.We describe herein the structure–activity relationship(SAR)for 8a,and the characterization of potent analogs for cytotoxicity/cytostatic property,the preliminary mechanism of action,and the absorption,distribution,metabolism and excretion(ADME)properties.The SAR revealed a few pharmacophore features conferring optimal antiviral profile,including the 5-OH,the N-1 benzyl,at least one–CH_(2)−in the linker,and a di-halogen substituted phenyl ring in the amide moiety.In the end,we identified numerous analogs with sub-micromolar antiviral potency and good selectivity index.The preliminary mechanism of action characterization used a pUL89-C biochemical endonuclease assay,a virus entry assay,a time-of-addition assay,and a compound withdrawal assay.ADME profiling measuring aqueous solubility,plasma and liver microsomal stability,and parallel artificial membrane permeability assay(PAMPA)permeability demonstrated largely favorable drug-like properties.Together,these studies validate the N-benzyl hydroxypyridone carboxamide as a viable chemotype for potent and mechanistically distinct antivirals against HCMV.

Human cytomegalovirus RNA2.7 inhibits RNA polymeraseⅡ(PolⅡ)Serine-2 phosphorylation by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9)

Human cytomegalovirus(HCMV)is a ubiquitous pathogen belongs to betaherpesvirus subfamily.RNA2.7 is a highly conserved long non-coding RNA accounting for more than 20%of total viral transcripts.In our study,functions of HCMV RNA2.7 were investigated by comparison of host cellular transcriptomes between cells infected with HCMV clinical strain and RNA2.7 deleted mutant.It was demonstrated that RNA polymeraseⅡ(PolⅡ)-dependent host gene transcriptions were significantly activated when RNA2.7 was removed during infection.A145 nt-in-length motif within RNA2.7 was identified to inhibit the phosphorylation of PolⅡSerine-2(PolⅡS2)by reducing the interaction between PolⅡand phosphorylated cyclin-dependent kinase 9(pCDK9).Due to the loss of PolⅡS2 phosphorylation,cellular DNA pre-replication complex(pre-RC)factors,including Cdt1 and Cdc6,were significantly decreased,which prevented more cells from entering into S phase and facilitated viral DNA replication.Our results provide new insights of HCMV RNA2.7 functions in regulation of host cellular transcription.

Histone Deacetylase Inhibitor SAHA Induces Expression of Fatty Acid-Binding Protein 4 and Inhibits Replication of Human Cytomegalovirus

Suberoylanilide hydroxamic acid(SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic cutaneous T-cell lymphomas. In addition to its anticancer activity,SAHA has significant effects on the growth of many viruses. The effect of SAHA on replication of human cytomegalovirus(HCMV) has not, however, been investigated. Here, we showed that the replication of HCMV was significantly suppressed by treatment with SAHA at concentrations that did not show appreciable cytotoxicity. SAHA reduced transcription and protein levels of HCMV immediate early genes, showing that SAHA acts at an early stage in the viral life-cycle. RNAsequencing data mining showed that numerous pathways and molecules were affected by SAHA. Interferon-mediated immunity was one of the most relevant pathways in the RNA-sequencing data, and we confirmed that SAHA inhibits HCMV-induced IFN-mediated immune responses using quantitative Real-time PCR(qRT-PCR). Fatty acid-binding protein 4(FABP4), which plays a role in lipid metabolism, was identified by RNA-sequencing. We found that FABP4 expression was reduced by HCMV infection but increased by treatment with SAHA. We then showed that knockdown of FABP4 partially rescued the effect of SAHA on HCMV replication. Our data suggest that FABP4 contributes to the inhibitory effect of SAHA on HCMV replication.

Cytomegalovirus glycoprotein B sequence variation in Chinese liver transplant recipients

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.

Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14^+CD16^+Monocytes in a Population of Weifang,China

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.

应用PCR技术对孕妇HCMV感染的前瞻性研究

应用聚合酶链反应（ＰＣＲ）技术对１５６ 例孕妇尿中ＨＣＭＶ 进行检测，并追踪观察１１４ 例新生儿脐血中ＨＣＭＶ 垂直传播情况。结果表明：１１ 例孕妇尿ＨＣＭＶＤＮＡ 检测阳性，孕期感染率为７－０５％ ，有异常妊娠史孕妇感染率（１４－７１ ％ ）较正常孕妇（４－９２ ％） 明显升高。９ 例ＨＣＭＶ 阳性孕妇有４ 例观察新生儿脐血ＨＣＭＶＤＮＡ 阳性，占４４％ ，而ＨＣＭＶＤＮＡ阴性孕妇无１ 例新生儿脐血出现阳性结果。我们认为ＰＣＲ方法是检测ＨＣＭＶ宫内感染的可靠而灵敏的指标，对ＨＣＭＶＤＮＡ阳性的孕妇应进一步检查羊水，如羊水ＨＣＭＶＤＮＡ也阳性，应终止妊娠。