人巨细胞病毒先天性潜伏感染再激活致小鼠肠炎

目的:建立人巨细胞病毒(HCMV)先天性潜伏感染再激活致出血性坏死性肠炎BALB/c小鼠模型,为HCMV致小肠炎发病机制及临床特异性诊断和抗病毒治疗奠定基础.方法:将HCMV先天性感染的小鼠饲养于SPF级屏障系统中,18mo后,分别取病毒感染组18只、细胞对照组9只,并按随机原则分别给病毒感染组中9只小鼠以及细胞对照组小鼠注射环磷酰胺,建立HCMV潜伏再激活感染鼠模型.采用无菌操作技术分别取细胞对照组、病毒潜伏组和病毒潜伏再激活组小鼠小肠组织,进行病理组织学检查、体外细胞共培养分离病毒、PCR和RT-PCR检测HCMVUL83基因DNA及mRNA,间接免疫荧光鉴定等.结果:病毒潜伏感染再激活组细胞共培养病毒分离实验发现小鼠小肠组织内病毒分离阳性,在镜下可见HCMV在HF细胞内增殖产生特征性细胞病变,病毒分离共培养物PCR检测到HCMVUL83DNA,间接免疫荧光检测可见苹果绿色阳性信号;小鼠小肠组织PCR和RT-PCR分别检测到HCMVUL83DNA和相应mRNA;HE染色镜检发现部分肠组织有非特异性小肠炎性病变,有局灶性坏死及出血.而病毒潜伏组仅小肠组织PCR检测到HCMVUL83DNA,出血和坏死现象不是很明显;细胞对照组均是阴性.结论:成功构建了HCMV先天潜伏感染再激活小鼠小肠炎模型,病理表现为出血性坏死性肠炎.

HCMV IE1和pp65在人神经胶质瘤U_(251)细胞中的时序表达

目的:研究人神经胶质瘤U251细胞对人巨细病毒(human cytomegalovirus,HCMV)的容许性,并探讨病毒蛋白IE1及pp65的时序表达特点。方法:HCMV感染U251细胞,以透射电镜观察U251细胞的形态学改变,以PCR方法检测HCMV核酸,于不同的时间点分别用抗蛋白IE1及蛋白pp65单克隆抗体进行免疫细胞化学染色,检测IE1及pp65的表达水平。结果:受染的U251细胞出现了明显的形态学改变,并用PCR方法检测到了HCMV核酸。免疫细胞化学染色表明,蛋白IE1在感染后30min～2h无表达,4h开始表达,14h达高峰,随后下降;蛋白pp65在感染后1h为入侵型pp65,4～96h一直呈低水平表达,至120h表达急剧升高。结论:U251细胞是HCMV的容许细胞,HCMV在其中的表达具有时序性,但是其时序表达较在人成纤维细胞中延迟。

Human Cytomegalovirus UL138 Open Reading Frame Is Highly Conserved in Clinical Strains

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.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

MODULATION OF THE FREQUENCY OF HUMANCYTOMEGALOViRUS-INDUCED CHROMOSOMEABERRATIONS BY CAMPTOTHECIN

The effects of selected DNA repair inhibitors on the frequency of human cytomegalovirus (HCMV)-induced chromosome aberrations in human peripheral blood lymphocytes (PBLs) were evaluated. Trealment of HCMV-infected PBLs with camptothecin (0.05 to 0.3μg/ml), an inhibitor of topoisomerase I, for 30 hr resulted in a significant (P<0.01) synergistic enhancement of the frequency of HCMV-induced chromosome damage, on the other hand, a significant increase in the frequency of chromosome damage was not noted for infected PBLs treated with either 3-aminobenzamide (3-AB) (3 to 30μg/ml), an inhibitor of poly (ADP-ribose) poiymerase, ur novobiocin 3 to 30 |ig/ml) an inhibitor of topoiso-merase I or excision repair processes for 30 hr. chromatid-type breaks including chromosome exchanges were the predominant type of chromosome aberrations observed in the HCMV-infected cells treated with camptothecin suggesting that HCMV infection is associated with the induction of single-strand DNA breaks. Furthermore, these findings suggest that HCMV infection does rol inflict dircc: DNA damage which is repaired through 3-AB- or novobiocinsensitive pathways.Human cytomegalovirus (HCMV) is a common pathogen which irferfs about 80% of the world's population causing, for the most part, persistent subcliniclil infeclions (Wellcr, 1971). A relatively small percentage of otherwise healthy immunologically competent people experience clinical HCMV disease (Cohen et al., 1985). Generalized HCMV infection, however, is the bane of individuals whose immune system is compromised (Schooley, 1990) Rubin, 1990). Molecular epidemiological studies strongly suggest that HCMV is one of the most frequent cause of congenital infections and that these infections result in a high incidence of birth defects and developmental abnormalities (Alford et al., 1990). Several studies (Nachtigal et al., 1978; Luleci et al., 1980; AbuBakar et al., 1988) have shown that HCMV infectior can result in an increase in the frequency of chromosome aberrations. Since the ability to cause chromosome damage may be significant in the induction of birth defects and possibly in HCMV-induced malignancy, we undertook the present investigation to evaluate the mechanisms by which HCMV may cause chromosome damage. In ths study, the effects of selected DNA repair inhibitors on the frequency of HCMV-induced chromosome aberrations were evaluated. The results indicae that the presence of camptothecin, but nut 3-aminobenzamidc (3-AB) ro novobiocin, results in a concentration-dependent increase in the frequent) of chromosome aberrations in HCMV-infected peripheral blood lymphocytes (PBLs). Accordingly, it is proposed that HCMV-induced chromosome damage in PBLs does not substantially involve DNA repair activities sensitive to inhibition of poly ADP-ribosylation or excision repair processes, but is related to camp of thccin-sersitive DNA repair, conceivably involving the activity of topoisomrrasc I .

HCMV Infection Depress NGF Expression in Human Glioma Cells

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.

Murine brain tissues with human cytomegalovirus infection: a proteomic study

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.

Diagnostic value of human cytomegalovirus late mRNA detection in active intrauterine infection

OBJECTIVE: To study the diagnostic value of human cytomegalovirus (HCMV) late mRNA detection in active intrauterine infection. METHODS: The HCMV late mRNA in peripheral blood of 42 HCMV IgM positive pregnant women and their fetal attachments (such as chorionic villi, amniotic fluid, umbilical blood and placenta) were detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Late mRNA was detected in 23 of 42 HCMV IgM positive cases, a rate of 54.3%. Fetal appendages in 13 cases of late mRNA positive mothers were also tested, of which 7 were positive, with a vertical transmission rate of 53.8%. In 12 late mRNA negative mothers, only 1 case of fetal appendages tested was positive, with a vertical transmission rate of 8.3%. There was significant difference between the transmission rates of these two groups. CONCLUSIONS: Positive results of HCMV IgM cannot accurately reflect the activity of HCMV at the time of testing. However, the activity of HCMV is closely related to the mother-fetus vertical transmission rate. As an indicator of active HCMV infection, late mRNA can not only reflect the mother-fetus transmission rate during active HCMV infection, but also provide some information about HCMV activity in fetal tissue.

Human cytomegalovirus infection dysregulates neural progenitor cell fate by disrupting Hes1 rhythm and down-regulating its expression

Human cytomegalovirus(HCMV) infection is a leading cause of birth defects, primarily affecting the central nervous system and causing its maldevelopment. As the essential downstream effector of Notch signaling pathway, Hes1, and its dynamic expression, plays an essential role on maintaining neural progenitor/stem cells(NPCs) cell fate and fetal brain development. In the present study, we reported the first observation of Hes1 oscillatory expression in human NPCs, with an approximately1.5 hour periodicity and a Hes1 protein half-life of about 17(17.6 ± 0.2) minutes. HCMV infection disrupts the Hes1 rhythm and down-regulates its expression. Furthermore, we discovered that depleting Hes1 protein disturbed NPCs cell fate by suppressing NPCs proliferation and neurosphere formation, and driving NPCs abnormal differentiation. These results suggested a novel mechanism linking disruption of Hes1 rhythm and down-regulation of Hes1 expression to neurodevelopmental disorders caused by congenital HCMV infection.

Multipotent mesenchymal stromal cells are fully permissive for human cytomegalovirus infection

Congenital human cytomegalovirus(HCMV) infection is a leading infectious cause of birth defects.Previous studies have reported birth defects with multiple organ maldevelopment in congenital HCMV-infected neonates. Multipotent mesenchymal stromal cells(MSCs) are a group of stem/progenitor cells that are multi-potent and can self-renew, and they play a vital role in multiorgan formation. Whether MSCs are susceptible to HCMV infection is unclear. In this study, MSCs were isolated from Wharton's jelly of the human umbilical cord and identified by their plastic adherence, surface marker pattern, and differentiation capacity. Then, the MSCs were infected with the HCMV Towne strain, and infection status was assessed via determination of viral entry,replication initiation, viral protein expression, and infectious virion release using western blotting,immunofluorescence assays, and plaque forming assays. The results indicate that the isolated MSCs were fully permissive for HCMV infection and provide a preliminary basis for understanding the pathogenesis of HCMV infection in non-nervous system diseases, including multi-organ malformation during fetal development.

Diagnostic value of human cytomegalovirus late mRNA detection in active intrauterine infection

To study the diagnostic value of human cytomegalovirus (HCMV) late mRNA detection in active intrauterine infection Methods The HCMV late mRNA in peripheral blood of 42 HCMV IgM positive pregnant women and their fetal attachments (such as chorionic villi, amniotic fluid, umbilical blood and placenta) were detected by reverse transcription polymerase chain reaction (RT PCR) Results Late mRNA was detected in 23 of 42 HCMV IgM positive cases, a rate of 54 3% Fetal appendages in 13 cases of late mRNA positive mothers were also tested, of which 7 were positive, with a vertical transmission rate of 53 8% In 12 late mRNA negative mothers, only 1 case of fetal appendages tested was positive, with a vertical transmission rate of 8 3% There was significant difference between the transmission rates of these two groups Conclusions Positive results of HCMV IgM cannot accurately reflect the activity of HCMV at the time of testing However, the activity of HCMV is closely related to the mother fetus vertical transmission rate As an indicator of active HCMV infection, late mRNA can not only reflect the mother fetus transmission rate during active HCMV infection, but also provide some information about HCMV activity in fetal tissue