Effects Of Congenital Cytomegalovirus Infection On Encephalodysplasia And Its Mechanisms

BackgroundHuman cytomegalovirus (HCMV) is the leading infectious cause of congenital anomalies of the central nervous system (CNS) in fetus, which badly impact the life quality of survival children. Therefore, it is a very important social and economical significance to find out the mechanism of neural injure caused by congenital HCMV infection for establishing the strategies of preventing and treating them. However, the exact pathogenesis of these brain abnormalities has not yet been fully elucidated due to the following reasons. Firstly, CMV is strict species specific which makes it impossible to establish the heterogeneous animal models by using HCMV. Secondly, CMV is the largest viruses containing 230 to 240 kb of DNA in its genome with at least 190 genes and also is a big protein complex. HCMV proteins are synthesized sequentially from corresponding mRNAs whose transcription is regulated in a temporal cascade and only a small number of HCMV proteins have been identified. Thirdly, CMV has an intrinsic affinity for developing brain cells, so that it became difficult to choose an appropriate temporal and spatial time for studying them during the developing process of brain.Murine cytomegalovirus (MCMV) has some similar biological properties of HCMV and also causes the similar pathological changes of central nervous system in experimentally congenital infected mice, which is therefore considered as a good model for studying the mechanism of neural injure caused by congenital CMV infection.CNS damage of CMV infection is mainly seen in fetal life, which was usually ascribed to the immature of immunity in fetus. Recently, it was revealed that CMV had an intrinsic preference for infection of developing brain cells, independent of the developmental status of the systemic immune system in controlling CMV infection. Allow for the consequence of CMV infection in central neural system of fetus is strongly related to the developing status of brain, the exploration of neural pathogenesis caused by congenital CMV infection should be investigated during the developing process of neural system.ObjectivesThe goals of the present project are to understand the molecular and cellular mechanisms involved in brain abnormalities of congenital cytomegalovirus infection and open up a new field to develop new intervention strategy. In particular, we are focusing on the following three parts:1. Establish an animal model of MCMV congenital infection to evaluate the relationship of different gestational age of infection and viral dose to perinatal outcome and the pathological characteristics of central nervous system damage.2. Set up two cell models that could mimic the developing process of neural system in vitro by means of inducting embryonic stem (ES) cells or neural stem cells (NSCs) into neuron cells.3. Investigate the effects of MCMV infection on NSCs, ES cells and the different phases of neural precursor cells in vitro and further reveal the signal pathway involved in brain abnormalities caused by congenital cytomegalovirus infection.Methods1. Animal model1μl recombinant MCMV RM461 strain (1×10~2,1×10~3, 1×10~4PFU), which was inserted a report gene of Lac Z, was injected into uterus of pregnant BALB/c mice on day 8 (E8d) and 13.5 (E13.5d) of gestation, respectively. Other pregnant mice injected with the same volume of DMEM cell culture media in the same way served as the controls. The embryos were allowed to develop to day 18.5 of gestation and then were taken out from uterus of mothers. The different tissues of the offspring were analyzed using the histological examination and X-gal staining methods. The perinatal outcome and neural pathological features were observed. 2. The effects of MCMV infection on differentiating and developing model of neural stem cells in vitroNSCs were prepared from fetal brain of BALB/c mouse on day 13.5 of gestation, and were cultured and identified by detecting specific markers (HRP-DAB assay). The 3 to 5 generations of NSCs were infected with recombinant MCMV RM461 at different multiplicity of infection (MOI). X-gal staining was used to monitor MCMV infected condition. The morphology of NSCs was observed under inverted microscope (IM) and transmission electron microscope (TEM). The productive infection was verified by finding viral articles by TEM, detecting MCMV DNA in infected NSCs or cultural supernatant by PCR and observing cytopathic effect (CPE) of mouse embryonic fibroblasts (MEF) inoculated with cultural supernatant of infected NSCs. The influence of MCMV infection on proliferation of NSCs was determined by drawing growth curve and MTT assay. The effects of MCMV infection on differentiation of NSCs was investigated by detection of the ratio of nestin, GFAP, NSE or NF-200 positives cells with flow cytometry. Apoptosis of infected NSCs were detected by flow cytometry with FITC-Annexin V and PI double staining. Finally, the transcription or translation of Wnt-1 and neurogenin 1 (Ngn1) genes, which play a determining role in regulating the processes of neural differentiation, were analyzed by RT-PCR and western blot.3. The effects of MCMV infection on differentiating and developing model of ES cells in vitroFirstly, mouse ES cells line-D3 was induced to differentiate into neurons based on 4-/4+ all-trans retinoic acid (ATRA) inducing protocol. Secondly, a new method was used to induce ES cells differentiating into neural stem/progenitor cells and neurons by using 2-/2+ ATRA protocol combined with astrocytes as inducing stromal layer in vitro. Totipotency of ES cells was identified by observation of cell morphology, formation of teratoma in immunocompromised mice, AKP staining and detection of ES marker Oct-4 mRNA by RT-PCR. The quantity and purity of NSCs derived from ES cells were analyzed by detection of cell markers with immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Finally, the influence of MCMV infection on the ES cells and their differentiation into different phases of neural precursor cells were investigated.Results1. An animal model of MCMV congenital infection1) An animal model of MCMV congenital infection had been successfully established by injection of MCMV through uterus wall of pregnant mouse. Significant more still birth and litter resorption could be found in high dose of MCMV group and only few fetuses survived (5.9%). Although the survival rate (47.1%) of low dose of MCMV group was higher than that of high dose group, the viral positive rate (12.5%) of brain tissues is lower yet. And 10~3 PFU was the appropriate dose to establish an animal model of MCMV congenital infection, the survival and viral positive rates were 27.8% and 80%, respectively.2) When using 10~3PFU dose of virus, the survival rate (26.9%) and fetus weight of E8d group were lower than those of DMEM control group, but the rate of litter resorption (55.8%) was higher than that of control group. While the survival rate (52%) of E13.5d group was lower than that of DMEM controls, but the rate of litter resorption (14%) was similar to that of DMEM controls. Comparison of two infected groups, the survival rate of E8d group was lower than that of E13.5d group, but the rate of litter resorption was higher than that of E13.5d group. Furthermore, three mice with microcephaly were found in E8d infected group.3) By X-gal staining, the positive cells were detected in the brain, heart, liver, spleen, lung and kidney of infected fetuses, and moreover, virus could be isolated from these tissues. In the brain, X-gal-positive cells were mainly located in marginal regions of the brains, hippocampus, ventricular and subventricular zones.2. The effect of MCMV infection on neural stem cells in vitro1) NSCs isolated from embryonic mouse brains could be continuously passaged and remained the ability to form neurospheres after subculture and strongly expressed nestin, a specific marker of NSCs. The cultured NSCs also kept the plasticity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes.2) NSCs were found to have productive MCMV infection, which were swollen and enlarged in size after 48h post-infection (p.i.). The cells at the edge of the neurospheres were fallen off and even dead. The change of cell morphology became more obvious as the MOI was increased. X-gal staining showed that the expression of Lac Z gene in NSCs could be detected at 24h p.i. and reached peak at 48h p.i. By 72h p.i., MCMV DNA was detected in infected NSCs and cultural supernatant by PCR, viral particles and swollen organellae in the plasma of infected cells were observed under TEM. When the supernatant was seeded on MEF cells, typical CPE appeared 3 days later.3) MCMV infection could markedly inhibit the proliferation of NSCs. The inhibitory effect was significantly enhanced as the MCMV MOI increased.4) The neuronal differentiation was also significantly inhibited by MCMV infection. The differentiation cells were analyzed by flow cytometry on 2 days p. i.. It was showed that the ratio of nestin positive cells in infected group was higher than that of normal group, (62.2±1.8) % vs (37.2±2.4) % (P<0.01), meanwhile, the ratio of GFAP and NSE positive cells in infected group were lower than that of normal group, (37.4±1.6) % vs (50.3±1.8) % (P<0.01) and (8.9±0.8) % vs (23.4±1.3) % (P<0.01), respectively.5) There was no significant difference in the early and late apoptosis rate between infected group and normal group, but the death rate of infected group gradually increased since 48h p.i. and reached (15.2±3.8) % by 5d p.i, which is obviously higher than (5.1±0.3) % of normal control.6) MCMV could inhibit the expression of Wnt-1 and Ngn1 genes of infected NSCs. Compared with normal cells, the mRNA level of Wnt-1 was significantly lower at 12h, 24h, 48h p.i. (P<0.01), the protein expression of Wnt-1 was obviously lower at 24h and 48h p.i. (P<0.01), and the expression of Ngn1 gene was markedly lower at 12h, 24h, 48h, 72h p.i. (P<0.01) in infected cells. 3. Effects of MCMV infection on ES cells in vitro1) If the ES line-D3 cells were cultured on MEF cells served as breeding layer, they could keep the potentiality to differentiating into cellular derivations of all three primary germ layers after continuous passages, which was confirmed by their becoming positive of AKP staining and expressing Oct-4 gene (RT-PCR).2) ES cells could be induced by ATRA to differentiate along a neuronal pathway. Inducing by 4-/4+ ATRA protocol, the ratio of NF-200 positive cells reached (27.3±1.2)% and only (2.1±0.4)% in bland control (P<0.01).3) By using 2-/2+ ATRA protocol combined with astrocytes as inducing stromal layer, up to 91.4% of nestin positive cells could be obtained. Nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with FCS. The results of differentiating test showed that rates of NF-200 and NSE positive cell were (42.7±2.6) % in ATRA/astrocyte group and only (11.2±1.8) % in 2-/2+/4- ATRA control (P<0.01).4) ES cells are non-permissive to MCMV infection in the undifferentiated state and acquire permissive for productive infection after differentiation. Furthermore, MCMV infection did not affect the growth of undifferentiated ES cells.5) The number of neurons differentiated from ES cells in MCMV infected group was less than that of the control group, the rates of NF-200 cells were (10.2±0.3)% and (29.3±0.8)%, respectively (P<0.01).Conclusions1. Animal model and cell models of MCMV congenital infection were successfully established by intrauterine infection and induction of ES Cells or NSCs into neuron cells, respectively, which will be of great benefit to study mechanisms of neural damage in congenital MCMV infection.2. Congenital MCMV infection could cause significant central nervous system abnormality. The perinatal outcome was related with viral dose and gestational age of infection. The fetus of middle gestational stage was more sensitive to MCMV infection than that of late gestational stage.3. NSCs were permissive for productive MCMV infection in vitro. The proliferation and differentiation of NSCs were significantly inhibited after MCMV infection, while the death rate was increased in MCMV infected cells. The decreased expression of wnt-1 and Ngn1 genes might be explained for the inhibition effect of MCMV infection on the differentiation of NSCs, and also involved in the pathogenesis of encephalodysplasia in congenital cytomegalovirus infection.4. Astrocytes not only promoted production of NSCs derived from ES cells but also improved differentiation of NSCs toward neuronal lineage.5. ES cells are non-permissive to MCMV infection in the undifferentiated state and acquire permissive for productive infection during the period of differentiation. There might be some factors within ES cells that could protect them from CMV infection. However, the differentiation of ES cells towards neural pathway could be inhibited by MCMV infection.