Apoptosis of Murine T-Cell Lymphoma EL4 Cells Induced by Murine Cytomegalovirus and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect whether the murine T-cell lymphoma cell line EL4 could be infected by murine cytomegalovirus (MCMV), and to observe the morphological changes and apoptosis of El4 cells before and after infection.</p><p><b>METHODS</b>EL4 cells were infected with MCMV smith strain with 1, 10 and 100 multiplicity of infection (MOI) respectively. The morphology of the cells was observed by light microscopy and Wright's-Giemsa staining. The survival rate was calculated by trypan blue staining. RT-PCR-based assay was used to detect the copy number of MCMV-DNA in the infected ELA cells. Flow cytometry was used to detect apoptosis. RT-qPCR was used to detect the mRNA expression levels of P53, P21, cFlip and Caspase 8. The protein expression levels of Caspase8, P53, BAX, BCL-2 and Cleaved Caspase3 proteins were detected by Western blot.</p><p><b>RESULTS</b>After Wright-Giemsa staining, it was found that the infected EL4 cells displayed larger volume, irregular nuclei and the folded twist under light microscopy. Compared with the normal control group, the survival rate of EL4 cells decreased, and the apoptosis rate statistically significantly (P<0.05) increased with the increasing MOI and the infected time in each group. While, the level of apoptosis protein P53, BAX/BCL-2, Cleaved-caspase3 and Caspase8 were up-regulated. And the survival rate, apoptosis rate and the apoptosis protein level of infected EL4 cells with MOI=10 were the most obvious at the 5day. Compared with MCMV infection group (MOI=60), the content of MCMV DNA in EL4 cells was decreased in MCMV+GGV group ［MOI=60, GCV 25 (g/ml)］, and the cell apoptosis rate and apoptosis protein expression of P53, Caspase8, BAX/BCL-2 were decreased (P<0.05).</p><p><b>CONCLUSION</b>Murine T-cell lymphoma cell line EL4 can be infected by MCMV and displayes an obvious apoptosis phenomenon. MCMV may up-regulate the expression levels of apoptosis protein P53, BAX-BCL-2, Cleaved-caspase3 and Caspase8 in EL4 cells. The drug ganciclovir reduces the copy mumber of MCMV DNA in infected EL4 cells and inhibited the killing effect of MCMV on EL4 cells.</p>

Protein interactions in the murine cytomegalovirus capsid revealed by cryoEM

Cytomegalovirus (CMV) is distinct among members of the Herpesviridae family for having the largest dsDNA genome (230 kb). Packaging of large dsDNA genome is known to give rise to a highly pressurized viral capsid, but molecular interactions conducive to the formation of CMV capsid resistant to pressurization have not been described. Here, we report a cryo electron microscopy (cryoEM) structure of the murine cytomegalovirus (MCMV) capsid at a 9.1 Å resolution and describe the molecular interactions among the ∼3000 protein molecules in the MCMV capsid at the secondary structure level. Secondary structural elements are resolved to provide landmarks for correlating with results from sequence-based prediction and for structure-based homology modeling. The major capsid protein (MCP) upper domain (MCPud) contains α-helices and β-sheets conserved with those in MCPud of herpes simplex virus type 1 (HSV-1), with the largest differences identified as a "saddle loop" region, located at the tip of MCPud and involved in interaction with the smallest capsid protein (SCP). Interactions among the bacteriophage HK97-like floor domain of MCP, the middle domain of MCP, the hook and clamp domains of the triplex proteins (hoop and clamp domains of TRI-1 and clamp domain of TRI-2) contribute to the formation of a mature capsid. These results offer a framework for understanding how cytomegalovirus uses various secondary structural elements of its capsid proteins to build a robust capsid for packaging its large dsDNA genome inside and for attaching unique functional tegument proteins outside.

Identification of proteins that interact with murine cytomegalovirus early protein M112-113 in brain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Murine cytomegalovirus (MCMV) early protein M112-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis. To investigate the biological function of M112-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus (CMV), a screening for proteins interacting with M112-113 was performed by a yeast two-hybrid system.</p><p><b>METHODS</b>Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast. After confirmation of the expression of MCMV M112-113 in yeast, the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M112-113. Interactions between M112-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.</p><p><b>RESULTS</b>Two proteins interacting with M112-113 were identified, including metastasis-associated 1 (MTA1) and zinc finger, CCHC domain containing 18 (ZCCHC18). M112-113 protein could interact with MTA1 or ZCCHC18 in yeast and mammalian cells.</p><p><b>CONCLUSION</b>The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.</p>

Influence of allitridin on transcription, expression and function of IL-12 genes in mice infected by murine cytomegalovirus / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate whether allitridin could interfere with the effects of murine cytomegalovirus (MCMV) infection on the transcription, expression and function of IL-12 genes in order to further explore the mechanism of allitridin against MCMV.</p><p><b>METHOD</b>Sixty mice were randomly divided into allitridin treated group, placebo and blank controls. Allitridin was intra-peritoneal injected to mice in treated group once a day with general dosage (25 mg x kg(-1)) at 24 hours after MCMV infection, and the same dosage of physiological saline were given to placebo and blank groups. Four experimental mice were sacrificed at 3, 5, 7, 10, 14 days after treatment (n = 4 per time point), respectively. The expression of IL-12 p70 and IFN-gamma in supernatant of spleen cell cultures were measured by double-antibody sandwich ELISA, and IL-12 p35 and p40 mRNAs in spleen cells were analyzed by RT-PCR.</p><p><b>RESULT</b>In systemic infection mice, the expression of both IL-12 p70 protein and p35 mRNA significantly increased on day 3 post-infection (pi); then rapidly and markedly decreased on day 5 pi and later. The level of IFN-gamma reached the peak on day 3 pi, then gradually dropped and returned to normal levels during the period of day 10 to 14 pi, and IL-12 p40 mRNA level was persistently and significantly higher after infection. In allitridin treated mice, the levels of IL-12 p70 protein, IL-12 p35 and p40 mRNAs reached the peak on day 3 after treatment (P < 0.05), and then rapidly dropped to the normal levels during the period of 5-14 days. Level of IFN-gamma was also reached the peak on day 3 after treatment; however, it dropped a little on day 5 and then gradually increased and was much higher than those of both placebo and bland controls during the period of day 7 to 14 after treatment (P < 0.01).</p><p><b>CONCLUSION</b>Allitridin could completely correct the disturbance of expression of IL-12 gene caused by MCMV and persistently promote IFN-gamma expression, which was useful for enhancing the specific cellular immune reactions against CMV and clearance of CMV viruses from host. The result suggests another mechanism of allitridin against CMV.</p>

A murine model with murine cytomegalovirus infection resulting in colon inflammation after allogeneic skin transplantation / 病毒学报

<p><b>UNLABELLED</b>To provide a reliable animal model for study of human CMV disease in gastrointestinal track, we tried to infect with murine cytomegalovirus (MCMV) in mice that were received allogenetic skin transplantation under immunosuppression. (1) Skin transplantation was performed between 18 donor C57BL/6 mice and 72 recipient BALB/c mice. (2) All recipient mice were then given Cyclosporine at 12 mg/kg daily for 2 weeks by intraperitoneal injection. Mice were randomly divided into 3 groups. Two experimental groups were received MCMV-infected mouse embryonic fibroblasts (MEF) at 10(4) PFU and 10(5) PFU respectively, and the control group received MEF only. We observed any possibly pathophysiological behavior changes and recorded the changes in body weight. The mice were sacrificed at 5d, 9d, 14d, 21d post infection and colon tissue was collected for analysis.</p><p><b>RESULTS</b>Mice infected with MCMV at 10(5) PFU group showed anorexia, lethargy and degression in locomotor activity. This group of mice showed significant decrease in body weight than that of other groups. Colon tissues were collected 14 days after infection. Histological examination revealed that the mucous layer became thinner in the proximal colon and increased number of lymphoid follicles in distal colon in infected animals. The changes in the mucosal structure was most prominent in the group 10(5) PFU MCMV. Viral DNA was present in the colon by in situ hybridization for IE1 gene, and viral gB transcript was positive by RT-PCR. One of the viral major proteins, pp65, was widely distributed in the colon by immunohistochemistry. These data demonstrated that MCMV established infection in colon of the mice after allogenetic skin transplantation. Electron microscopy showed that there were herpes virus particles in the colon tissue.</p><p><b>CONCLUSION</b>Infection with MCMV in mouse after allogenetic skin transplantation by nasal cavity inoculation resulted in the pathological changes in colon tissue similar to that of inflammation in human colon. The small animal model of colon inflammation may provide a platform for further study of pathogenesis as well as medical intervention of HCMV involved inflammation of human bowel.</p>

Infection of the mononuclear cell subpopulations in murine bone marrow with murine cytomegalovirus / 中国实验血液学杂志

This study was aimed to explore the infection characteristics of murine mononuclear cell subpopulations in bone marrow with murine cytomegalovirus (MCMV). Subpopulations of mononuclear cells, including lin(+), lin(-), lin(-)CD117(+) and lin(-)CD117(-) cells, were infected with MCMV after being separated by MACS, and induced to differentiation by adding cytokines or inducer, then nucleic acid and proteins were detected. The results indicated that the MCMV DNA, IE transcripts and IE protein could be detected in the lin(+) cells infected with MCMV; no virus products were detected in infected lin(-) cells without adding any stimulating factors, while IE and E transcripts and proteins were detected after adding GM-CSF, rhEPO or phorbol ester in the lin(-) cells infected with MCMV. Furthermore, no IE or E gene transcripts were detected in the lin(-)CD117(+) and lin(-)CD117(-) cells, but the cell colony formation of lin(-)CD117(+) hematopoietic stem and progenitor cells was inhibited after MCMV infection and expression of CD117 antigen on cell surface of the lin(-) cells was downregulated. It is concluded that MCMV can latently infect subpopulations of mononuclear cells in the murine bone marrow. Cells which are of characteristics of primitive stem and progenitor cells are not susceptible to MCMV, but infection of these cells with MCMV can inhibit functions of cells and downregulate the expression of antigen on cells surface.

Fluvastatin's effect on atherogenesis in apolipoprotein-E knockout mice infected by cytomegalovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>The goal of this study was to investigate whether murine cytomegalovirus (MCMV) is able to exacerbate the atherosclerotic process in apolipoprotein E knockout (apoE -/-) mice, and the effect of fluvastatin on the atherogenesis.</p><p><b>METHODS</b>The apoE-/- mice kept on a west diet were given low dosage of MCMV. At 14,18 and 24 weeks post infection, AS lesion were measured on aorta. The fluvastatin was administered, and AS lesion were measured accordingly above.</p><p><b>RESULTS</b>We observed that in the chronic phase of the infection, AS lesion area was significantly increased. MCMV gB mRNA was not amplified by real-time PCR from the arterial wall. The IgG antibody level of MCMV in blood plasma and the content of virus DNA in salivary gland were not correlated with AS lesions. After the administration of fluvastatin, there was no significant difference of AS lesions between MCMV infected group and mock-infected group.</p><p><b>CONCLUSION</b>MCMV may aggravate the AS lesion in apoE -/- mice in the chronic phase of infection, and promote more severe type of AS lesions. But it might not be the direct effects of mechanism of MCMV on the local lesion of AS. Fluvastatin could meliorate the progression of AS after MCMV infection, but this was not accomplished by decreasing MCMV duplication.</p>

The effects of murine cytomegalovirus on the maturation, fertilization, cleavage and blastula formation of mouse oocytes in vitro / 华中科技大学学报（医学）（英德文版）

To study the effects of mouse cytomegalovirus (MCMV) on the in vitro maturation, fertilization, cleavage and blastula formation of mouse oocytes, the immature oocytes were infected in vitro by MCMVs of different dosages (100 TCID(50), 10 TCID(50) and 1 TCID(50)). The oocytes were then observed for in vitro maturation, fertilization, cleavage and blastula formation and the ultrastructural changes after the culture with the viruses. Our results showed that no significant differences were found in IVM, IVF, cleavage and blastula formation among the groups treated with of virus of various dosages. And ultrastructural abnormality was observed in the oocytes treated by 100 TCID(50) of viruses. It is concluded that MCMV did not have any conspicuous effects on IVM, IVF, cleavage and blastula formation of murine immature oocytes.

Inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation and its mechanisms / 中华儿科杂志

<p><b>OBJECTIVE</b>Cytomegalovirus (CMV) is the leading infectious cause of congenital anomalies of the central nervous system caused by intrauterine infection. However, the exact pathogenesis of these brain abnormalities has not been fully elucidated. It has been reported that periependymitis, periventricular necrosis and calcification are the most frequent findings in the brains of congenital CMV infection. Because a number of multipotential neural stem cells (NSCs) have been identified from ventricular zone, it is possible that NSCs in this area are primary targets for viral infection, which seems to be primarily responsible for the generation of the brain abnormalities. Therefore, the objective of the present study was to investigate the effect and mechanism of murine cytomegalovirus (MCMV) infection on neural stem cells' differentiation in vitro and its role in the mechanisms of brain abnormalities caused by congenital cytomegalovirus infection.</p><p><b>METHODS</b>NSCs were prepared from fetal BALB/c mouse and were infected with recombinant MCMV RM461 inserted with a report gene LacZ at 1 multiplicity of infection (MOI = 1). The effect of MCMV infection on neural stem cells' differentiation was observed by detecting the ratio of nestin, GFAP and NSE positive cells with immunohistochemistry and flow cytometry on day 2 postinfection. The effects of MCMV infection on gene expression of Wnt-1 and neurogenin 1 (Ngn1) related to neural differentiation were detected by RT-PCR.</p><p><b>RESULTS</b>NSCs isolated from embryonic mouse brains strongly expressed nestin, a specific marker of NSCs and had the capacity to differentiate into NF-200 and NSE positive neurons or GFAP positive astrocytes. At MOI = 1, the results of flow cytometry assay showed that nestin positive cells' proportion in the infection group [(62.2 +/- 1.8)%] was higher than that in the normal group [(37.2 +/- 2.4)%] (t = 4.62, P < 0.01). At the same time, the rates of GFAP and NSE positive cells' in the infection group were significantly lower than those in the normal group (P < 0.01). The scanning densities of Wnt-1 was 0.14 +/- 0.03 in the infection group while 0.32 +/- 0.04 in the control group (t = 7.21, P < 0.01). The scanning densities of Ngn1 were 0.09 +/- 0.01 and 0.21 +/- 0.02 in the two groups (t = 10.7, P < 0.01).</p><p><b>CONCLUSIONS</b>These results suggest that MCMV infection could inhibit neuronal differentiation, which may be primary causes of disorders of brain development in congenital CMV infection. The decreased expression of Wnt-1 and Ngn1 may be involved in the inhibitory effect of murine cytomegalovirus infection on neural stem cells' differentiation, which may lead to a new strategy for preventing and treating brain abnormalities caused by CMV infection through regulating these two signal pathways.</p>

BALB/c mice model system of cytomegalovirus-induced myocarditis / 中华心血管病杂志

<p><b>OBJECTIVE</b>To establish a BALB/c mice model system of cytomegalovirus-induced myocarditis.</p><p><b>METHODS</b>Twenty five specific pathogen-free inbred female BALB/c mice (5 weeks old, 16 - 18 g, seronegative for MCMV) were infected with 1 x 10(4) PFU MCMV by the intraperitoneal (i.p.) route. All experimental mice were sacrificed at 3, 5, 7, 10, 14 days i.p. (n = 5 per time point). Hearts were removed under aseptic conditions, and were transected along the midline. One part of each heart was processed with Bouin's fixative for histological examination. The other part of each heart was immediately frozen in liquid nitrogen and stored at -80 degrees C until MCMV titre was determined by plaque assay. Serum cTnI level was assayed by ELISA.</p><p><b>RESULTS</b>MCMV was detected in the hearts at extremely low levels on 3 days i.p. and could not be detected on 10 days i.p. A mixed cellular infiltrate composed of polymorphonuclear neutrophils and mononuclear lymphocytes was observed on 3 days, which reached a peak at 7 to 10 days after MCMV infection and was maintained for at least 3 - 4 months postinfection. Serum cTnI levels were elevated on 3 days i.p., reaching a peak at 7 to 10 days i.p..</p><p><b>CONCLUSIONS</b>These data highlight the possible therapeutic uses of antiviral drugs in viral myocarditis as well as further elucidating the pathogenic nature of the disease.</p>

Experimental study of mouse cytomegalovirus infected mice / 华中科技大学学报（医学）（英德文版）

In order to investigate the human cytomegalovirus (HCMV) infection, the mouse cytomegalovirus (MCMV) infected mice were experimentally studied. 6 to 8 week old female BALB/C mice with immunosuppression were selected to undergo the MCMV inoculations: intracranial inoculation and peritoneal inoculation. MCMV of the infected mice in various organs and tissues were detected by using beta-gal staining and in situ nucleic acid hybridization assay. The pathological changes were observed in HE staining paraffin-embedded sections. It was found that all the MCMV infected mice showed the retardation of growth and development, and feather looseness. Both intracranial inoculation of 10(4) PFU viruses or peritoneal inoculation of 10(6) PFU viruses resulted in the pathological changes, to some extent, of various organs and tissues in the mice. The pathological changes in liver were consistent with the amount of beta-gal staining positive cells, indicating the liver lesions were mainly caused by viral proliferation. It was also found that the viruses in the immunosuppressed mice subjected to intracranial inoculation could spread to whole body organs, while the viruses in the immunosuppressed mice subjected to intrapeitoneal inoculation couldn't spread to the brain, suggesting blood-brain barrier could prevent the virus from spreading to the brain.

Experimental study of mouse cytomegalovirus infected mice / 华中科技大学学报（医学）（英德文版）

In order to investigate the human cytomegalovirus (HCMV) infection, the mouse cytomegalovirus (MCMV) infected mice were experimentally studied. 6 to 8 week old female BALB/C mice with immunosuppression were selected to undergo the MCMV inoculations: intracranial inoculation and peritoneal inoculation. MCMV of the infected mice in various organs and tissues were detected by using beta-gal staining and in situ nucleic acid hybridization assay. The pathological changes were observed in HE staining paraffin-embedded sections. It was found that all the MCMV infected mice showed the retardation of growth and development, and feather looseness. Both intracranial inoculation of 10(4) PFU viruses or peritoneal inoculation of 10(6) PFU viruses resulted in the pathological changes, to some extent, of various organs and tissues in the mice. The pathological changes in liver were consistent with the amount of beta-gal staining positive cells, indicating the liver lesions were mainly caused by viral proliferation. It was also found that the viruses in the immunosuppressed mice subjected to intracranial inoculation could spread to whole body organs, while the viruses in the immunosuppressed mice subjected to intrapeitoneal inoculation couldn't spread to the brain, suggesting blood-brain barrier could prevent the virus from spreading to the brain.

Lesiones respiratorias y alteraciones en el material del lavado pulmonar en ratones expuestos a la inhalación de basuco / Respiratory injury and alterations in the broncoalveolar lavage in mice exposed to basuco inhalation

Desde 1970, por su menor costo, se ha popularizado el uso de formas intermedias, menos purificadas de cocaína (base de cocaína), especialmente para ser fumadas. Sólo existen informes aislados de manifestaciónes patológicas respiratorias asociadas con fumar base de cocaína, se desarrolló el presente estudio para tratar de caracterizar mejor las lesiones que en el pulmón origina la inhalación de basuco. Se utilizaron dos grupo de veintidos ratones; uno se expuso a basuco y el otro sirvió como control. La dosis de basuco se calculó de acuerdo con la cantidad promedio utilizada por un consumidor diariamente. Se sacrificaron paulatinamente durante un año; se realizó lavado pulmonar y los pulmones fueron extraídos, insuflados y fijados en formol para ser procesados completamente. Los hallazgos de los ratones control fueron normales. En el grupo expuesto, la bronquiolitis linfoplasmocitaria fué el hallazgo más común (44 por ciento), seguido por la neumonitis intersticial (22 por ciento), inflamación linfocitica perivascular (22 por ciento) y bronquiolitis folicular (17 por ciento). La distribución citológica diferencial de los lavados broncoalveolares se correlacionó con los hallazgos histopatológicos. Se demuestra con claridad que la inhalación de basuco induce cambios histopatológicos en el pulmón de ratón, así como en el recuento citológico diferencial del lavado broncoalveolar.