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Background Childhood neuromyelitis optica spectrum disorders (NMOSDs) may cause visual impairment and brain or spinal cord damage, and the effects may be permanent if left untreated. Since the incidence of NMOSD cases in children is relatively low, the understanding of NMOSD among children is inadequate. Methods This investigation examined the clinical and neuroimaging characteristics of childhood NMOSD. We retrospectively analyzed the clinical information of 11 NMOSD patients admitted to our centre from 2012 to 2021. The disease status was assessed by the Expanded Disability Status Scale (EDSS) score. Results The two major symptoms observed in the study cohort were optic neuritis (ON) (9/11) and encephalopathy (7/11). Antibody tests were performed on 8 children, 2 of whom showed serum aquaporin 4 (AQP4) antibody positivity, and another 2 presented with serum myelin oligodendrocyte glycoprotein (MOG) antibody positivity. All patients showed white matter hyperintensity on magnetic resonance imaging (MRI) scans. Interestingly, a rare radiological sign, enlarged perivascular space (PVS), which is more commonly observed in the elderly or adults, was found in 4 participants with more severe clinical manifestations. Conclusions While NMOSD in children is less commonly diagnosed through clinical evaluations, the symptoms of ON and encephalopathy should raise the possibility of the disease. As the diagnosis of NMOSD in children is relatively difficult, enlarged PVS may represent a promising biomarker for the diagnosis and evaluation of NMOSD.

Autophagy is involved in the pathogenesis of multiple pathogen infection. Previous studies have reported that human cytomegalovirus (CMV) activates autophagy in the early stage of infection and then inhibits autophagy. Little is known about the role of autophagy in murine CMV (MCMV) infection, especially in MCMV-induced hepatitis. The purpose of this study is to investigate the role of autophagy in MCMV hepatitis. BALB/c mice were infected with MCMV and a series of experiments involving western blot, immunofluorescence, immunohistochemistry, HE (Hematoxylin and Eosin) staining and quantitative real-time polymerase chain reaction were performed in this study. The expression of SQSTM1/p62, PI3K, the ratio of phosphorylated Akt to total Akt, and the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR were increased, and the expression of light-chain 3 (LC3)-II were decreased in the livers of infected mice on days 3 and 7 postinfection (p.i.). Compared with the untreated infected group, increased transcription level of MCMV glycoprotein B (gB), increased expression levels of interleukin1-β (IL-1β), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreased expression level of type I interferon α (IFN-α), as well as aggravated liver pathological injury were detected in starvation-treated infected group on days 3 and 7 p.i.; whereas decreased transcription level of MCMV gB, decreased expression levels of IL-1β, AST and ALT, increased expression level of type I IFN-α, as well as alleviated liver pathological injury were detected in chloroquine (CQ)-treated infected group on day 3 p.i. In conclusion, autophagy is inhibited through activating the PI3K/Akt/mTOR pathway in the liver of BALB/c mice during MCMV infection, and autophagy may promote MCMV replication and aggravate liver pathological damage and inflammation. Further understanding of the interactions between autophagy and MCMV infection and its potential mechanism may bring new important cues to the control of MCMV infection and antiviral therapy.

The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

Background Cytomegalovirus (CMV) induced autophagy affects virus replication and survival of the infected cells. The purpose of this study was to investigate the role of autophagy inhibition by 3-methyladenine (3-MA) on murine cytomegalovirus (MCMV) replication and whether it is associated with caspase-3 dependent apoptosis. Methods The eyecup isolated from adult C57BL/6J mice (6-8 weeks old) and mouse embryo fibroblast cells (MEFs) were infected with MCMV K181 strain, followed by the treatment of 3-methyladenine (3-MA), chloroquine(CQ) or rapamycin to block or stimulate autophagy. Results In cultured MEFs, the ratio of LC3I/II was reduced at 24 hours post infection (h.p.i.), but was increased at 48h.p.i. In the eyecup culture, LC3I/II ratio was also decreased at 4 and 7days post infection (d.p.i.). In addition, caspase-3 cleavage was increased at 48h.p.i. in MEFs and also elevated in MCMV infected eyecups at 4, 7, 10 and 14d.p.i. 3-MA treatment significantly inhibited the virus replication in MEFs and eyecups. The expression of early antigen (EA) of MCMV was also decreased in MEFs and eyecups. Meanwhile, cleaved caspase-3 dependent cell death was promoted with the presence of 3-MA in MCMV infected MEFs and eyecups, while RIPK1/RIPK3/MLKL pathway was inhibited by 3-MA in eyecups. Conclusions Inhibition of autophagy by 3-MA restricts virus replication and promotes caspase-3 dependent apoptosis in the eyecup and MEFs with MCMV infection. It can be explained that during the early period of MCMV infection, suppressed autophagy process directly reduced virus release, but later caspase-3 dependent apoptosis dominated and resulted in decreased virus replication. This article is protected by copyright. All rights reserved.

Absent in melanoma 2 (AIM2) inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA (dsDNA). Through establishing models of disseminated murine cytomegalovirus (MCMV) infection in BALB/c and C57BL/6 mice, we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication, trying to figure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV. BALB/c and C57BL/6 mice were sacrificed on day 0, 1, 3, 7, 14 and 28 post infection. Expression levels of AIM2, pro-caspase-1, caspase-1 p20, pro-IL1β and mature IL1β in primary peritoneal macrophages (PMs) and spleens were detected by Western blotting. Contents of IL18 in the serum were detected by ELISA. Pathological examinations of livers were performed, and mRNA levels of MCMV glycoprotein B (gB) in salivary glands also assessed. Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3, even continued to day 28; caspase-1 p20 and mature IL1β increased on day 7, 14 and 28; the persistently high expression of IL18 in the serum started on day 1, showing a double peak curve. As for BALB/c mice, expression of AIM2 in PMs increased on day 1 and day 7, while contents of AIM2 in spleens increased on day 1 and day 3; caspase-1 p20 and mature IL1β merely increased 7 days fter infection. Thereafter, expression levels of AIM2, caspase-1 p20, mature IL1β and IL18 were limited; the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice. The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV. In conclusion, the activation of AIM2 inflammasome in BALB/c mice was short-lived, which is quite possibly related to the chronicity of MCMV infection.

Increasing evidence has revealed that maternal cytomegalovirus (CMV) infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 (TLR2) and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL-10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV (MCMV) infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide (LPS)-treated and uninfected mice were used as controls. At E13.5, E14.5 and E18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 μg/kg could decrease the EL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more proinflammatory cytokine IL-6. High dose of LPS stimulation (50 μg/kg) during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

Background Cytomegalovirus (CMV) infection occurs frequently and is widespread globally. Numerous studies have shown that various types of immune cells play roles in mediating the response to CMV infection. CD11c, a commonly used dendritic cell (DC) marker, is expressed by other immune cells as well, such as T cells. This study analyzed the immune cells that express CD11c and monitored the expression level of their specific cell surface markers in the lung following a disseminated murine (M)CMV infection. Methods Mouse models of disseminated MCMV infection were used; uninfected and lipopolysaccharide (LPS)-treated mice were used as controls. At 1, 3 and 7 days following infection, single cell suspensions prepared from freshly digested lung tissue were stained for CD11c, CD86 and MHC II. Stained cells were analyzed using flow cytometry. Peripheral blood and single cell suspensions from spleen were sorted as well. Then these cells were subjected to analyze the CD11c expression pattern on natural killer (NK) cells and T cells. Results This assay showed that after MCMV infection, the expression of CD86 on pulmonary CD11chiMHC-IIhi cells (encompassing conventional DCs) was higher at 3 days post-infection than at 1 or 7 days post-infection, accompanied by a downregulation of MHC II. In addition, expression of CD11c was greatly increased in the MCMV infection group at 7 days post infection. This study also detected a large population of cells displaying an intermediate level of expression of CD11c (CD11cint); these cells were in the MCMV groups exclusively, and were subsequently identified as CD8+ T cells. In lung, spleen and blood, different proportions of CD11cint cells among the NK cell and T cell populations were observed between the BALB/c and C57BL/6 mice with or without MCMV infection. The expression level of NKp46 in NK cells dropped to a lower level after MCMV infection. Conclusions The findings collectively indicate that CD11cintCD8+ T cells might play a key role in anti-MCMV adaptive immune response in lungs, as well as in spleen and blood. B220+CD11cint NK cells might be a more effective type of NK cell, participating in anti-MCMV infection. The downregulation of NKp46, in particular, might be linked with the immune evasion of MCMV. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0801-x) contains supplementary material, which is available to authorized users.

Aim: This study aimed to explore the potential role of IL-17 in the pathological damage to the salivary gland during CMV infection. Materials & methods: For all studies, 4-week-old female BALB/c mice were used. Mouse embryo fibroblast cells were isolated from embryos of pregnant mice. The Smith murine CMV (MCMV) strain was propagated and obtained from homogenates of the salivary glands. BALB/c mice were randomly divided into two groups. Mice in one group were infected with Smith MCMV to establish disseminated infection; mice in the other group were controls. Four mice of each group were randomly chosen to be harvested on days 3, 7, 14 and 28. Viral titers in salivary gland tissues were determined using a standard plaque assay; IL-17+ T cells in the spleen were analyzed by flow cytometry; the expressions of IL-17 and IL-17 receptor (IL-17R) mRNA was measured by reverse transcription PCR; and the pathology of the salivary glands in infected mice were assessed by hematoxylin and eosin staining. Results: The viral titers in salivary gland tissues, which were much higher than those of other organs, elevated on day 7 and peaked on day 14, then gradually declined on day 28. The percentages of CD4+IL-17+ T cells were increased in MCMV-infected mice compared with controls, with the main increase of CD4+IL-17+ T cells being Th17 cells (p < 0.05). In salivary gland tissues, the expression of IL-17 and IL-17R mRNA increased significantly and achieved the highest level on day 14, which was significantly higher than that of the controls (p < 0.05). Compared with controls, serious inflammatory cellular infiltrates in the salivary glands were present on day 7 after MCMV infection, and on day 14, many of the cellular infiltrate became larger and coalesced with neighboring foci. We found that IL-17 and IL-17R were enriched in the salivary glands when the tissues were mainly injured. Conclusion: The results illustrated that the inflammatory factors, IL-17 and IL-17R, possibly contributed to the process of viral infection and played a part in inducing pathological damage of the salivary gland.

Background AIM2, a cytosolic DNA sensor, plays an important role during infection caused by pathogens with double-stranded DNA; however, its role in human cytomegalovirus (HCMV) infection remains unclear. Previously, we showed an increase in AIM2 protein levels during the early stage of HCMV infection and a decrease 24 h post infection. Because HCMV has developed a variety of strategies to evade host immunity, we speculated that this decline might be attributed to a viral immune escape mechanism. The tegument protein pUL83 is an important immune evasion protein and several studies have reported that pUL83 binds to specific cellular proteins, such as AIM2-like receptor IFI16, to affect their functions. To determine whether pUL83 contributes to the variation in AIM2 levels during HCMV infection, we investigated the pUL83/AIM2 interaction and its impact on the AIM2 inflammasome activation. Methods We constructed plasmids expressing recombinant pUL83 and AIM2 proteins for two-hybrid and chemiluminescence assays. Using co-immunoprecipitation and immunofluorescent co-localization, we confirmed the interaction of pUL83/AIM2 in THP-1–derived macrophages infected with HCMV AD169 strain. Furthermore, by investigating the expression and cleavage of inflammasome-associated proteins in recombinant HEK293T cells expressing AIM2, apoptosis-associated speck-like protein (ASC), pro-caspase-1 and pro-IL-1β, we evaluated the effect of pUL83 on the AIM2 inflammasome. Results An interaction between pUL83 and AIM2 was detected in macrophages infected with HCMV as well as in transfected HEK293T cells. Moreover, transfection of the pUL83 expression vector into recombinant HEK293T cells stimulated by poly(dA:dT) resulted in reduced expression and activation of AIM2 inflammasome-associated proteins, compared with the absence of pUL83. Conclusions Our data indicate that pUL83 interacts with AIM2 in the cytoplasm during the early stages of HCMV infection. The pUL83/AIM2 interaction deregulates the activation of AIM2 inflammasome. These findings reveal a new strategy of immune evasion developed by HCMV, which may facilitate latent infection.

This study investigated the effects of allitridin compound on murine cytomegalovirus (MCMV)-induced regulatory T cell (Treg; CD4(+) CD25(+) Foxp3(+) ) amplification in vivo and in vitro. One hundred twenty MCMV-infected mice were allocated at random into two groups for treatment with allitridin or placebo. Another 120 mock-infected mice were randomly allocated as controls for the allitridin treatment and placebo treatment groups. The mice were euthanized at various time points after infection (out to 120 days) to evaluate the effects of treatment on Treg presence and function, as well as MCMV infective load. Co-culture with mouse embryo fibroblasts (MEF) and MCMV was performed to evaluate allitridin-mediated Treg and anti-CMV effects. The maximum tolerance concentration (MTC) of allitridin was used to treat cells for 3 days. Changes in Foxp3 mRNA and protein levels, percentages of T cell subsets, and Treg-related cytokines (IL-10 and TGF-β) were measured. Allitridin treatment did not influence Foxp3 expression and Treg proportion in uninfected mice, but did down-regulate each in infected mice during the chronic infection period. Additionally, allitridin treatment reduced the MCMV load in salivary glands. MTC allitridin treatment of co-cultures partially blocked MCMV induction of Foxp3 mRNA and protein expression. In vitro treatment with allitridin also increased significantly the percentages of Tc1, Tc2, and Th1, reduced the secreted levels of IL-10 and TGF-β1, and significantly suppressed viral loads. In conclusion, allitridin can promote MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression. Therefore, allitridin may be useful as a therapeutic agent to enhance the specific cellular immune responses against CMV.

Absent in melanoma 2 (AIM2) inflammasome is a multiprotein complex which plays a pivotal role in the host immune response to multiple pathogens. The role of AIM2 in human cytomegalovirus (HCMV) infection is poorly studied. Thus, using a small inference RNA (siRNA) approach and THP-1 derived macrophage cells infected with HCMV AD169 strain, we investigated the impact of HCMV infection on AIM2-mediated molecular events. Compared to wild-type cells, AIM2-defiecient macrophages showed a limited ability to activate caspase-1, process IL-1β, and induce cell death. In addition, AIM2-defiecient cells were unable to efficiently control HCMV infection, as the transcription of virus DNA polymerase gene UL54 and major tegument protein gene UL83 were higher compared to wild-type cells. In conclusion, HCMV infection induces an AIM2 inflammasome response, which negatively influences viral life cycle. This article is protected by copyright. All rights reserved

This study investigated the effects of allitridin on acute and chronic mouse cytomegalovirus (MCMV) infections in vivo. The results demonstrated that allitridin reduced the titers of MCMV in salivary glands, and reductions in viral loads were confirmed by determining viral DNA and RNA levels in susceptible organs during the acute infection phase. Although allitridin did not eliminate MCMV, treatment reduced viral levels and facilitated healing of pathologic lesions in organs, particularly during the chronic infection phase. The results presented in this report suggest that allitridin could act as an effective agent against MCMV infections in vivo.

Cytomegaloviruses (CMVs) cause common viral infectious diseases and are difficult for the host immune system to eliminate, which leads to persistent or chronic infection. To investigate the T cell immune response stimulated by murine cytomegalovirus (MCMV) infection and the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) in this process, T cells containing various proportions of Tregs were co-cultured with MCMV-infected mouse embryo fibroblasts (MEFs). MCMV infection stimulated proliferation of effector T cells as well as differentiation to Tregs, which consequently increased the expression of TGF-beta and IL-10. The proliferation of Tc1 (CD3(+)CD8(+)IFN-gamma(+)), Th1 (CD3(+)CD4(+)IFN-gamma(+)), and Tc2 (CD3(+)CD8(+)IL-4(+)) subsets was significantly suppressed with an increased proportion of Tregs in the co-culture system. Treg-depleted T cells inhibited viral load when co-cultured with MCMV-infected MEFs, however, this inhibitory effect was diminished when an increased proportion of Tregs was introduced. The suppressing effects of Tregs on effector T cells were attenuated by the addition of monoclonal antibody to TGF-beta, but not the one to IL-10, suggesting that TGF-beta is a major messenger involved in the immune suppressing effect of Tregs.

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.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.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.The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.

OBJECTIVE The effect of allitridin on the transcription levels of immediate-early (ie), early(e) and late (1) genes of human cytomegalovirus (HCMV) was investigated in order to explore the mechanism of allitridin against HCMV. METHOD Established the models of HCMV AD169 strain infected cells and AD169 strain infected cells treated with allitridin (9.6 mg x L(-1)), and they were compared with the appropriate dose(2.3 mg x L(-1)) of ganciclovir (GCV). All groups of cells were infected at 2.5 multiplicity of infection (MOI), using SYBR Green real-time PCR method to detect the dynamic change of ul122, ul123, ul54 and ul83 mRNA expression at 0.5, 2, 4, 6, 12, 24 h post-infection. RESULT The mRNA levels of ul122 and ul123 in AD169 infected cells treated with allitridin at all time points were markedly lower than those of AD169 infected controls (P

To investigate the effect of allitridin on murine cytomegalovirus (MCMV) induced regulatory T cells (Treg) amplification in vitro.A co-culture system of T cells and MCMV infected mouse embryo fibroblasts (MEF) was established. A maximum tolerance concentration (MTC) of allitridin was added into the co-culture system. After 3 days, the change of Foxp3 mRNA was measured by real-time PCR. And the percentages of Tc1 (CD3(+)CD8(+)IFN-γ(+)), Tc2 (CD3(+)CD8(+)IL-4(+)), Th1 (CD3(+)CD8(-)IFN-γ(+)) and Th2 (CD3(+)CD8(-)IL-4(+)) were analyzed by flow cytometry. The production of IL-10 and TGF-β in supernatants was detected with double-antibody sandwich ELISA while the viral load of culture quantified by plaque assay. All results were compared with those of the placebo group.The MTC of allitridin was 9.83 µg/ml in MEF. The treatment of 9.83 µg/ml allitridin could partly block the MCMV induction of Foxp3 mRNA expression [(87 ± 5) vs (114 ± 8), P0.01]. The percentages of Tc1, Tc2 and Th1 significantly increased to the levels of (12.42 ± 1.23)%, (4.28 ± 0.56)% and (13.25 ± 0.68)% respectively. They showed statistic differences with those of placebo controls [(6.85 ± 0.92)%, (2.34 ± 0.42)% and (9.32 ± 0.86)%; all P0.01]. Meanwhile, the levels of IL-10 and TGF-β1 in supernatants also significantly decreased to (29.98 ± 3.15) pg/ml and (3.48 ± 0.23) ng/ml by allitridin treatment as compared with placebo controls [(38.21 ± 4.02) pg/ml and (5.31 ± 0.59) ng/ml, all P0.05]. In addition, the MCMV plaque assays showed that allitridin significantly suppressed the viral loads by one order of magnitude.Allitridin can partly retrieve MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression so as to enhance the specific cellular immune responses against CMV.