Cellular responses to Sindbis virus infection of neural progenitors derived from human embryonic stem cells
© Xu et al.; licensee BioMed Central Ltd. 2014
Received: 28 July 2014
Accepted: 10 October 2014
Published: 24 October 2014
Sindbis virus (SINV) causes age-dependent encephalitis in mice, and therefore serves as a model to study viral encephalitis. SINV is used as a vector for the delivery of genes into selected neural stem cell lines; however, the toxicity and side effects of this vector have rarely been discussed. In this context, we investigated the cellular responses of human embryonic stem cell (hESCs) derived neural progenitors (hNPCs) to SINV infection by assessing susceptibility of the cells to SINV infection, analyzing the effect of infection on cell proliferation and cell death, and examining the impact of SINV infection on hNPCs markers of stemness.
We found that hNPCs are highly susceptible to SINV infection. Upon infection, the viruses induced apoptosis to hNPCs while not affecting the expression of cell proliferation markers. Lastly, SINV infections result in significant changes in the expression of key regulators of hNPCs’ plasticity and homeostasis.
The robust and versatile signaling, proliferation, and other cell responses of hESCs-derived hNPCs to virus infection demonstrated that it is a good model to study the pathogenesis of viral-induced neurodevelopmental and degenerative diseases. On the other hand, the toxicity of SINV to hNPCs cells cannot be ignored, and therefore extra care should be taken when using SINV as a vector to deliver genes into human stem cell lines.
KeywordsStem cell infection Human neural progenitors Sindbis virus
Sindbis virus (SINV), a positive strand RNA virus in the genus Alphavirus of the Togaviridae family, causes rash and fever in humans and age-dependent encephalitis in mice. The virus has long served as a model to study viral encephalitis induced by viruses in the Togaviridae family, as well as other neurovirulent viruses [1, 2]. More importantly, SINV has been widely used to express a variety of genes in cultured neurons and in vivo as it provides fast onset and high level of expression of foreign genes . The high and rapid expression of foreign genes from SINV vector is accomplished at the cost of shutting off protein nsP2 synthesis in the infected host cells. As nsP2 are commonly encoded in the SINV vector, whether this machinery leads to the toxicity of the SINV vector in cells are unknown . In recent studies, SINV was used as vector to deliver HIV gp120 into hNPCs and showed a lytic effect to cells, while others using wild-type HIV did not [5, 6]. The mechanisms governing pathogenic outcome and extent of SINV replication in human cells are not well characterized [7–9]. As SINV gains popularity in neurotherapy as an ideal vector for gene transfer into neural stem/progenitor cells, the toxicity, as well as other side effects of these vectors, needs to be addressed [3, 10].
Recently, hNPCs have been developed commercially as a model to study developmental neurotoxicity and neurotherapy . In this study, we used hNP1 cells (ArunA Biomedical), an hNPC line derived from the NIH-approved H9 (WiCell Research Institute’s H9 (WA09)) human embryonic stem cell line . Undifferentiated hNP1 cells stably express the stemness markers Nestin and SOX2, and the cells have the capacity to differentiate into multiple neuronal subtypes, including cholinergic, dopaminergic, and GABAergic neurons, glia, and oligodendrocytes. hNP1 cells have not been immortalized or otherwise transformed, and therefore the potential caveats of transformation are not an issue when using these cells. In addition, hNP1 can grow as a monolayer without fibroblast support, another advantage over other primary neuronal stem cells. Lastly, hNP1 cells have been successfully used in the study of radiation sensitivity, neurotoxicity screening, neurophysiology, tissue engineering, and translational medicine [13–19]. Better still, hNP1 cells have been recently applied to neurotherapies of multiple sclerosis patients; techniques such as SINV-based vector-mediated gene transfer were intensively utilized . Thus, the study of the cellular responses of hNP1 cells to SINV infection will serve as a reference for the application of commercialized hNPCs in stem cell infection and translational medicine field.
Here, we show that hNPCs are highly susceptible to SINV infection with a cytopathic effect (CPE) starting at 24 hours post infection (h.p.i). SINV replicated and disseminated effectively as virus titer increased by 100 fold in hNPC cells, and the percentage of infected cells reached over 85% at later time points. Besides these developments, the virus also triggered apoptosis while not affecting the expression of cell proliferation markers. In addition, reduced expression of hNPCs stemness marker Nestin was observed throughout the infection time course. Close scrutiny suggested that SINV upsets the dedicated balance of hNPC cell signaling, such as STAT3, but not NF-kB and pIRF3. Thus, we added this commercialized hNPC line into another type of human neural stem cells that are susceptible to SINV infection. SINV establish lytic replication cycles in hNPCs, and thus extra care should be taken when using SINV-based vector for gene delivery in stem cell therapy.
Material and methods
hNP1™ Neural Progenitor cells (hNPC’s) (ArunA Biomedical) derived from the WA09 hESCs were cultured in Matrigel™ (BD Bioscience) coated tissue culture. Cells were maintained in complete neural expansion medium composed of Neuro-X™ (Jeevan Biosciences, Inc. (Dunwoody, Georgia)) medium supplemented with 20 ng/ml Leukemia Inhibitory Factor (LIF, Millipore), 2 mM L-Glutamine, 0.5 U/ml penicillin, 0.5 U/ml streptomycin (both from Invitrogen), 20 ng/ml basal human fibroblast growth factor (bFGF, Millipore) at 37°C and 5% CO2. Culture medium was changed every other day, and hNP1 cells were passaged every 3–4 days using either a cell scraper or manual pipetting. For all the experiments described below, passage 6–10 cells were used. Baby hamster kidney (BHK-21) cells for preparing SINV stock were obtained from the American Type Culture Collection (ATCC) and grown and maintained at 35°C in Dulbecco’s Modified Eagle Medium (D-MEM, Cellgro) with 5% fetal bovine serum (FBS), as described previously .
Virus stock preparation
To prepare SINV stock, 80% confluent BHK cells were infected with the SINV HR strain at a multiplicity of infection (m.o.i) of 0.1. Culture medium was collected at 2 days post infection (d.p.i) when CPE was obvious in the culture. Cell-free (clarified) virus stock was prepared by collecting supernatant of such medium after high speed centrifugation. hNP1 cells were infected as follows: after the removal of culture media, cells were washed once with phosphate buffered saline (PBS) with calcium and magnesium (KCl 2.68 mM, KH2PO4 1.47 mM, NaCl 136.89 mM, Na2HPO4 8.1 mM, CaCl2 0.9 mM, MgCl2 0.49 mM) and infected with SINV stock at the appropriate m.o.i. The plates were incubated at 37°C for one hour to allow adsorption. The inoculum was removed and replaced by complete neural expansion medium supplemented with growth factors. Culture medium was collected at appropriate times to test for the presence of virus by standard plaque assay on Vero cells.
SINV growth curve characterization
To characterize SINV growth kinetics, hNP1 cells at 60-70% confluency were infected with SINV at an m.o.i of 1, and the medium was collected at 4, 8, 12, 24, and 48 hours post infection. The amount of infectious SINV in the medium collected from infection hNP1 cells was titered by plaque assay. Plaque assay was performed as previously described with minor modifications .
Immunofluorescent assay (IFA)
Standard immunocyto-fluorescence was performed. hNPCs grown on coated glass-coverslips (80% confluence) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 10 minutes at room temperature, rinsed twice with PBS and then permeabilized with 0.2% TritonX-100 diluted in PBS for 5–7 min. Primary antibodies used were directed against Nestin (a neural stem marker; 1:450; Neuromics), SINV-NSP (a SINV antigen, 1:100, Eptomics), active-caspase 3 (1:500; Cell signaling), and β-tubulin (cytoskeleton marker, 1:200, Abcam). Secondary antibodies were anti-rabbit/mouse Alexa Fluor 488 and anti-rabbit/mouse Alexa Fluor 594 (1:2000–4000; Molecular Probes-Invitrogen Life Technologies). Fluorescence images were acquired on a Zeiss Axioplan epifluorescence wide-field microscope and processed with AxioVision software. For each condition within the same experiment, at least 3 fields were analyzed. For image quantification, at least three fields in the same experiment were analyzed.
The percentage of cells expressing virus antigens or lineage specific markers was determined by flow cytometry. hNPCs were harvested and washed twice with 2%FBS/PBS (staining buffer) and then fixed by BD Cytofix/Cytoperm solution according to manufacturer’s instruction (BD Bioscience), in aliquots of 100,000 cells in replicate for each antigen. Each aliquot was stained with one or two of the selected cell marker antibodies for 1 hr on ice. Antibodies used were: anti active Caspase-3 (conjugated with V450, BD Pharmingen) and anti-Nestin (conjugated with PE, BD Pharmingen). Cells stained with isotype (mouse IgG V450 or mouse IgG PE, BD Pharmingen) were used as controls. Flow cytometry was performed using a BD LSR Fortessa (BD Bioscience). Data analysis was performed using FACS Diva software (BD Bioscience). The percentage of cells expressing fluorescence intensity greater than the control cells was calculated.
For the assessment of the effect of SINV on hNPCs differentiation, hNP1 cells were seeded onto 60 mm2 plates in a density of 2 × 106 cell/plate and infected with SINV at an m.o.i of 1. Mock infected and SINV infected samples were collected at 12, 24, and 48 hours post infection and subjected to an EdU incorporation assay (Invitrogen) by flow cytometry for assessment of cell cycle and proliferation.
Western Blot analysis was performed on cell lysates of hNPCs, either mock infected or SINV infected, at 4, 12, 24, 36, and 48 hours post infection, using protocols described previously . Primary antibodies used were anti-GAPDH (1:5000; Abcam), anti-NF-kB p65 (1:200; Santa Cruz); anti-phospho-STAT3 (1:200; Cell signaling), anti-phospho-IRF3 (1:1000; Eptomics); anti-Nestin (1:500; Neuromics); anti-neuro-filament M (NF-M; 1:500; Neuromics); anti-Tuj1 (1:1000; Abcam); anti-PCNA (1:1000, Santa Cruz) and anti-cleaved-caspase 3 (1:1000; Cell Signaling). For quantification of western blot, films of immunoblot were scanned with a flat-bed scanner, and digital images were imported and quantified using Image J software . Then, the intensities of bands were compared according to their grayscale (http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html).
hNPCs are fully permissive to SINV infection
SINV inhibits hNPCs proliferation by inducing apoptosis, but not cell cycle arrest
Thus, SINV limited hNPC proliferation primarily by inducing cell death/apoptosis instead of promoting cell cycle arrest.
Proliferating hNPCs loose cellular multipotency/stemness upon SINV infection
As hNPCs exhibited a neuron-like elongated cell shape at the end of SINV infection time course, we wanted to investigate whether SINV induces premature differentiation or loss of cellular multipotency upon infection. The percentage of Nestin-positive cells was analyzed at 24 hours after SINV infection (Figure 3A). SINV infection decreased Nestin-positive cells by 30% compared to the uninfected control (91% ±1.3% of uninfected vs. 62.2% ±3.33% of SINV infected (Figure 3B). Expression of Nestin was also monitored on the protein level by Western Blot (Figure 3C and D). A similar decrease in Nestin expression was noticed, strongly suggesting that proliferating hNPCs lost their multipotency/stemness during SINV infection. Reduction of Nestin levels was not seen until 24 h.p.i, which is the time point SINV reached its highest infection rate, indicating that alteration in cell multipotency also correlated with virus replication (Figure 3D). To investigate if a premature differentiation was initiated, we probed the cells against lineage specific markers such as Tuj-1, A2B5 and NF-M, however, none of these markers showed a positive staining to hNPCs at 48 h.p.i (data not shown). This suggested that SINV infection impaired hNPCs differentiation potential.
SINV modulates multiple cell signaling pathways of hNPCs during infection
In this study, we addressed the susceptibility of hNPCs to SINV, and their cellular responses to infection. Although SINV does not produce CNS anomalies in human, there are still multiple reasons to study the virus effects on hNPCs: (a) SINV is a model alphavirus that produces age-dependent encephalitis in mice, providing a parallel reference for viral induced human CNS degenerative disease study [27, 28]. (b) SINV is a common test analytic in the class of viral agent for various in vitro models . hESC-derived hNPCs are rarely used in viral pathogenesis studies, and another goal of this study is to assess the feasibility of hNPCs as a model to study viral infection. (c) SINV-based vectors have been widely employed in stem cell infection and tumor therapies in human species, however, little is known about the toxicity of the vector itself. Therefore, in the current study, we evaluate the potential effect of using SINV as vector in the studies of human cells as well as in gene therapies of cancer . For the first time in the Alphavirus genus, we report that SINV infects hNP1 cells and significantly attenuates cell proliferation. The virus induces robust cell death and abruptly terminates hNPC multipotency. The result of our studies could shed light on multiple aspects discussed above.
We have shown that hNPCs are fully permissive for SINV infection. The virus is able to establish productive replication and dissemination in culture, since over 90% of cells were infected at the time peak virus titer achieved, which corresponds to its neurotropic nature. Highly efficient cell entry of SINV is probably due to the abundant availability of the cell surface receptor, laminin, on hNPCs, which has been shown as a receptor for SINV in mammalian cells . SINV infection of human cells has not been extensively studied; however, there are several reports that human brain cells are susceptible to SINV infection. In a comparison of oncolytic potential with eight other viruses, SINV was shown to infect nearly 100% of human glioblastoma cells (U-87MG) and induce apoptosis immediately upon its entry. The spread of SINV has also been demonstrated in human brain microvascular endothelial cells (HBMECs) where the virus infection renders cells hypersensitive to the inflammatory inducer Bradykinin [9, 32]. Here, we also demonstrated sufficient virus replication of SINV in hNPCs and its potential to deplete said cell pool. Therefore, it is intriguing why the virus is not neurovirulent in human CNS. SINV has also been shown to replicate in human peripheral mononuclear cells (PBMC) and decrease cell adhesion . Thus, it is highly possible that the virus is eliminated by the immune system before entering the CNS, as the virus was shown to enter the brain through hematogenous route in mice model .
Several viruses actively induce apoptosis at late stages of infection, thus allowing the dissemination of progeny viruses, while avoiding host inflammatory and immune responses . In mice models, NPCs are very susceptible to SINV induced apoptosis as early as one day post infection . It has been recently shown that Coxsackie virus B (CVB3) and Cytomegalovirus (CMV) can both induce apoptosis in hNPCs, and thus achieve optimal viral dissemination [34, 35]. Consistent with these reports, we observed a strong induction of apoptosis in SINV-infected hNPCs, as illustrated by an increase in the active-caspase 3 positive cell population and protein expression at late stage of infection. Further validation of proliferation damage in hNPCs with progressive infection was obtained by EdU incorporation analysis, which clearly demonstrated that SINV impacted cell metabolism since there was a significant decrease in EdU uptake compared to the uninfected control. Interestingly, we did not observe any reduction of cell cycle progression in hNPCs as the relative expression of PCNA was consistent even at a later time points of infection, indicating that cell cycle arrest does not contribute to the reduced NPC population following infection. Thus, considering similarities between human NPC and murine NPC in their responses to SINV infection, our study validated that hNP1 cells, as an established cell line, retained their capacities to respond effectively and efficiently, and generated genuine response to SINV infection.
Next, we sought to determine if SINV impacts differentiation of hNPCs. However, as the virus almost depleted the hNPC pools in 2 days, it is thus impossible to initiate any type of differentiation in presence of the virus. But still, we were able to capture a significant reduction of stemness marker Nestin expression in proliferation hNPCs after infection, indicating the virus infection triggered abnormal neural precursor development. Reduced Nestin expression has been highlighted in a recent study in which the researchers reported that a SINV-based vector expressing HIV envelope protein (SIN-HIVenv) could impair murine neural stem cell survival and expression of Nestin . The role of HIVenv in damaging NPCs was ruled out by other groups, as it was reported that few apoptosis or TUNNEL positive NPCs were detected following exposure of the cells to either high concentration of gp120, or in the SGZ of gp120 transgenic mice [36, 37]. Thus, it is the SINV vector that disturbs Nestin expression observed in the neural stem cells. Taking these studies together with ours, we showed that SINV impacts the multipotency of NPCs, and therefore extra care should be taken when using SINV-based vector to deliver genes into NPCs. Further studies should focus on determining if the reduced expression of Nestin is directly triggered by SINV, or by a bystander effect, as well as characterizing the cell lineage that hNPCs tend to differentiate into after SINV infection.
In the last part of our study, we took advantage of the strong cell responses that SINV triggered to investigate cell-signaling profile of hNPCs, which regulated its diverse response to pathogens.
The JAK/STAT pathway plays a pivotal role in balancing hNPCs lineage specific differentiation . The reduced pSTAT3 level in SINV-infected hNCPs indicates that the virus probably impaired cell specific differentiation into this cell type. Extracellular signal-regulated kinases (ERK1/2) regulate multiple cell events, including apoptosis in hNPCs [26, 32]. In our study, we observed that SINV infection induced the phosphorylation of both ERKs at early time points post infection. The level of pERK1/2 was decreased later on, suggesting that the virus interplays with cellular apoptosis signaling with progression of infection, possibly to achieve its optimal replication and dissemination in culture. Consistent with our study, these changes of ERK1/2 expression upon SINV infection were also reported in HMBCs . NF-kB plays a central role in cellular stress responses and in inflammation by controlling the expression of a network of inducers and effectors; in this way, it defines the response to a specific pathogen [39, 40]. Surprisingly, our results showed that expression of NF-kB was not altered by SINV infection, an indication of a lack of an inflammatory response during infection. IRF3 mediates Type I-IFN induction and signaling. Type-I IFN signaling was enhanced during hNPCs differentiation, suggesting maturation of the innate immune system during fetal development . However, in our study, we did not observe any changes of IRF3 induction upon virus infection, implying a lack of IFN responses to SINV infection. On the other hand, we may have simply verified the immaturity of innate immune system in hNPCs .
In summary, we showed that SINV establishes productive infection to hNPCs, induces massive apoptosis/cell death, and alters stem cell marker expression. This is in combination with the virus’ ability to avoid cell inflammation responses by upsetting specific cell signaling and taking advantage of hNPCs’ intrinsic immaturity in innate immune system. In particular, the latter may contribute to the age dependence of neurological disease seen in the SINV-mouse model. The robust and diverse cell responses of hESC-derived hNPCs to SINV infection demonstrated that it is a good model to study stem cell infection. We are adding hNP1 cells to a novel type of human stem cells that are susceptible to SINV infection, and prove its cytopathic effect in stem cell lineages. Therefore, extra care should be taken when utilizing SINV-based vectors for human disease gene therapy and stem cell infection.
JX is a Postdoctoral Fellow in Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine.
This research was supported by a grant from NIH (AI21389) to TKF. We thank Christie Sleigher for technical support on tittering SINV. Sümeyra Naz Usta from Jeevan Bioscience for technical support and instructions on using Neuro-X media. Dr. Yumei Zhou for her suggestions in initiating this study.
- Lewis J, Wesselingh SL, Griffin DE, Hardwick JM: Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence. J Virol. 1996, 70: 1828-1835.PubMedPubMed CentralGoogle Scholar
- Nargi-Aizenman JL, Griffin DE: Sindbis virus-induced neuronal death is both necrotic and apoptotic and is ameliorated byN-methyl-d-aspartate receptor antagonists. J Virol. 2001, 75: 7114-7121. 10.1128/JVI.75.15.7114-7121.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Ehrengruber MU, Schlesinger S, Lundstrom K: Alphaviruses: Semliki forest virus and Sindbis virus vectors for gene transfer into neurons. Curr Protoc Neurosci. 2011, Chapter 4: Unit 4.22. doi:10.1002/0471142301.ns0422s57. PubMed PMID: 21971849Google Scholar
- Atasheva S, Gorchakov R, English R, Frolov I, Frolova E: Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J Virol. 2007, 81: 5046-5057. 10.1128/JVI.02746-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Schwartz L, Major EO: Neural progenitors and HIV-1-associated central nervous system disease in adults and children. Curr HIV Res. 2006, 4: 319-327. 10.2174/157016206777709438.PubMedView ArticleGoogle Scholar
- van Marle G, Antony JM, Silva C, Sullivan A, Power C: Aberrant cortical neurogenesis in a pediatric neuroAIDS model: neurotrophic effects of growth hormone. AIDS. 2005, 19: 1781-1791. 10.1097/01.aids.0000189854.06194.87.PubMedView ArticleGoogle Scholar
- Garmashova N, Gorchakov R, Frolova E, Frolov I: Sindbis virus nonstructural protein nsP2 Is cytotoxic and inhibits cellular transcription. J Virol. 2006, 80: 5686-5696. 10.1128/JVI.02739-05.PubMedPubMed CentralView ArticleGoogle Scholar
- Recillas-Targa F: Gene transfer and expression in mammalian cell lines and transgenic animals. Methods Mol Biol. 2004, 267: 417-433.PubMedGoogle Scholar
- Rust NM, Papa MP, Scovino AM, da Silva MMC, Calzavara-Silva CE, Marques ETDA, Peçanha LMT, Scharfstein J, Arruda LB: Bradykinin enhances Sindbis virus infection in human brain microvascular endothelial cells. Virology. 2012, 422: 81-91. 10.1016/j.virol.2011.10.003.PubMedView ArticleGoogle Scholar
- Jeromin A, Yuan LL, Frick A, Pfaffinger P, Johnston D: A modified Sindbis vector for prolonged gene expression in neurons. J Neurophysiol. 2003, 90: 2741-2745. 10.1152/jn.00464.2003.PubMedView ArticleGoogle Scholar
- Breier JM, Gassmann K, Kayser R, Stegeman H, De Groot D, Fritsche E, Shafer TJ: Neural progenitor cells as models for high-throughput screens of developmental neurotoxicity: State of the science. Neurotoxicol Teratol. 2010, 32: 4-15. 10.1016/j.ntt.2009.06.005.PubMedView ArticleGoogle Scholar
- Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM: Embryonic stem cell lines derived from human blastocysts. Science. 1998, 282: 1145-1147.PubMedView ArticleGoogle Scholar
- Acharya MM, Lan ML, Kan VH, Patel NH, Giedzinski E, Tseng BP, Limoli CL: Consequences of ionizing radiation-induced damage in human neural stem cells. Free Radic Biol Med. 2010, 49: 1846-1855. 10.1016/j.freeradbiomed.2010.08.021.PubMedView ArticleGoogle Scholar
- Cheng K, Kisaalita WS: Exploring cellular adhesion and differentiation in a micro-/nano-hybrid polymer scaffold. Biotechnol Prog. 2010, 26: 838-846. 10.1002/btpr.391.PubMedView ArticleGoogle Scholar
- Dodla MC, Mumaw J, Stice SL: Role of astrocytes, soluble factors, cells adhesion molecules and neurotrophins in functional synapse formation: implications for human embryonic stem cell derived neurons. Curr Stem Cell Res Ther. 2010, 5: 251-260. 10.2174/157488810791824520.PubMedView ArticleGoogle Scholar
- Jin K, Xie L, Mao X, Greenberg MB, Moore A, Peng B, Greenberg RB, Greenberg DA: Effect of human neural precursor cell transplantation on endogenous neurogenesis after focal cerebral ischemia in the rat. Brain Res. 2011, 1374: 56-62.PubMedPubMed CentralView ArticleGoogle Scholar
- Lai B, Mao XO, Greenberg DA, Jin K: Endothelium-induced proliferation and electrophysiological differentiation of human embryonic stem cell-derived neuronal precursors. Stem Cells Dev. 2008, 17: 565-572. 10.1089/scd.2007.0124.PubMedPubMed CentralView ArticleGoogle Scholar
- Wu ZZ, Kisaalita WS, Wang L, Zachman AL, Zhao Y, Hasneen K, Machacek D, Stice SL: Effects of topography on the functional development of human neural progenitor cells. Biotechnol Bioeng. 2010, 106: 649-659. 10.1002/bit.22715.PubMedView ArticleGoogle Scholar
- Young A, Machacek DW, Dhara SK, Macleish PR, Benveniste M, Dodla MC, Sturkie CD, Stice SL: Ion channels and ionotropic receptors in human embryonic stem cell derived neural progenitors. Neuroscience. 2011, 192: 793-805.PubMedPubMed CentralView ArticleGoogle Scholar
- Cristofanilli M, Cymring B, Lu A, Rosenthal H, Sadiq SA: Cerebrospinal fluid derived from progressive multiple sclerosis patients promotes neuronal and oligodendroglial differentiation of human neural precursor cells in vitro. Neuroscience. 2013, 250: 614-621.PubMedView ArticleGoogle Scholar
- Frolova EI, Gorchakov R, Pereboeva L, Atasheva S, Frolov I: Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol. 2010, 84: 11679-11695. 10.1128/JVI.01441-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Matthews J, Morgan R, Sleigher C, Frey T: Do viruses require the cytoskeleton?. Virol J. 2013, 10: 121-10.1186/1743-422X-10-121.PubMedPubMed CentralView ArticleGoogle Scholar
- Matthews JD, Tzeng WP, Frey TK: Determinants in the maturation of rubella virus p200 replicase polyprotein precursor. J Virol. 2012, 86: 6457-6469. 10.1128/JVI.06132-11.PubMedPubMed CentralView ArticleGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Meth. 2012, 9: 671-675. 10.1038/nmeth.2089.View ArticleGoogle Scholar
- Rajan P: STATus and context within the mammalian nervous system. Mol Med. 2011, 17: 965-973.PubMedPubMed CentralView ArticleGoogle Scholar
- Shioda N, Han F, Fukunaga K: Role of Akt and ERK signaling in the neurogenesis following brain ischemia. Int Rev Neurobiol. 2009, 85: 375-387.PubMedView ArticleGoogle Scholar
- Griffin DE: Role of the immune response in age-dependent resistance of mice to encephalitis due to Sindbis virus. J Infect Dis. 1976, 133: 456-464. 10.1093/infdis/133.4.456.PubMedView ArticleGoogle Scholar
- Reinarz AB, Broome MG, Sagik BP: Age-dependent resistance of mice to sindbis virus infection: viral replication as a function of host age. Infect Immun. 1971, 3: 268-273.PubMedPubMed CentralGoogle Scholar
- Thach DC, Shaffer KM, Ma W, Stenger DA: Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus. Biosens Bioelectron. 2003, 18: 1065-1072. 10.1016/S0956-5663(02)00246-4.PubMedView ArticleGoogle Scholar
- Wollmann G, Tattersall P, van den Pol AN: Targeting human glioblastoma cells: comparison of nine viruses with oncolytic potential. J Virol. 2005, 79: 6005-6022. 10.1128/JVI.79.10.6005-6022.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang KS, Kuhn RJ, Strauss EG, Ou S, Strauss JH: High-affinity laminin receptor is a receptor for Sindbis virus in mammalian cells. J Virol. 1992, 66: 4992-5001.PubMedPubMed CentralGoogle Scholar
- Nuttall JR, Oteiza PI: Zinc and the ERK kinases in the developing brain. Neurotox Res. 2012, 21: 128-141. 10.1007/s12640-011-9291-6.PubMedPubMed CentralView ArticleGoogle Scholar
- Levine B: Apoptosis in viral infections of neurons: a protective or pathologic host response?. Curr Top Microbiol Immunol. 2002, 265: 95-118.PubMedGoogle Scholar
- Feuer R, Pagarigan RR, Harkins S, Liu F, Hunziker IP, Whitton JL: Coxsackievirus targets proliferating neuronal progenitor cells in the neonatal CNS. J Neurosci. 2005, 25: 2434-2444. 10.1523/JNEUROSCI.4517-04.2005.PubMedView ArticleGoogle Scholar
- Kosugi I, Shinmura Y, Kawasaki H, Arai Y, Li RY, Baba S, Tsutsui Y: Cytomegalovirus infection of the central nervous system stem cells from mouse embryo: a model for developmental brain disorders induced by cytomegalovirus. Laboratory investigation. J Tech Meth Pathol. 2000, 80: 1373-1383. 10.1038/labinvest.3780145.View ArticleGoogle Scholar
- Das S, Basu A: Viral infection and neural stem/progenitor cell’s fate: implications in brain development and neurological disorders. Neurochem Int. 2011, 59: 357-366. 10.1016/j.neuint.2011.02.020.PubMedView ArticleGoogle Scholar
- Okamoto S-I, Kang Y-J, Brechtel CW, Siviglia E, Russo R, Clemente A, Harrop A, McKercher S, Kaul M, Lipton SA: HIV/gp120 decreases adult neural progenitor cell proliferation via checkpoint kinase-mediated cell-cycle withdrawal and G1 arrest. Cell Stem Cell. 2007, 1: 230-236. 10.1016/j.stem.2007.07.010.PubMedView ArticleGoogle Scholar
- Pleschka S: RNA viruses and the mitogenic Raf/MEK/ERK signal transduction cascade. Biol Chem. 2008, 389: 1273-1282.PubMedView ArticleGoogle Scholar
- Vitiello M, Galdiero M, Finamore E, Galdiero S, Galdiero M: NF-kappaB as a potential therapeutic target in microbial diseases. Mol Biosyst. 2012, 8: 1108-1120. 10.1039/c2mb05335g.PubMedView ArticleGoogle Scholar
- Widera D, Mikenberg I, Kaltschmidt B, Kaltschmidt C: Potential role of NF-kappaB in adult neural stem cells: the underrated steersman?. Int J Dev Neurosci. 2006, 24: 91-102. 10.1016/j.ijdevneu.2005.11.017.PubMedView ArticleGoogle Scholar
- Farmer JR, Altschaefl KM, O’Shea KS, Miller DJ: Activation of the type i interferon pathway is enhanced in response to human neuronal differentiation. PLoS One. 2013, 8: e58813-10.1371/journal.pone.0058813.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.