Later Passages of Neural Progenitor Cells from Neonatal Brain Are More Permissive for Human Cytomegalovirus Infection

Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development, as well as the state of differentiation of susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. IMPORTANCEHuman cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study, we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells, which are similar to those present in the developing neural tube, do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS. Human cytomegalovirus (HCMV) is the most important infectious cause of congenital developmental birth defects, with the central nervous system (CNS) being a main target. The incidence of fetal transmission is between 1 and 4% of live births. At birth, 5 to 10% of in utero-infected infants display symptoms of CMV inclusion disease, which can range from mild to severe and is often lethal. Neural developmental abnormalities i...

supporting

confidence: 53%

Section: 05)mentioning

confidence: 61%

Human Cytomegalovirus Infection of Human Embryonic Stem Cell-Derived Primitive Neural Stem Cells Is Restricted at Several Steps but Leads to the Persistence of Viral DNA

Belzile

Stark

Yeo

et al. 2014

“…The Wuhan Institute of Virology Institutional Review Board approved (WIVH10201202) the isolation of primary human NPCs from postmortem fetal embryonic tissue and waived the need for consent. The isolated NPCs are maintained in our laboratory and complied with the rule that NPCs must be less than nine passages (49).…”

Section: Methodsmentioning

confidence: 99%

“…After incubation, the cells were washed with fresh medium three times to remove free virus; the cultures were then continued at 37°C until harvesting. At 10 hpi, the cells were fixed with 4% paraformaldehyde (PFA), permeabilized, and subsequently incubated with mouse anti-gE antibody (9H8; Abcam) and Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (A11032; Invitrogen) as described previously (49). Cell nuclei were counterstained with Hoechst 33342 (catalog no.…”

Section: Deletion Of Vzv-orf7 Affects Viral Envelopmentmentioning

confidence: 99%

ORF7 of Varicella-Zoster Virus Is Required for Viral Cytoplasmic Envelopment in Differentiated Neuronal Cells

Jiang

Wang

Jiang

et al. 2017

Self Cite

Although a varicella-zoster virus (VZV) vaccine has been used for many years, the neuropathy caused by VZV infection is still a major health concern. Open reading frame 7 (ORF7) of VZV has been recognized as a neurotropic gene in vivo, but its neurovirulent role remains unclear. In the present study, we investigated the effect of ORF7 deletion on VZV replication cycle at virus entry, genome replication, gene expression, capsid assembly and cytoplasmic envelopment, and transcellular transmission in differentiated neural progenitor cells (dNPCs) and neuroblastoma SH-SY5Y (dSY5Y) cells. Our results demonstrate that the ORF7 protein is a component of the tegument layer of VZV virions. Deleting ORF7 did not affect viral entry, viral genome replication, or the expression of typical viral genes but clearly impacted cytoplasmic envelopment of VZV capsids, resulting in a dramatic increase of envelopedefective particles and a decrease in intact virions. The defect was more severe in differentiated neuronal cells of dNPCs and dSY5Y. ORF7 deletion also impaired transmission of ORF7-deficient virus among the neuronal cells. These results indicate that ORF7 is required for cytoplasmic envelopment of VZV capsids, virus transmission among neuronal cells, and probably the neuropathy induced by VZV infection.IMPORTANCE The neurological damage caused by varicella-zoster virus (VZV) reactivation is commonly manifested as clinical problems. Thus, identifying viral neurovirulent genes and characterizing their functions are important for relieving VZV related neurological complications. ORF7 has been previously identified as a potential neurotropic gene, but its involvement in VZV replication is unclear. In this study, we found that ORF7 is required for VZV cytoplasmic envelopment in differentiated neuronal cells, and the envelopment deficiency caused by ORF7 deletion results in poor dissemination of VZV among neuronal cells. These findings imply that ORF7 plays a role in neuropathy, highlighting a potential strategy to develop a neurovirulenceattenuated vaccine against chickenpox and herpes zoster and providing a new target for intervention of neuropathy induced by VZV.

“…Although the clinical spectrum of neurologic disease following congenital HMCV infection is well documented, the variability in the phenotypes of central nervous system (CNS) sequelae coupled with limited availability of pathological specimens has resulted in an incomplete understanding of the mechanism(s) of CNS disease. The susceptibility of the developing CNS to CMV infection has been suggested to be a determinant of pathogenic outcomes in humans and animal models (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In rodent models, CNS structural damage is associated with infection early in development (19).…”

mentioning

confidence: 99%

Tumor Necrosis Factor Alpha-Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in Murine Cytomegalovirus-Infected Newborn Mice

Seleme

Kosmac

Jonjić³

et al. 2017

Self Cite

Congenital human cytomegalovirus (HCMV) infection is a significant cause of abnormal neurodevelopment and long-term neurological sequelae in infants and children. Resident cell populations of the developing brain have been suggested to be more susceptible to virus-induced cytopathology, a pathway thought to contribute to the clinical outcomes following intrauterine HCMV infection. However, recent findings in a newborn mouse model of the infection in the developing brain have indicated that elevated levels of proinflammatory mediators leading to mononuclear cell activation and recruitment could underlie the abnormal neurodevelopment. In this study, we demonstrate that treatment with tumor necrosis factor alpha (TNF-␣)-neutralizing antibodies decreased the frequency of CD45 ϩ Ly6C hi CD11b ϩ CCR2 ϩ activated myeloid mononuclear cells (MMCs) and the levels of proinflammatory cytokines in the blood and the brains of murine CMVinfected mice. This treatment also normalized neurodevelopment in infected mice without significantly impacting the level of virus replication. These results indicate that TNF-␣ is a major component of the inflammatory response associated with altered neurodevelopment that follows murine CMV infection of the developing brain and that a subset of peripheral blood myeloid mononuclear cells represent a key effector cell population in this model of virus-induced inflammatory disease of the developing brain.IMPORTANCE Congenital human cytomegalovirus (HCMV) infection is the most common viral infection of the developing human fetus and can result in neurodevelopmental sequelae. Mechanisms of disease leading to neurodevelopmental deficits in infected infants remain undefined, but postulated pathways include loss of neuronal progenitor cells, damage to the developing vascular system of the brain, and altered cellular positioning. Direct virus-mediated cytopathic effects cannot explain the phenotypes of brain damage in most infected infants. Using a mouse model that recapitulates characteristics of the brain infection described in human infants, we have shown that TNF-␣ plays a key role in brain inflammation, including recruitment of inflammatory mononuclear cells. Neutralization of TNF-␣ normalized neurodevelopmental abnormalities in infected mice, providing evidence that virus-induced inflammation is a major component of disease in the developing brain. These results suggest that interventions limiting inflammation associated with the infection could potentially improve the neurologic outcome of infants infected in utero with HCMV.