Failure to infect embryos after virus injection in mouse zygotes

f Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.

mentioning

confidence: 99%

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

Pan

Liu

et al. 2013

“…The ability of the virus to infect the developing fetal brain is a key factor in its neuropathogenesis (3)(4)(5)(6)(7)(8). While considerable experimental data were obtained from newborn and embryonic mouse models (6,(9)(10)(11)(12)(13)(14)(15), the strict species specificity precludes animal models of HCMV. Recent studies in human neuronal progenitor cells (NPC) derived from fetal and neonatal brains have revealed productive HCMV infection of NPC, with resultant functional alterations (16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

Transition toward Human Cytomegalovirus Susceptibility in Early Human Embryonic Stem Cell-Derived Neural Precursors

Berger

Gil

Panet

et al. 2015

Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of plateletderived growth factor receptor alpha (PDGFR␣) as a determinant of the developmentally acquired HCMV susceptibility. Human cytomegalovirus (HCMV) is a leading cause of congenital infection (1), associated with neurodevelopmental disabilities (2, 3). The ability of the virus to infect the developing fetal brain is a key factor in its neuropathogenesis (3)(4)(5)(6)(7)(8). While considerable experimental data were obtained from newborn and embryonic mouse models (6, 9-15), the strict species specificity precludes animal models of HCMV. Recent studies in human neuronal progenitor cells (NPC) derived from fetal and neonatal brains have revealed productive HCMV infection of NPC, with resultant functional alterations (16)(17)(18)(19)(20). Notably, NPC do not represent early neural embryonic development but rather a more advanced stage along the neuronal differentiation route. Hence, the earliest events defining the susceptibility of the developing human nervous system to HCMV have remained largely unknown.Human embryonic stem cells (hESC) provide an opportunity to study early human neural development (21-23). We have developed highly reproducible protocols for controlled induction of differentiation of hESC toward a neural lineage, giving rise to enriched populations of proliferating, developmentally multipotent early NPC (24,25).Here, we have employed experimental HCMV infection in a dynamic model of controlled differentiation of hESC into neural precursors, to gain insight into the molecular events mediating HCMV infection during early human neurodevelopment.We first sought to track the earliest stage of neural differentiation at which the cells become susceptible to HCMV. To this end, differentiation of hESC was induced in floating cell clusters toward the formation of neural spheres (Fig. 1A) as detailed previously (24, 25). The emerging neural spheres were infected at sequential time points along the neural differentiation process with the broadly tropic HCMV TB40/E strain expressing green fluorescent protein (GFP) (26,27) (Fig. 1B). In accordance with previous studies (25), immunostaining analysis for the pluripotent stem cell marker TRA 1-81 and the neural differentiation marker PSA-NCAM showed that the majority of the cells underwent early neural differentiation within 11 days (Fig. 1C). Importantly, we have identified a transition toward HCMV susceptibility occurring between 4 and 11 days post-differentiation induction (Fig. 1B). A similar susceptibility pattern was observed for low-passage-number, cell-associated, clinic...

“…Many studies using murine and human CMV in cells ranging from embryonic stem (ES) cells to neural progenitor/stem cells to carcinogenic cell lines have been performed in an attempt to more clearly define this neuropathogenesis (1,15,(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). Significantly, in parallel to the observations of Tsutsui and coworkers, our earlier studies showed that ϳ10% of neuronal cells differentiated from neonatal neural progenitor cells (NPCs) appeared to be persistently infected (45), suggesting that these cells may serve as a reservoir of infectivity within the brain.…”

mentioning

confidence: 99%

Maintenance of Large Numbers of Virus Genomes in Human Cytomegalovirus-Infected T98G Glioblastoma Cells

Duan

Zavala

et al. 2014

After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection IMPORTANCEOur previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.