Human induced pluripotent stem cell (iPSC) lines are a promising model for the early phase of human embryonic development. Here, their contribution to the still incompletely understood pathogenesis of congenital virus infections was evaluated. The infection of iPSC lines with miscarriage-associated coxsackievirus B3 (CVB3) and measles virus (MV) was compared to the efficient teratogen rubella virus (RV). While CVB3 and MV were found to be cytopathogenic on iPSC lines, RV replicated without impairment of iPSC colony morphology and integrity. This so far outstanding course of infection enabled maintenance of RV-infected iPSC cultures over several passages and their subsequent differentiation to ectoderm, endoderm, and mesoderm. A modification of the metabolic profile of infected iPSC lines was the only common aspect for all three viruses. This study points toward two important aspects. First, iPSC lines represent a suitable cell culture model for early embryonic virus infection. Second, metabolic activity represents an important means for evaluation of pathogen-associated alterations in iPSC lines.

The study of congenital virus infections in humans requires suitable ex vivo platforms for the species-specific events during embryonal development. A prominent example for these infections is rubella virus (RV) which most commonly leads to defects in ear, heart, and eye development. We applied teratogenic RV to human induced pluripotent stem cells (iPSCs) followed by differentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages. In the presence of RV, lineage-specific differentiation markers were expressed, indicating that lineage identity was maintained. However, portrait analysis of the transcriptomic expression signatures of all samples revealed that mock- and RV-infected endodermal cells were less related to each other than their ecto- and mesodermal counterparts. Markers for definitive endoderm were increased during RV infection. Profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons were found, especially after endodermal differentiation of RV-infected iPSCs. Moreover, the eye field transcription factors RAX and SIX3 and components of the gene set vasculogenesis were identified as dysregulated transcripts. Although iPSC morphology was maintained, the formation of embryoid bodies as three-dimensional cell aggregates and as such cellular adhesion capacity was impaired during RV infection. The correlation of the molecular alterations induced by RV during differentiation of iPSCs with the clinical signs of congenital rubella syndrome suggests mechanisms of viral impairment of human development.

Rubella virus (RV) infection impacts cellular metabolic activity in a complex manner with strain-specific nutritional requirements. Here we addressed whether this differential metabolic influence was associated with differences in oxidative stress induction and subsequently with innate immune response activation. The low passaged clinical isolates of RV examined in this study induced oxidative stress as validated through generation of the reactive oxygen species (ROS) cytoplasmic hydrogen peroxide and mitochondrial superoxide. The addition of the cytoplasmic and mitochondrial ROS scavengers N-acetyl-l-cysteine and MitoTEMPO, respectively, reduced RV-associated cytopathogenicity and caspase activation. While the degree of oxidative stress induction varied among RV clinical isolates, the level of innate immune response and interferon-stimulated gene activation was comparable. The type III IFNs were highly upregulated in all cell culture systems tested. However, only pre-stimulation with IFN β slightly reduced RV replication indicating that RV appears to have evolved the ability to counteract innate immune response mechanisms. Through the data presented, we showed that the ability of RV to induce oxidative stress was independent of its capacity to stimulate and counteract the intrinsic innate immune response.

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The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.