Infection of iPSC Lines with Miscarriage-Associated Coxsackievirus and Measles Virus and Teratogenic Rubella Virus as a Model for Viral Impairment of Early Human Embryogenesis

Abstract

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.

Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate with Strain-Specific Rubella Virus Phenotypes for Cell Migration and Induction of Apoptosis

“…Closure of the ductus arteriosus requires migration of undifferentiated smooth muscle cells [ 52 ]. The analysis of cell mechanics in the context of RV-associated alterations of developmental processes will be possible in future studies through the availability of iPSC-based model systems [ 53 ]. Our mechanical and functional studies emphasize that the parameters investigated in this study are required to fully elucidate RV-induced pathogenic alterations during both perinatal and postnatal infections.…”

Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation

“…Additionally, a more systematic analysis of RV strains on a cell culture model with relevance to RV teratogenicity is required. We have recently introduced induced pluripotent stem cells (iPSCs) as such a model [ 52 ]. They also represent a suitable in vitro model for RV persistence, which is established shortly after infection of the developing embryo and will be used to follow up the questions raised in this study.…”

Rubella Viruses Shift Cellular Bioenergetics to a More Oxidative and Glycolytic Phenotype with a Strain-Specific Requirement for Glutamine

“…Proteins were extracted from cell monolayers with radioimmunoprecipitation assay buffer (c.c.pro GmbH) according to the manufacturer's instructions. Standardized SDS-PAGE and electroblotting were performed as described previously (23). Briefly, after incubation with the primary antibody overnight at 4°C, the polyvinylidene difluoride membrane was transferred to secondary antibody and finally to ECL solution (LI-COR Biosciences) for chemiluminescence analysis.…”

“…Due to the Warburg effect, tumor cell lines such as A549 rely on glycolysis, which was indicated by a rather low OCR/ECAR value of 2.6 Ϯ 0.8 for A549 compared to 3.7 Ϯ 1.3 for Vero cells and 4.1 Ϯ 1.8 for HUVEC cells (Table 1). A comparative analysis of the metabolic activity of these three cell lines, especially the high glycolytic rate in A549 cells, was illustrated in a recent publication (23).…”

“…Assessment of cytotoxicity. Cytotoxicity based on a MultiTox-Fluor multiplex cytotoxicity assay (Promega) was determined as described previously (23). The ratio of live to dead cells was determined after fluorescence measurements on a Victor X3 microplate reader.…”