Infection of iPSC Lines with Miscarriage-Associated Coxsackievirus

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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 Denise Huebner, Kristin Jahn, Sandra Pinkert, Janik Böhnke, Matthias Jung, Henry Fechner, Dan Rujescu, Uwe Gerd Liebert, and Claudia Claus ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00103 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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ACS Infectious Diseases

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 Denise Hübner1, Kristin Jahn2§, Sandra Pinkert3$, Janik Böhnke1, 2, Matthias Jung4, Henry Fechner3, Dan Rujescu4, Uwe Gerd Liebert1, Claudia Claus1*

1

Institute of Virology, University of Leipzig, Johannisallee 30, 04103 Leipzig,

Germany 2

Institute of Virology, University of Leipzig; Faculty of Life Sciences, University of

Leipzig, Talstrasse 33, 04103 Leipzig; §Current address: Institute of Biology and SIKT, University of Leipzig, Philipp-Rosenthal-Str. 55, 04103 Leipzig, Germany 3

Department of Applied Biochemistry, Institute of Biotechnology, Technische

Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany; $Current address: Charité, University Medicine Berlin, Institute of Biochemistry, Charitéplatz 1, 10117 Berlin, Germany 4

Department of Psychiatry, University of Halle-Wittenberg, Julius-Kühn-Str. 7, 06112

Halle, Germany

* Corresponding author [email protected]

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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 towards 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.

Keywords: rubella virus; coxsackievirus; measles virus; induced pluripotent stem cells; in vitro model for embryogenesis


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The unlimited capacity for self-renewal and differentiation into various somatic cell types are highly valuable properties of pluripotent stem cells (PSC), which comprise embryonic and induced pluripotent stem cell (ESC and iPSC, respectively) lines. Human (h)iPSCs were initially derived from skin fibroblasts through the introduction of just four transcription factors acting as reprogramming inducers (OCT4 [octamerbinding transcription factor 4; also known as OCT3/4], SOX2 [SRY (sex determining region Y)-box 2], KLF4 [Kruppel-like factor 4], and c-myc) 1. iPSC lines are now not only regarded as a functional amendment to ESCs

2, 3

they also possess several

beneficial properties. They are less controversial regarding ethical issues than ESCs, which are derived from inner cell mass of late-stage human blastocysts

4

.

Additionally, multiple iPS cell lines with great donor diversity match the genetic diversity found in the human population and enable the generation of so called iPSCderived somatic human cell types. iPSC lines are also an in vitro cell culture model for the early steps of human development, representing the blastocyst stadium until a gastrulation-like stage

5, 6

. Both, iPSC as well as ESC lines were already used for

embryotoxicity assessment of known chemical teratogens, including ethanol and thalidomide (traded as Contergan)

7, 8

. The transcriptomic changes observed in

differentiating hESCs after exposure to thalidomide comprised differential expression of transcription factors associated with limb development and thus mirror the symptoms observed in malformed children 7. In the field of virology iPSC-derived hepatocytes, cardiomyocytes, and neural progenitor cells or even neurons, have provided cellular models for the infection with various viruses including hepatotropic viruses, coxsackieviruses, as well as Zika virus and

herpesviruses

(varicella

cytomegalovirus), respectively

zoster

9, 10

virus,

herpes

simplex

virus,

and

. This already promising approach holds the 3

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potential to be extended towards the study of congenital (intrauterine) viral infections and the viral impact on early human embryogenesis. This is not only an underdetected, but also poorly evaluated aspect in virus-associated pathologies

11, 12

. The

availability of such a model might even contribute to the assessment of vaccine safety during early pregnancy

13

. Moreover, cell survival as well as cell death signals

in PSCs, especially under virus infections, are not well studied 14. For this study three viral pathogens were chosen as representative agents for in utero viral infections in humans: mainly miscarriage-associated measles virus (MV) and coxsackievirus B3 (CVB3)

15, 16, 17, 18, 11

as opposed to teratogenic rubella virus

(RV). Additionally, CVB3 infection during early gestation induces a high rate of abortions in mice of pregnancy

19

. It is important to note, that RV can infect the fetus at any stage

20, 21, 22

. In the first trimester congenital rubella syndrome (defined as

the classical triad of cataracts, cardiac abnormalities, sensorineural deafness) and only occasionally miscarriages develop, but defects are rarely seen after the 16th week of gestation 21, 23. Our research focus is set on elucidation of RV teratogenicity. CVB3 as well as MV lack teratogenic malformations, hence they are useful to distinguish RV-specific from rather general viral alterations on iPSC lines. Through this study metabolic alterations were identified as a common factor for cytopathogenic CVB3 and MV as well as noncytopathogenic RV replication. RV allowed passaging of infected iPSC colonies and subsequent differentiation into ectoderm, endoderm, and mesoderm. Data presented provide evidence that iPSC lines are an important tool for the study of the so far under-characterized field of virus-associated alterations during the early and highly vulnerable phase of human development.


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Results and discussion Pluripotent stem cell technology holds great potential for developmental biology as it offers recapitulation of human development “in a dish” 6. Here we have used this approach to study so far under-characterized viral alterations during the very early phase of human embryogenesis. CVB3 and MV are in vivo associated with a high rate of miscarriages

15, 16, 17

the first day of fertilization

. They were contrasted to RV, which is teratogenic from

20, 21, 22

. Figure 1A illustrates the experimental approach

that was undertaken in this study for assessment of viral impairment of iPSC lines.

Fig. 1. Outline of the experimental approach and characterization of the iPSC lines chosen for this study (A) The experimental outcomes that were anticipated for the characterization of viral alterations in iPSC lines are summarized. (B) Nuclear extracts obtained from iPSC lines and terminally differentiated Vero cells were subjected to Western blot analysis for assessment of the pluripotency markers OCT4 and SOX2. PCNA protein band was used as loading control. (C) and (D) Extracellular flux measurement based on the medium conditions of the Mito Stress Test Kit was applied to the indicated cell lines at 1 or 2 days after plating (A549 n=2; HUVEC n=2; HEK n=2; Vero n=2; IMR90 iPSC n=3; A18945 iPSC n=3). (C) The ratio of basal oxygen consumption rate (OCRbasal) and extracellular acidification rate (ECARbasal) was determined. A low OCRbasal/ECARbasal ratio indicates a preference for glycolysis. 5 ACS Paragon Plus Environment


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(D) Illustration of the ratio of the maximum OCR value (OCRmax) reached under stressed conditions after injection of FCCP and basal OCR. A high OCRmax/ OCRbasal ratio reflects a higher respiratory capacity of the given cell line. The two iPSC lines A18945 and IMR90 were used in this study as they were generated through two different approaches, plasmid- and retroviral vector-based expression of pluripotency factors, respectively. To strengthen their dual application in the verification of more general, not cell line-restricted viral effects, the expression level of OCT4 and SOX2 as pluripotency markers and metabolic activity as an important parameter of the cellular phenotype were determined. Expression levels of pluripotency markers were comparable between both iPSC lines (Fig. 1B). Reflecting the state found in the embryo, iPSCs rely on glycolysis instead of oxidative phosphorylation for energy generation

24, 25

. This aspect can be highlighted by

extracellular flux analysis through the ratio of basal oxygen consumption rate (OCR) to basal extracellular acidification rate (ECAR) under normal/unstressed conditions, which is low in cell lines with a high glycolytic rate

26

. OCR/ECAR ratios of the two

iPSC lines used were similar and indicated a high glycolytic activity. Within the analyzed somatic cell lines, a low OCR/ECAR ratio was only found for primary HUVEC cell line (Fig. 1C). Among human primary endothelial cells including human coronary artery endothelial cells (HCAEC), HUVEC were reported to have a low metabolic rate

27

. For further evaluation of the metabolic profile of the examined cell

lines, the ratio of maximal OCR (after injection of the electron uncoupler FCCP) to basal OCR was determined (Fig. 1D). The OCRmax to OCRbasal ratio of the A549 cell line and the two iPSC lines was lower than for the remaining cell lines. A low OCRmax to OCRbasal ratio is indicative for a low mitochondrial reserve capacity and a reduced reliance on mitochondrial metabolism (Fig. 1D). Similar to the cancer cell line A549, iPSC lines are known to rely on glycolysis.


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Evaluation of the susceptibility of human iPSC lines to coxsackievirus, measles and rubella virus reveals a virus- and cell-line specific replication Due to their growth in compact colonies iPSCs could restrict accessibility to virus infections. Thus the distribution of virus-infected cells within iPSC colonies was determined. Recombinant CVB3-EGFP encodes enhanced green fluorescent protein (EGFP) and allows direct visualization of coxsackievirus-infected cells. This offers an advantage for the study of CVB3 on iPSC lines, as on differentiated cells CVB3 displays a fast and lytic replication cycle. For MV, the Edmonston vaccine-like strain and for RV the laboratory strain Therien and the clinical isolate Wb-12 were applied to iPSC lines. Immunofluorescence analysis was used for tracking MV P protein, an essential part of the viral RNA polymerase complex, and for RV E1, the envelope glycoprotein. The cell death response of terminally differentiated cells to Therien differs from the one observed for Wb-12

28

. As the focus of this study was set on

characterization of teratogenic RV, both RV strains were used to take any cell culture adaptations into account.



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Fig 2. Susceptibility of human iPS cell lines A18945 and IMR90 to coxsackievirus, measles and rubella virus. (A) EGFP expression was monitored microscopically in iPSCs at 6 and 24 hpi after infection with CVB3-EGFP virus. Colony morphology is highlighted through phase contrast (PH) imaging. (B) Titer of CVB3-EGFP in iPSCs was determined at the indicated time points. The schematic illustration highlights the experimental set-up of infection after 1 and 3 days after plating and the time point of sample collection. (C) Immunofluorescence analysis of A18945 iPSC at 1 dpi with an antibody against MV P protein (shown in red). (D) Titer of MV in iPSCs at 1 and 2 dpi was determined by plaque assay. (E) A18945 iPSC were infected after one day of cultivation with indicated RV strains. After three days of incubation samples were processed for immunofluorescence analysis with an antibody against E1 protein (shown in red), DNA counterstain is shown in blue. (H) Viral titer was determined by standard plaque assay for supernatants collected at indicated time points. 8 ACS Paragon Plus Environment

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The course of CVB3-EGFP infection appeared to be influenced by iPSC colony size (Fig. 2A). On both iPS cell lines, smaller colonies (upper panel, diameter less than 200 µm) were almost exclusively EGFP-positive in the rim region. Bigger colonies (lower panels, diameter above 200 µm) were not only EGFP positive in the rim, but also throughout the entire colony. To analyze the influence of iPSC colony size on CVB3-EGFP replication, iPSCs were infected at two different time points after plating, one (small-sized colonies are dominating at this time point) and three days. The scheme of infection is illustrated in Fig. 2B. Under both conditions viral titer obtained for CVB3-EGFP was up to one order of magnitude higher on IMR90 as compared to A18945 iPSC (Fig. 2B). In contrast to CVB3-EGFP, infection with MV and RV was not influenced by colony size and did only initiate at the rim region, from where infection was progressing (Fig.2C and E). Such a course of virus infection on iPSCs was also noted for Zika virus

10

. First, the structure of iPSC colonies appears to restrict their accessibility for

virus infections. Second, CVB3-EGFP is rather exceptional through its course of infection on bigger-sized iPSC colonies. MV infection of A18945 iPSC resulted in extensive syncytium formation (Fig 2C), a hallmark of its course of infection in differentiated cells. Similar to CVB3-EGFP, maximum viral titer was generated on IMR90 iPSC. A notable difference between CVB3-EGFP and MV as compared to RV was the iPS cell line-dependent replication of the first two viruses, while RV titers were comparable between both iPS cell lines (Fig. 2F). Coxsackievirus impairs iPSC viability in an iPSC cell line-dependent manner and at a higher rate than cytopathogenic measles and non-cytopathogenic rubella virus



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The assessment of distribution of virus-infected cells within iPS colonies was followed by studies on cellular viability, as especially CVB3-EGFP and MV display a cytopathogenic infection on differentiated cells. Alkaline phosphatase (AP) staining was performed on CVB3-EGFP, MV and RV-infected iPSC cultures to visualize colony size and distribution as compared to the mock-infected control. After infection with CVB3-EGFP, a notable reduction in the number and size of iPSC colonies was present at 3 dpi (Fig. 3A). This loss of colony mass was independent from the age of the iPSC colonies at the time point of infection, but differed among the two iPS cell lines used, being more pronounced on IMR90 iPSC. Microscopic analysis confirmed that CVB3-EGFP-infected colonies were positive for AP expression. This indicates that loss of iPSC colony mass was the cause for the reduction in AP signal. Microscopic analysis revealed cytopathogenic replication of CVB3-EGFP through disintegration of infected cells from iPSC colonies (Fig 3B).


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Fig 3. Analysis of iPSC viability after coxsackievirus, measles and rubella virus infection. (A) After one and three days of cultivation A18945 and IMR90 iPSCs were infected with CVB3-EGFP. At 3 dpi cultures were subjected to alkaline phosphatase assay. (B) Fluorescence and phase contrast microscopy of CVB3EGFP infected A18945 iPSCs reveals cytopathogenic alterations of iPSC colonies, resulting in disintegration and floating of cells in the supernatant (so called floaters). (C) A18945 iPSC were infected with MV and subjected at 3 dpi to AP assay. (D) MVinfected iPSC were analyzed through phase contrast microscopy at 2 dpi. [a] arrow head: enlarged nuclei. [b] balloon-like structures, with (open arrow) or without (closed arrow) the association with syncytial debris. The insets are a high-powered image of the boxed area. (E) Alkaline phosphatase assay was performed for Wb-12- and Therien-infected A18945 iPSC at passage 4. (F) Phase contrast microscopy of Therien (P3)-infected A18945 iPSC after 5 days of cultivation. (G) Viral titer was determined by standard plaque assay for supernatants collected at the sixth day of cultivation directly before passaging. (H) Western blot analysis of the expression of RV E1 protein in cytoplasmic fractions of passaged A18945 iPSC with cofilin as loading control.



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MV replication was also associated with loss of colony mass, but at a rate less profound than the one observed for CVB3-EGFP-infected iPSCs (Fig. 3C). MVassociated cytopathogenic effects were noted as enlargement of nuclei (Fig. 3D[a]), which was also reported for ectoderm differentiation of ESCs after treatment with bone morphogenetic protein-4 (BMP4)

29

. This points towards a transitional state

within syncytia with notable alterations in nuclear morphology. The inset in Fig. 3D[b] depicts balloon-like structures as an important morphological feature of MV infection. They were associated with syncytial debris (open arrow) or floating freely in the supernatant (closed arrow). These structures could be a means of viral spread and appear to be cell-line specific. They were also described for mouse ECSs, but only after knock-down of moesin 30. In contrast to CVB3-EGFP and MV, RV displayed a non-cytopathogenic replication in iPSCs. This enabled maintenance of infected cultures over multiple passages. Passaging of transduced cells was reported to increase the number of viral vectorpositive cells

31

. This was also the case for RV, producing a homogenously infected

culture, achieving about 60% to 80% E1-positive cells at passage 3. AP staining revealed that RV-infected iPSC colonies were comparable in size and number to the mock control, even at passage 4 (Fig. 3E). Additionally, no change in iPSC colony morphology was detected after passaging of RV-infected iPSC cultures was identical to the mock-infected control (Fig. 3F). They were characterized by a constant viral titer and E1 expression level as determined by plaque assay (Fig. 3G) and Western blot analysis (Fig. 3H), respectively. Due to the similar infection pattern found in A18945 and IMR90 iPSCs, both were used alternatively in subsequent experiments Caspase activation contributes to iPS cell line-specific effects observed after infection with coxsackievirus 12 ACS Paragon Plus Environment

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The cytopathogenic effect observed for CVB3-EGFP differed among iPS cell lines and was more pronounced on IMR90 iPSC (Fig. 3A). To explore these differences, the three main influencing factors were examined for both iPS cell lines. First, the initial infection rate was determined by flow cytometry through the number of EGFPpositive cells. Second the expression rate of the coxsackievirus and adenovirus receptor (CAR) was analyzed by flow cytometry after staining with the respective antibody. Third, the rate of apoptotic cell death was determined through the activity of caspase 3/7 and cytotoxicity assessment. In the first approach, two subpopulations were identified in CVB3-EGFP-infected iPSCs by forward and sideward scattering (Fig. 4A). These subpopulations were also present after analyzing just the cells floating in the supernatant (the so called floaters). Analysis of forward and side scatter intensity area (FSC-A and SSC-A) revealed that the set sort gate 1 did contain cell clumps and probably resulted from floater aggregates in the supernatant. Fig. 4A highlights that for the main subpopulation (indicated as gate 2) at 1 dpi a portion of 15% ± 2% EGFP-positive IMR90 iPSC was obtained after infection of three day old cultures as compared to 17% ± 7% EGFP-positive A18945 iPSC. This number was increasing over time of incubation until almost all iPSC colonies were positive for CVB3-EGFP infection. In summary, the rate of CVB3-EGFP infection was similar in the two iPSC lines and could not account for the observed differences in cytopathogenicity. In the second approach, flow cytometric analysis of the cell surface expression of the receptor CAR was performed as this could differ between hES cell lines

32

. The

marker M was set to identify the right-shifted graphs within the histogram, which represent the cell population with a high level of cell-surface expressed CAR (Fig. 4B). To get a better distinction between the two cell lines, the right-shifted population 13 ACS Paragon Plus Environment


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peaks were further subdivided by the markers M1 and M2 (Fig. 4B). M2 encompasses the cell population with the highest level of cell surface-expressed CAR. In the case of A18945 iPSC, the M2 marker within the right-shifted M population contained 24% ± 14% more cells than IMR90 iPSC. This indicates that the cell-surface expression level of CAR was higher in A18945 iPSC (Fig. 4B) and could thus not account for the higher cytopathogenicity of CVB3-EGFP on IMR90 iPSC. The third approach, apoptosis induction, was already described for CVB3-infected hESCs and their derived EBs

33, 34

the apoptosis program in iPSCs

. Thus caspase 3/7 activity as an essential part of

14

was used to assess CVB3-induced cell death

within iPSC colonies. Fig. 4C indicates that the extent of caspase 3/7 activation was higher in A18945 than in IMR90 iPSCs. This fluorescence-based assay excluded the use of CVB3-EGFP, which was also the case for the next assay, cytotoxicity assessment with the MultiTox-Fluor multiplex cytotoxicity assay. Cytotoxicity was not only compared between iPS cell lines, but also between viruses. Similar to cells after two days without medium change as a known stress factor for iPSCs 35, the course of replication of CVB3 and MV was accompanied by a significant increase in dead cell protease activity at 2 dpi (Fig. 4D). Dead cell protease activity was higher in CVB3infected A18945 cells as compared to IMR90 iPSC. This points towards an increased rate of clearing of CVB3-infected cells from A18945 iPS colonies through caspase activation, which could in turn support maintenance of colony mass, while virus replication was constantly progressing in IMR90 iPSC. To support this notion, the integration-free DF19-9-7t iPSC line was included in the analysis of CVB3 infection in iPSC lines (Fig. S1). Comparable to IMR90 iPSC, a high rate of infection was already present at 1 dpi (Fig. S1A and B). The obtained viral titer on DF19-9-7t (Fig. S1B) was even slightly higher than the one on IMR90 iPSC (Fig. 2A). Accordingly, CVB314 ACS Paragon Plus Environment

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EGFP-associated cell death rate was higher in DF19-9-7t as compared to IMR90 and A18945 iPSC, which resulted in loss of the entire iPSC culture at 3 dpi (Fig. S1C). This was accompanied by a low level of activated caspase 3/7 (Fig. S1D). In summary, activation of caspase 3/7 activity appears to contribute to maintenance of CVB3-EGFP-infected iPSC cultures through disintegration of infected cells.

Fig 4. Analysis of the higher cell death rate observed for coxsackievirusinfected A18945 iPSC as compared to IMR90 iPSC. (A) The number of CVB3EGFP-infected iPSCs was quantified at 1 dpi through flow cytometric analysis of EGFP expression. Infection was initiated on three day old iPSC colonies. The green line indicates the CVB3-EGFP-infected cell population; the black line reflects the mock-infected control. FSC and SSC, forward and side wards scatter (B) Flow cytometric analysis of the CAR expression level in iPSCs after 5 days of cultivation. 15 ACS Paragon Plus Environment


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(C) and (D) The use of fluorescence-based assays required infection with recombinant CVB3 instead of CVB3-EGFP virus. (C) Activation of caspase 3/7 (through the FAM-DEVD-FMK probe) in CVB3-infected iPSCs was assessed at 2 dpi. (D) The dead to live cell protease signal ratio was determined for cells without medium change for 2 days and for virus-infected iPSCs at 2 dpi (CVB3 and MV) and after 2 days of cultivation (passaged RV) by the MultiTox-Fluor multiplex cytotoxicity assay. The ratio was expressed as fold changes as compared to the mock control. Infection with cytopathogenic coxsackievirus and measles virus and noncytopathogenic rubella virus does not impair expression of pluripotency markers The expression level of pluripotency markers as an important characteristic of iPSCs was determined after virus infection and compared to the mock-infected control. To assess OCT4 or SOX2 expression levels in CVB3-EGFP-, MV- and RV-infected iPSCs immunofluorescence (Fig. 5A, C, and E, respectively) and Western blot (Fig. 5B, D, and F, respectively) analysis was performed. Only after cell death induction in CVB3-EGFP-infected cells a loss of OCT4 expression was noted (Fig. 5A, marked by asterisks). Otherwise expression levels of pluripotency markers were similar between virus- and mock-infected iPSCs.


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Fig 5. Impact of coxsackievirus, measles and rubella virus infection on the expression of pluripotency markers. (A) Expression of OCT4 (shown in red) in CVB3-EGFP-infected A18945 iPSC at 1 dpi was verified through immunofluorescence analysis and revealed constant (closed arrow) or slightly reduced (open arrow) expression of OCT4. Asterisks indicate lack of OCT4. Area of the insets is depicted. (B) Three day old IMR90 iPSCs were infected with CVB3EGFP to obtain nuclear and cytoplasmic extracts at 1 dpi. Western blot analysis was performed with anti-OCT4 and anti-EGFP antibodies. (C) At 1 dpi iPSC colonies were processed for immunofluorescence analysis with antibodies against SOX2 (shown in green). MV infection was monitored with anti-P antibody (shown in red). (D) Nuclear extracts obtained at 2 dpi from mock- and MV-infected IMR90 iPSCs were subjected to Western blot analysis for assessment of the pluripotency marker OCT4. (E) RVpassaged iPSC cultures were processed for immunofluorescence analysis with antibodies against E1 protein (shown in red) and SOX2 (shown in green). (F) Expression of pluripotency markers OCT4 in nuclear extracts as assessed by Western blot analysis. (A, C, E) DNA counterstain is shown in blue. (B, D, F) For Western blot analysis of nuclear and cytoplasmic extracts, anti-PCNA and anti-alpha tubulin were used as loading control, respectively.



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Viral infection by cytopathogenic coxsackievirus and measles virus and noncytopathogenic rubella virus alters metabolic activity Metabolism influences the developmental potential of a cell, as the conversion from primed to the naïve state is characterized by a shift from predominant glycolytic to a bivalent metabolism employing both, glycolysis and oxidative metabolism

36, 37

. Thus

metabolic activity was determined through extracellular flux measurement of OCR indicative for mitochondrial respiration. OCR was increased at 1 dpi under basal and stressed conditions after infection with all three viruses studied here, CVB3-EGFP (Fig. 6A), MV (Fig. 6B), as well as RV (Fig. 6C). Moreover, an increase in basal OCR, respiratory capacity, and ATP production was not only noted for all three viruses, but also after lack of daily medium change as another important cause of stress in iPSCs (Fig. 6A)

35

. To our knowledge, this is the first report on an altered metabolic activity

in iPSCs during a virus infection, most notably after cytopathogenic as well as noncytopathogenic viral infection. In summary, mitochondrial metabolism appears to be valuable for the analysis of cytopathogenic alterations in iPSCs. This is an important notion, as so far parameters for assessment of pathogenic alterations in iPSCs are hardly available and characterized. This is in line with a very recent publication on the metabolic response of iPSCs to alterations in cellular physiology 38.


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Fig 6. Impact of coxsackievirus, measles and rubella virus infection on the metabolic activity of iPSCs. OCR was measured under stressed conditions using the Mito Stress Test Kit for (A) CVB3-EGFP-infected IMR90 iPSC at 1 dpi and control IMR90 iPSC after one day without medium change, (B) MV-infected IMR90 iPSC at 1 dpi, and (C) RV (Therien strains)-infected and passaged A18945 iPSC colonies were measured at 3 days after plating. Rubella virus-positive iPSC cultures retain capacity to differentiate into all three germ layers While CVB3-infected iPSC lines could only be maintained for 2 to 3 days after initiation of passaging at 1 dpi (data not shown), RV-infected iPSCs were passaged 19 ACS Paragon Plus Environment


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multiple times. This enabled directed differentiation of an RV-enriched iPSC culture through application of the STEMdiff trilineage differentiation kit. Cultures were maintained for 7 (ectoderm) and 5 days (endo- and mesoderm). The expression level of specific markers was determined in ectoderm, endoderm, and mesoderm cells in comparison to undifferentiated A18945 iPSC (Fig. S2A). While expression of OCT4 was notably reduced in cells of all three germ layers, expression of SOX2 was maintained, but as expected only in ectoderm cells. The remaining low level expression of OCT4 in endoderm and mesoderm cells is in agreement with a report on OCT4 expression level in primitive endodermal and mesodermal lineages

39

.

Immunofluorescence analysis verified expression of SOX2, FOXA2, and NESTIN as representative marker proteins in Wb-12-positive cells of ectodermal, endodermal and mesodermal lineage, respectively (Fig. 7A). Additionally, the expression level of delta like non-canonical notch ligand 1 (DLK1, ectoderm cells), GATA binding protein 4 (GATA4, endoderm cells), and CD56 (mesoderm cells) was comparable between lineages derived from uninfected controls (mock) and RV-enriched iPSC cultures (Fig. S2B). Thus the continued presence of RV in iPSC cultures did not alter the capacity for directed differentiation. Although the STEMdiff trilineage differentiation kit as an endpoint assay does not allow for further differentiation in distinct downstream cell populations, it represents an important functional assay. Future analysis will enable elucidation of cell populations that are possibly affected by RV.


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Fig 7. Differentiation of rubella virus-infected iPSCs after multiple passages. (A) Immunofluorescence analysis of Wb-12 (P4) A18945 iPSCs with polyclonal RV antibody and antibodies against indicated lineage marker proteins after induction of differentiation into ectoderm, endoderm, and mesoderm. (B) Titer determination by plaque assay for RV Wb-12-infected and passaged A18945 iPSCs at 5 (endo- and mesoderm) and 7 (ectoderm) days after initiation of differentiation into embryonic germ layers. (C) Summary of the main findings of the study: impact of a virus infection on iPSC colony functionality reveals metabolic impairment as a common factor. Non-cytopathogenic replication of RV enables directed differentiation. Timeline is depicted in gestational weeks. ICM, inner cell mass A slightly higher number of RV-positive cells (Fig. 7A) and an increased viral titer (Fig. 7B) were observed for Wb-12 iPSC-derived mesoderm. Descendants of this lineage give rise to vascular cells

40

. This might help to explain the potential of RV to

infect almost any organ of the fetus and to cause congenital (cardio) vascular defects. 21 ACS Paragon Plus Environment


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To strengthen the unique capacity of RV-enriched iPSC cultures to undergo directed differentiation, the experimental set-up of this process was applied to MV-infected iPSC cultures. For this approach MV-infected A18945 iPSC were plated at 1 dpi before onset of syncytium formation. Already 1 day after plating of MV-infected iPSC cultures, a high expression level of viral P protein and extensive syncytium formation were noted in undifferentiated MV-infected iPSC at passage 1 (Fig. S3A). Despite a lower level of cytopathogenicity of MV on iPSC cultures as compared to CVB3, a high level of cytopathic effect induction was noted after initiation of differentiation through formation of balloon-like structures (Fig. S3B and S3C, closed arrow). In contrast to MV-infected undifferentiated iPSC cultures, syncytia were smaller and present at a lower number (Fig. S3C, open arrow). In summary, directed differentiation of MVinfected A18945 iPSC was not possible as the presence of an extensive cytopathic effect led to loss of the cultures before the differentiation process could be completed. Fig. 7C summarizes the data shown here and sets the ability of RVinfected and passaged iPSCs to undergo directed differentiation in a broader context of iPSCs as an in vitro cell culture model for pathogenic alterations during the early phase of human embryogenesis. Conclusion In our work, we established two stem cell lines (IMR90 and A18945 iPSCs) as a model for systematic investigations of three specific virus types, CVB3, MV, and RV, which influence embryogenesis. Although pluripotent stem cells are only present in a short time frame during embryonal development, they enable analysis of the blastocyst stage until gastrulation-like events. Viral infection could impair implantation of the blastocyst or disturb progression of embryogenesis. Moreover, RV can be teratogenic from the first day of fertilization

20, 21, 22

which includes blastocyst stage 22


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and gastrulation. In contrast to RV, congenital varicella syndrome is mainly noted after maternal infection during weeks 8 and 20 of gestation 11, which is in agreement with the reported lack of susceptibility of pluripotent stem cells to varicella zoster virus 11

. However the infection of hESCs with herpes simplex virus, whose role in

miscarriage events is still under discussion, resulted in a strong cytopathic effect 12, 41, 42

.

In contrast, hESC lines are more or less resistant to human cytomegalovirus

which causes brain malformations as a later stage congenital birth defect 43, 44. The unanswered questions in the characterization of viral impairment of human development overlap virology, innate immunity, and developmental biology

45

.

Antiviral response pathways in PSCs differ in multiple ways from their differentiated somatic counterparts

46, 47

. Most importantly, the interferon response pathway as one

of the most powerful antiviral countermeasures is attenuated in PSCs

47, 48

. Virus-

infected iPSCs, especially by non-cytopathogenic RV, could reveal important insights into the immunobiology of ESCs at the molecular and cellular level

45

. A lytic or

cytopathogenic infection as shown here for CVB3 and MV does probably initiate apoptotic cell death before anti-viral cellular pathways are activated. This stands in contrast to RV-infected iPSCs, which can be maintained in culture without impairment of viability and allow for initiation of differentiation. This enables further research into iPSCs as a platform for the elucidation of RV teratogenic mechanisms and subsequently of the activation of antiviral countermeasures in iPSCs. Data presented have set the basis for these studies and already point to a different replication rate of RV within the three primary germ layers.



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Methods Reagents The primary antibodies against octamer transcription factor 3/4 (OCT3/4, thereafter referred to as OCT4 [sc-5279]), stage-specific embryonic antigen 4 (SSEA4 [sc21704]), NANOG (sc-30331), SOX2 (sc-17320), NESTIN (sc-23927), FOXA2 (HNF3β, sc-374376), against CAR (coxsackievirus and adenovirus receptor; H-300, sc15405), GATA4 (sc-377543), DLK1 (sc-376755), VIMENTIN (sc-6260), cofilin (sc33779), and against proliferating cell nuclear antigen (PCNA, sc-9857) were purchased from Santa Cruz Biotechnologies. Additionally, the antibody against CD56 (NCAM1) was from StemCell Technologies, the goat polyclonal antibody to RV (HPV77) from Advanced Biotechnologies Inc. (ABI), and primary antibodies against RV E1 (MAB925, EMD Millipore Corp for Western blot analysis and mAb anti-E1 from Viral Antigens for immunofluorescence), MV Phosphoprotein (P) (Covalab, mab0041P) were used. Donkey IgG anti-mouse IgG (H+L)-Cy3 and donkey IgG anti-goat IgG (H+L)-Alexa Fluor 488 as secondary antibodies were from Dianova. Cell cultivation Permanent cell lines Vero (green monkey kidney epithelial cell line), HeLa (human epithelial cervix carcinoma), A549 (human lung carcinoma epithelial cells), and HEK 293T/17 (referred to as HEK, human embryonic kidney) were obtained from American Type Culture Collection (ATCC). Vero, A549, and HEK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific) with high glucose and GlutaMAX, supplemented with 10% fetal calf serum (FCS) and antibiotics. HeLa cells were cultivated in DMEM, supplemented with 5% FCS, 1% nonessential amino acids (NEAA, Gibco, Thermo Fisher Scientific), 2% HEPES buffer (Gibco, Thermo 24 ACS Paragon Plus Environment

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Fisher Scientific) and antibiotics. Human umbilical vein endothelial cells (HUVEC, pooled, PromoCell) were maintained in endothelial cell basal medium 2. iPSC lines Three human iPSC lines were used in this study, which are referred to as iPSC or iPSC line. The zero-footprint, viral-integration-free episomal A18945 iPSC line (catalogue number A18945) was purchased from Thermo Fisher Scientific, the lentiviral IMR90-1 iPSC line from WiCell, Madison (thereafter noted as IMR90 iPSCs). Additionally, the vector-free iPSC line DF19-9-7T from WiCell was used, which was generated by a non-integrating reprogramming method. iPSC lines were cultured on growth factor reduced Matrigel™ (BD Biosciences, performed in DMEM/F-12)-coated plates in mTeSR™1 medium (StemCell Technologies) with 10 µg/ml gentamycin. Under all culture conditions, medium was changed daily. iPSC colonies were enzymatically passaged in clumps using collagenase type IV (Thermo Fisher Scientific, dissolved in DMEM-F12) every 5-7 days at a splitting ratio of 1:4 to 1:6. Colonies with signs of spontaneous differentiation were removed. Some experiments required plating of iPSC colonies as single cells at a density of 50,000 cells/cm2 after incubation with Accutase (Sigma Aldrich). Y-27632 ROCK inhibitor was added at 10 µM during plating of dissociated colonies. Induction of directed differentiation For directed differentiation the STEMdiffTM trilineage differentiation kit (StemCell Technologies) as an endpoint assay was used according to manufacturer’s instructions. For analysis of lineage specificity of cells derived from the directed differentiation kit, the following lineage-specific markers were used based on the molecular analysis of theses cultures provided by the manufacturer: SOX2 for



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undifferentiated and ectoderm cells; NESTIN and DLK1 for ectoderm cells; FOXA2 and GATA4 for endoderm cells, and CD56 (NCAM1) for mesoderm cells. Virus infection The CVB3-EGFP virus was generated through transfection of the pEGFP-CVB3 plasmid (provided by Zhao-Hua Zhong, Department of Microbiology, Harbin Medical University, Harbin, China) into HeLa cells. Correspondingly, CVB3 was generated from the plasmid pCVB3. The CVB3 virus encoding EGFP in its genome (not fused to a viral sequence) was described previously and possesses a slightly attenuated phenotype as compared to the wild-type CVB3

49, 50

. Virus-containing supernatants

were centrifuged at 500×g for 10 min and passed through a 0.45 µm membrane filter to remove cellular debris. All viral supernatants were concentrated through ultracentrifugation at 25,000 rpm for 120 min at 4°C with a 20% sucrose cushion and pellets were resuspended in mTeSR™1 medium. Viral titers were determined by standard plaque assays on Vero (RV and MV) and HeLa (CVB3) cells. Collagenase-treatment results in iPSC colony plating in clumps, thus it was not possible to clearly define, how many cells were present at the time point of infection. Moreover, cultivation of iPSCs does not generate a confluent monolayer and thus Matrigel remains exposed for interaction with virus particles, which as charged particles possibly stick to this surface coat

51

. This limits their free movement to iPSC

colonies and restricts the use of a multiplicity of infection (MOI). In subsequent infection studies of iPSCs, cultures were plated at high density to reach 40 to 50% confluency at the time of infection and to permit growth of colonies until sample analysis. Thus a virus-specific PFU was applied per well of a six well culture plate: 1.5 × 106 PFU of CVB3-GFP, 4 × 105 PFU of MV, and 1 × 107 PFU of RV were used per well of a six well culture plate. Virus inoculum was removed after an incubation


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period of 1 h for CVB3 and MV and 2 h for RV and fresh mTeSR™1 medium was added. Flow cytometry analysis iPSCs were detached with 0.05% trypsin and washed with PBS after centrifugation at 400×g at 4°C for 10 min and fixed with 2% (w/v) paraformaldehyde (PFA). For detection of CAR, 0.5 to 1× 106 cells were incubated with a 1:100 dilution of the H300 antibody.

Flow cytometric investigations were performed with an Accuri C6 by

detecting 10,000 events. For data analysis the BD Accuri™ C6 Software was used. Standard cell characterization assays: assessment of cell viability and cytotoxicity 1. Alkaline phosphatase (AP) activity was assessed through the AP staining kit from System Biosciences according to manufacturer’s instructions. 2. Apoptotic cell death was verified through fluorescence-tagged Annexin V (Thermo Fisher Scientific) according to manufacturer’s instructions. 3. Cytotoxicity was assessed through the MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega). A substrate for live and dead cell protease was added to the cell cultures and subsequent fluorescence measurements were performed on the Victor X3 microplate reader (PerkinElmer) to calculate the ratio of dead to live cells. 4. Activity of caspase 3 and 7 was determined using the CellEvent caspase-3/7 ReadyProbes reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. Assessment of caspase-3/7 activity was based on microscopic analysis of fluorescent events after cleavage of the non-fluorescent DEVD peptide. Metabolic assessment through extracellular flux analysis: analysis of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) 27 ACS Paragon Plus Environment


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OCR and ECAR were measured using an XFp Analyzer (Agilent Seahorse Technologies). Seahorse plates were coated with Matrigel for plating of iPSCs and HEK cells. Subsequently, 20,000 Vero, A549, HUVEC, 30,000 HEK cells or 15,000 (acute infection) to 30,000 (measurement at 1 day after plating) iPSCs were seeded into each well. Culture medium was replaced by XF Base Medium (Agilent Seahorse Technologies) supplemented with 2 mM sodium pyruvate, 2 mM L-glutamine, and 11 mM glucose with an adjusted pH of 7.4. Cells were then incubated at 37°C and 5% CO2 for one hour. Four compounds from the XFp Cell Mito Stress Test kit (Agilent Seahorse Technologies) were injected during the assay to achieve the following final concentrations: oligomycin (2 µM), FCCP (0.8 mM), and a mixture of antimycin A and rotenone (0.5 µM). The increase in OCR after application of FCCP reflects the respiratory capacity of a cell, the maximum respiratory rate that can be reached. For normalization, the protein content of each well was determined by standard Bradford assay. According to a previous publication

26

basal OCR and ECAR as determined

during the initial three measurement points of the XFp Cell Mito Stress Test kit were used to compare metabolic activities of various somatic cell lines with the one found in the two iPSC lines, IMR90 and A18945. Basal OCR and ECAR values given in the manuscript refer to the third of the three measurement points. Immunofluorescence analysis and microscopy Live-cell phase-contrast bright-field images were obtained using the inverted Olympus XM 10 and DMIRB (Leica) microscope. For immunofluorescence analysis cells

were cultivated on

Matrigel-coated 2-well

chamber-slides

(Lab-Tek™

Permanox™, Thermo Fisher Scientific) or Nunclon 24 well plates and fixed for 10 min at room temperature with 2% (w/v) PFA in PBS, followed by permeabilization with 0.1% Triton X-100 for 30 min at 37°C. Thereafter cells were blocked with 1% donkey 28 ACS Paragon Plus Environment

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serum (Santa Cruz Biotechnology) in PBS containing 0.02% Triton X-100 for 30 min at 37°C in a humidified chamber, followed by an incubation with 10% donkey serum in PBS for 30 min. Incubation with primary antibodies was performed for 90 min at 37°C or overnight at 4°C. All primary antibodies were diluted 1:100 in PBS, except the 1:200 dilution of the anti-E1 antibody, 0.1% Triton X-100. Samples were washed thrice with PBS and incubated with the secondary antibodies, diluted at 1:100 in PBS, 0.1% Triton X-100 for 60 min at 37°C. Thereafter plates were washed thrice with PBS. Hoechst bisbenzamide 33285 (5 µg/ml, Thermo Fisher Scientific) was used as DNA counterstain. Samples were mounted in Fluoromount G (Biozol). Images were taken with a Zeiss 510 confocal microscope and processed with CorelDRAW X7 with minimal alterations to brightness and contrast. Western blot analysis Cytoplasmic and nuclear protein fractions were extracted from iPSCs following manufacturer’s instructions (NE-PER nuclear and cytoplasmic extraction kit; #78835, Thermo Fisher Scientific). Standardized SDS-page was followed by electroblotting in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol). After blocking the blot with 5% nonfat dried milk powder in PBS and 0.1% Tween-20 the primary antibodies were added at their respective dilution (antibody against E1 1:500, all others at 1:200) and incubated at 4°C overnight. This was followed by incubation of the PVDF membrane with the corresponding secondary antibody and ECL solution for chemiluminescence analysis. Statistics Assays consisted of duplicates and triplicates (experimental replicates) and two to three independent experiments (biological replicates) were performed. Only the performed flux analysis per RV-infected and passaged iPSCs was based on three experimental replicates, as activity was determined per passage number for different 29 ACS Paragon Plus Environment


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iPSC cultures. All data in the diagrams are expressed as means ± standard deviation (SD). One-way ANOVA followed by Tukey’s post hoc analysis was applied to determine statistical significance using Graph Pad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). Asterisks indicate level of significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Supporting Information Figure S1, Characterization of the course of infection of coxsackievirus B3 on DF199-7t iPSC; Figure S2, Analysis of lineage-specific marker expression after endpoint differentiation of A18945 iPSC using the STEMdiff trilineage differentiation kit; Figure S3, Assessment of the capacity of measles virus-infected A18945 iPSC to differentiate into embryonic germ layers Acknowledgements The authors want to thank Bernadette Harwardt for technical assistance, Johannes Busch and Nicole Christin Bilz for careful reading of the manuscript, and Dr. B. Weißbrich, University of Wuerzburg, Germany for providing the Wb-12 strain of RV. This work was supported by grants from the DFG (CL 459/3-1 to C.C. and FE785/2-2 to H.F.) and the Roland Ernst foundation (2/14 to C.C.). Author contributions D.H., K.J., S.P., J.B., and C.C. performed the experiments and data analysis and prepared figures. M.J., H.F. and C.C. contributed to experimental design. D.R. and U.G.L. assisted on the experiments and provided resources. C.C. wrote the manuscript, conceived the project. All authors reviewed the manuscript. Notes 30 ACS Paragon Plus Environment

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The authors declare no competing financial interest. References

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“For Table of Contents Use Only”



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TOC graphic 81x25mm (300 x 300 DPI)


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Fig. 1 181x154mm (300 x 300 DPI)



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Fig. 2 175x235mm (300 x 300 DPI)


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Fig. 3 189x226mm (300 x 300 DPI)



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Fig. 4 196x210mm (300 x 300 DPI)


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Fig. 5 200x164mm (300 x 300 DPI)



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Fig. 6 185x218mm (300 x 300 DPI)


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Fig. 7 179x177mm (300 x 300 DPI)



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Fig. S1 158x156mm (300 x 300 DPI)


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Fig. S2 168x208mm (300 x 300 DPI)



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Fig. S3 123x104mm (300 x 300 DPI)


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Infection of iPSC Lines with Miscarriage-Associated Coxsackievirus

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