Image_1_Autophagy Benefits the Replication of Egg Drop Syndrome Virus in Duck Embryo Fibroblasts.TIF

<p>Egg drop syndrome virus (EDSV) is an economically important pathogen with a broad host range, and it causes disease that leads to markedly decreased egg production. Although EDSV is known to induce apoptosis in duck embryo fibroblasts (DEFs), the interaction between EDSV and its host needs to be further researched. Here, we provide the first evidence that EDSV infection triggers autophagy in DEFs through increases in autophagosome-like double-membrane vesicles, the conversion of LC3-I to LC3-II, and LC3 colocalization with viral hexon proteins. Conversely, P62/SQSTM1 degradation, LC3-II turnover, and colocalization of LAMP and LC3 confirmed that EDSV infection triggers complete autophagy. Furthermore, we demonstrated that inhibition of autophagy by chloroquine (CQ) and 3-methyladenine (3MA) or RNA interference targeting ATG-7 decreased the yield of EDSV progeny. In contrast, induction of autophagy by rapamycin increased the EDSV progeny yield. In addition, we preliminarily demonstrated that the class I phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway contributes to autophagic induction following EDSV infection. Altogether, these finding lead us to conclude that EDSV infection induces autophagy, which benefits its own replication in host cells. These findings provide novel insights into EDSV–host interactions.</p

MOESM1 of MicroRNA expression profiling of goat peripheral blood mononuclear cells in response to peste des petits ruminants virus infection

Additional file 1. Flow chart of the miRNA prediction and differentially expressed (DE) miRNA analysis from goat PBMC infected with PPRV at 1.0 multiplicity of infection (MOI). The 49nt sequence tags from Hiseq sequencing will go through the data cleaning analysis first, then the standard analysis will annotate the clean tags into different categories and take those which cannot be annotated to any category to predict the novel miRNA and seed edit of potential known miRNA. After getting miRNA result, target prediction for miRNA and GO enrichment and KEGG pathway for target genes will be analyzed

MOESM6 of MicroRNA expression profiling of goat peripheral blood mononuclear cells in response to peste des petits ruminants virus infection

Additional file 6. KEGG analysis of target genes annotated for miRNA differentially expressed in mock- and PPRV-infected goat PBMC. KEGG pathway annotation revealed that 10 364 background genes were annotated for 317 biological processes

Characterization of primary endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs).

<p>The cobblestone structure and expression of cytokeratin were shown in EECs (a, b) and the spindle-shape structure and vimentin expression were shown in ESCs (e, f). The negative control shown of cytokeratin and vimentin involved use of mouse IgG in place of primary antibody (c, g). Western blot analysis of estrogen receptor (ER) and progesterone receptor (PR) protein expression were shown separately in the endometrial cells (d, h). Results were quantified by densitometry analysis of the bands and normalization to β-actin protein. Data represent the mean ± standard error of the mean (SEM) from five independent experiments. Columns with different superscript letters are significantly different (<i>P</i> < 0.05). Scale bar = 30 µm.</p

MOESM2 of MicroRNA expression profiling of goat peripheral blood mononuclear cells in response to peste des petits ruminants virus infection

Additional file 2. Summary of deep sequencing data for small RNA (sRNA) in mock- and PPRV-infected goat PBMC. A total of 30 573 869 and 30 644 798 clean reads were obtained from the uninfected and infected groups, respectively. The clean reads were annotated and classified as snRNA, rRNA, snoRNA, Rfam other sncRNA, precursor miRNA, mature miRNA, intergenic, intron, exon, and repeats

Different developmental stages of goat embryos produced <i>in vitro</i>.

<p>Transferred IVF blastocysts (a) or SCNT blastocysts (b) showed clear inner cell masses (ICMs) and trphoectodermal cells (TE) under bright field at Day 7. Both the IVF blastocysts (c) and SCNT blastocysts (d) attached to the endometrial epithelial cells after co-cultured for 24 h. The trophoblast of IVF (e) and SCNT (f) underwent outgrowth on the epithelial cells monolayer after co-cultured for 72 h. Scale bar = 50 µm.</p

MOESM3 of MicroRNA expression profiling of goat peripheral blood mononuclear cells in response to peste des petits ruminants virus infection

Additional file 3. Details of differentially expressed known and novel microRNA (miRNA) in PPRV- versus mock-infected goat PBMC. A total of 316 miRNA (including 103 known miRNA and 213 novel miRNA) were differentially expressed in mock- and PPRV-infected groups. Among these 316 DEmiRNA, 147 miRNA were upregulated and 169 miRNA were downregulated in the PPRV-infected cells compared to the mock-infected cells

MOESM5 of MicroRNA expression profiling of goat peripheral blood mononuclear cells in response to peste des petits ruminants virus infection

Additional file 5. WEGO analysis of target genes annotated for DEmiRNA in mock- and PPRV-infected goat PBMC. WEGO analysis showed that a total of 12 065 target genes were successfully annotated for 103 known miRNA and 213 novel miRNA differentially expressed in two groups

The secretion and expression of cytokines by uterine DBA⁺ leukocytes in response to embryos and steroids.

<p>ELISA analyses of the concentrations of IFN-γ (a) and VEGF (c) secreted by uterine DBA⁺ leukocytes in response to cloned and fertilized embryos. Representative western blotting and densitometric analysis of IFN-γ (b) and VEGF (d) normalized to β-actin to correct for protein loading. <i>Columns</i> and <i>vertical bars</i> represent the mean ± SEM from at least five independent experiments, and analyzed by two-way ANOVA with treatment group and/or embryos as the independent variable(s). Columns with different superscript letters are significantly different from each other (<i>P</i> < 0.05).</p

Flow cytometry analysis of the expression of CD56⁺CD16^- on goat uterine leukocytes.

<p>Representative flow cytometry analysis of the percentage of CD56⁺CD16^- cells on isolated goat uterine leukocytes in response to cloned and fertilized embryos (a). The absolute numbers of uterine CD56⁺CD16^- leukocytes over 120-hr incubation with different conditioned mediums or control medium (b). Fold expansion of total numbers of isolated goat uterine leukocytes after 120-hr incubation with different conditioned mediums or control medium in comparison to the cells at the beginning of the culture period (c). <i>Columns</i> and <i>vertical bars</i> represent the mean ± SEM from at least five independent experiments, and analyzed by two-way ANOVA with treatment group and/or embryos as the independent variable(s). Columns with different superscript letters are significantly different from each other (<i>P</i> < 0.05).</p