Journal: Frontiers in Microbiology

Article Title: Enterovirus 71 Antagonizes Antiviral Effects of Type III Interferon and Evades the Clearance of Intestinal Intraepithelial Lymphocytes

doi: 10.3389/fmicb.2021.806084

Figure Lengend Snippet: IFN-λ effectively inhibits EV71 infection in intestinal epithelial cells. (A) HT29 and Jurkat T cells were uninfected or infected with EV71 at an MOI of 1, qRT-PCR was used to detect transcriptional upregulation of both IFN-β, IFN-λ1, and IFN-λ2 at 48 hpi. (B) At the same infection conditions, the production and secretion of IFN-β, IFN-λ1, and IFN-λ2 proteins in the supernatant were detected by ELISA. Mock was cells without infection. (C) HT29 and Jurkat T cells were stimulated without or with 10 ng/ml recombinant protein IFN-β or IFN-λ1 for 12 h. Then the expression levels of ISG54, ISG15, PKR, and OAS were measured by qRT-PCR. Calculated the expression level of each gene relative to the expression of GAPDH and normalized it to mock-treated cells. Control was cells without treatment. (D,E) HT29 cells were pretreated with 10 ng/ml IFN-λ or 10 ng/ml IFN-β for 6 h, respectively, and then infected with EV71 at MOI of 1 for 24 h. The virus replication was monitored by qRT-PCR and plaque experiments. Control was cells with infected but without IFN treatment. Dates were presented as mean ± SD of three replicates ( n = 3 independent experiments, ** P < 0.01, *** P < 0.001, *⁣*⁣** P < 0.0001 and ns, not significant).

Article Snippet: The secretion levels of IFN-β, IFN-λ1, and IFN-λ2 proteins in the supernatant were detected by commercially available enzyme-linked immunosorbent assay kits, including Human IFN-β ELISA Kit (LIANKE, EK1236), Human IL-28A ELISA Kit (BOSTER, EK0969), and Human IL-29 ELISA Kit (BOSTER, EK0964).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing

Journal: Frontiers in Microbiology

Article Title: Enterovirus 71 Antagonizes Antiviral Effects of Type III Interferon and Evades the Clearance of Intestinal Intraepithelial Lymphocytes

doi: 10.3389/fmicb.2021.806084

Figure Lengend Snippet: The antiviral function of IFN-λ is antagonized by EV71. (A) HT29 cells were infected with EV71 at an MOI of 1. The expression levels of IFN-β, IFN-λ1, and IFN-λ2 were detected at 0, 12, 24, 48, and 72 hpi by qRT-PCR. (B) At the same infection conditions, ISG54, ISG15, PKR, and OAS expression levels were detected by qRT-PCR. (C) Recombinant protein IFN-λ treatment (10 ng/ml) was performed 6 h before infection or 6 h after infection (MOI = 1). (D) After 24 hpi, the cellular RNA was extracted, and ISG54, ISG15, PKR, and OAS expression levels were measured by qRT-PCR. HT29 cells were mock-infected and were not treated by IFN-λ as “control (Ctrl)”. HT29 cells were infected with EV71 as “EV71”. The “IFN-λ + EV71” meant that HT29 was treated with IFN-λ 6 h before infection, and “EV71 + IFN-λ” meant that HT29 was treated with IFN-λ 6 h after infection. (E) The viral titers in the culture supernatant were monitored by plaque experiments at 24 hpi (F) . The intracellular viral protein (VP1) load was measured by confocal fluorescence microscopy. Dates were presented as mean ± SD ( n = 3 independent experiments, * P < 0.05, *** P < 0.001, *⁣*⁣** P < 0.0001 and ns, not significant).

Article Snippet: The secretion levels of IFN-β, IFN-λ1, and IFN-λ2 proteins in the supernatant were detected by commercially available enzyme-linked immunosorbent assay kits, including Human IFN-β ELISA Kit (LIANKE, EK1236), Human IL-28A ELISA Kit (BOSTER, EK0969), and Human IL-29 ELISA Kit (BOSTER, EK0964).

Techniques: Infection, Expressing, Quantitative RT-PCR, Recombinant, Fluorescence, Microscopy