Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted.</p></div

Effect of cyclosporin A (CsA) on type I interferon expression.

<p>Wa rotavirus-infected HT-29 cells were treated with CsA at indicated doses at 12 h post-infection. Total cell lysates were collected at 24 h post-treatment. Total cellular RNA extracted from the total cell lysates was subjected to the real-time RT-PCR to quantify IFN-α (A) and IFN-β (B). IFN-α/IFN-β expression is expressed as mRNA levels relative (fold) to the control (uninfected HT-29 cells without CsA treatment, which is defined as 1). Data shown are expressed as mean ± standard deviation of triplicate cultures, and 3 independent experiments were carried out (*<i>P</i><0.05, **<i>P</i><0.01).</p

Effect of CsA on SA11 rotavirus-infected neonatal mouse model.

<p>Neonatal mice were inoculated with SA11 rotavirus, and mice developed diarrhea during the 18–24 h of experimental observation. The mice scored for diarrhea were randomly divided into 3 groups (30 mice each group). Mice were subjected to PBS, Ribavirin (5 mg/kg/d), or CsA (2.5 mg/kg/d) treatments by oral gavages. (A) Healing time. (B) Rotavirus-antigen clearance rate (%) in stool. Data shown are expressed as mean ± standard deviation of triplicate cultures, and are representative of 3 experiments (*<i>P</i><0.05, **<i>P</i><0.01).</p

Jejunum changes as assessed by histology using a light microscope.

<p>(A) RV+PBS group. (B) RV+Ribavirin group. (C) RV+CsA group. (D) PBS group. (A-D) Morphologic changes in mouse jejunum pathology under a light microscope after 3 days of treatment (H&E, magnification: 200×). Red arrow: Villus vacuolar degeneration. Green arrow: Lesion on the epithelium. Blue arrow: Intestinal hydropsia.</p

CsA toxicity analysis in the animal model.

<p>Body weight growth curve (A) and survival curve (B) after CsA treatment of RV-infected BALB/c mice. Body weight or survival were monitored among the 4 groups of mice during the week following rotavirus infection challenge CsA or PBS treatment (mean ± SEM, n >10). (C) CD4/CD8 T-cell ratio analysis in spleens and thymus from RV-infected mice treated with CsA or Ribavirin. (D) Proliferation assay of splenocytes in RV infected and CsA treatment mice by CFSE labeling. Ribavirin treatment mice as positive control and PBS treatment mice as negative control. Proliferation index (PI) was 3.47±0.37, 3.16±0.47, 3.47±0.79, 3.49±0.67 respectively. (E) IL-2, IL-4, and IFN-γ analysis from cell culture supernatants by sandwich ELISA. Data are representative of at least 3 independent experiments (n = 10 mice/group). Error bars indicate the SD with technical triplicates. N.s., not significant (*<i>P</i><0.05, **<i>P</i><0.01).</p

Effect of cyclosporin A (CsA) on the expression of the type I interferon (IFN) signaling pathway regulators.

<p>Wa rotavirus-infected HT-29 cells were treated with CsA at indicated concentrations at 12 h post-infection. Total cell lysates were collected at 24 h post-treatment. (A) Interferon regulatory factor (IRF)-1, -3, -5, and -7 mRNA levels were analyzed by real-time PCR, and protein levels were measured by western blotting. GAPDH was used as an internal control for RT-PCR and a loading control for western blotting. (B) Suppressor of cytokine signaling (SOCS)-1, -2, and -3 mRNA levels were analyzed by real-time PCR, and protein levels were measured by western blotting. GAPDH was used as an internal control for RT-PCR and a loading control for western blotting. Data are expressed as mRNA levels relative (fold) to the control (without CsA treatment, which is defined as 1). Data shown are expressed as mean ± standard deviation of triplicate culture, and 3 independent experiments were carried out (*<i>P</i><0.05).</p

Two chromosome-level genomes of Smittia aterrima and Smittia pratorum (Diptera, Chironomidae)

Abstract Chironomids are one of the most abundant aquatic insects and are widely distributed in various biological communities. However, the lack of high-quality genomes has hindered our ability to study the evolution and ecology of this group. Here, we used Nanopore long reads and Hi-C data to produce two chromosome-level genomes from mixed genomic data. The genomes of Smittia aterrima (SateA) and Smittia pratorum (SateB) were assembled into three chromosomes, with sizes of 78.45 Mb and 71.56 Mb, scaffold N50 lengths of 25.73 and 23.53 Mb, and BUSCO completeness of 98.5% and 97.8% (n = 1,367), 5.68 Mb (7.24%) and 1.94 Mb (2.72%) of repetitive elements, and predicted 12,330 (97.70% BUSCO completeness) and 11,250 (97.40%) protein-coding genes, respectively. These high-quality genomes will serve as valuable resources for comprehending the evolution and environmental adaptation of chironomids

The adsorption behaviours of Pt adatom on pristine and defective bilayer graphene

<p>The structural stability and electronic property of metal Pt atom anchors on two typical substrates (including the pristine and defective bilayer graphene, PBG and DBG) are studied using the first-principles calculations. For the PBG sheets, the Pt atom at the bridge site of bottom layer has only one stable adsorption, which is more stable than other sites of the top layer. For the DBG sheets, the doped Pt below defective site has the larger adsorption energy than that of the upper one. Compared to the isolated graphene films, the Pt(111) substrate-supported graphene systems have effect on the adsorption energies of Pt adatom to some extent, but it does not affect the most preferable configurations. Moreover, the diffusion pathways and energy barriers of Pt adatom on PBG and DBG substrates are comparatively investigated. For the DBG sheets, the Pt dopant has smaller diffusion barrier on upper layer than that of the intercalation process through the defective site. Therefore, the Pt dopant prefers to diffuse on the top layer and then forms the metal impurity. This work provides valuable information on understanding the formation process and intercalation mechanism of metal adatom on graphene sheets.</p