Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity

Abstract

Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection.

Keywords: BmNPV; Bombyx mori; antivirus; thymosin.

Fig. 1.

Western blotting and qRT-PCR analysis of BmTHY expression and transcription in BmN cells challenged by wild-type BmNPV. (A) Western blotting was used to analyze the expression of BmTHY after being challenged with BmNPV in BmN cells. Ten micrograms of total protein extracts were loaded per lane. Tubulin was used as the control. (B) The mRNA expression of the target genes challenged with BmNPV in BmN cells. RNA were extracted from each group of BmN cells. Error bars represent the SEM in three replicates. Asterisk indicates significant difference between control group and experimental group (t-test, P<0.05).

Fig. 2.

Expression and purification of rBmTHY protein. (A) M: Protein Marker: (1) BL21 (pET-28a (+)–BmTHY) induced by 0.5 mmol/liter IPTG at 37 °C for 5 h; (2) the deposition of supersonic fragmentation of BL21 (pET-28a (+)–BmTHY) induced by 0.5 mmol/liter IPTG at 37 °C for 5 h; (3) the supernate of supersonic fragmentation of BL21 (pET-28a (+)–BmTHY) induced by 0.5 mmol/liter IPTG at 37 °C for 5 h; (4) BL21 (pET-28a (+)–BmTHY) without IPTG at 37 °C for 5 h and (B) M: Protein Marker: 1,2,3: samples collected from Wash Buffer and 4,5,6: samples collected from Elution Buffer.

Fig. 3.

Titration curve of extracellular BmNPV virus present after BmN cells infected with BmNPV. The titer was determined on BmN cells by end-point dilution in a 96-well plate according to the method of TCID₅₀.(A) BmN cells treated with PBS control and (B) BmN cells treated with endotoxin-free rBmTHY. Data represent mean±SEM of three independent experiments.

Fig. 4.

Evaluation of antiviral effects of rBmTHY on BmN cells against BmNPV infection. (A) Effects of rBmTHY on BmNPV-infected cell viability. BmN cells were infected with BmNPV and then treated with rBmTHY. Cell viability in each group was evaluated by MTT assays. Cell viability, OD₄₉₀ (experiment group)/OD₄₉₀ (negative group); Nc, negative control group; Vc, viral control group; Ldt, low dose rBmTHY (0.04 μg ml⁻¹) treatment group; Mdt, middle dose rBmTHY (0.4 μg ml⁻¹); Hdt, high dose rBmTHY (4 μg ml⁻¹) treatment group. Data represent mean±SEM of three independent experiments (two-way ANOVA * denotes P<0.05 vs. Vc). (B) Antiviral activity of rBmTHY on BmN cells against BmNPV infection. The antiviral activities of rBmTHY were calculated according to the formula: antiviral activity (%) = (OD_T490)v − (OD_C490)v/(OD_C490)mock − (OD_C490)v×100%. Data are presented as the mean ±SEM of three independent experiments. (C) Morphologies of BmN cells under electron microscope (scale bar=50 μm). (A1 and B1) Cells in negative control group at 48 or 72 h of incubation. (A2 and B2) Cells in drug treatment group (infected with BmNPV and then treated with 4 μg ml⁻¹ rBmTHY) at 48 or 72 h of incubation. (A3 and B3) Cells in viral control group (infected with BmNPV) at 48 or 72 h of incubation. Untreated cells had well-defined, intact shapes with smooth surfaces. Cell morphologies, such as integrity, transmission of light, and surface smoothness, in the drug treatment group were better than that of the viral control group.

Fig. 5.

Survival curve of BmNPV-infected fifth-instar larvae treatment with endotoxin-free rBmTHY. Nc, negative control: treatment with PBS; Vc, virus control: subcutaneous inoculation with wild-type BmNPV; Ldt, treatment with low dose (0.2 μg ml⁻¹) of rBmTHY and infection with BmNPV; Mdt, treatment with middle dose (2 μg ml⁻¹) of rBmTHY and infection with BmNPV; Hdt, treatment with high dose (20 μg ml⁻¹) of rBmTHY and infection with BmNPV. The experiments were carried out in quadruplicate, and each repeat consisted of 50 larvae for each group. Data are presented as the mean±SEM. Asterisk indicates significant difference between control group and experimental group (Gehan–Breslow–Wilcoxon Test, ***P<0.001).