Antiviral Activities of Carbazole Derivatives against Porcine Epidemic Diarrhea Virus In Vitro

Abstract

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, causes neonatal pig acute gastrointestinal infection with a characterization of severe diarrhea, vomiting, high morbidity, and high mortality, resulting in tremendous damages to the swine industry. Neither specific antiviral drugs nor effective vaccines are available, posing a high priority to screen antiviral drugs. The aim of this study is to investigate anti-PEDV effects of carbazole alkaloid derivatives. Eighteen carbazole derivatives (No.1 to No.18) were synthesized, and No.5, No.7, and No.18 were identified to markedly reduce the replication of enhanced green fluorescent protein (EGFP) inserted-PEDV, and the mRNA level of PEDV N. Flow cytometry assay, coupled with CCK8 assay, confirmed No.7 and No.18 carbazole derivatives displayed high inhibition effects with low cell toxicity. Furthermore, time course analysis indicated No.7 and No.18 carbazole derivatives exerted inhibition at the early stage of the viral life cycle. Collectively, the analysis underlines the benefit of carbazole derivatives as potential inhibitors of PEDV, and provides candidates for the development of novel therapeutic agents.

Keywords: antiviral drug; carbazole derivatives; porcine epidemic diarrhea virus.

Figure 1

Chemical structures of 18 synthetic carbazole alkaloids.

Figure 2

Evaluation anti-PEDV activities of 18 carbazole derivatives. Vero-81 cells were treated with 18 kinds (from No.1 to No.18) of carbazole derivatives, and then infected with EGFP-PEDV for 24 h. (a) The fluorescence in the cells were observed by fluorescence microscope. (b) RNA was extracted from PEDV-infected or non-infected vero-81 cells. The relative mRNA level of PEDV N was determined by qRT-PCR. * p < 0.05; ** p < 0.01; *** p < 0.001; ns not significant.

Figure 3

No.5, No.7, and No.18 carbazole derivatives were confirmed to anti-PEDV replication. (a–c) The vero-81 cells were incubated with No.5 (a), No.7 (b), or No.18 (c) carbazole derivatives at a concentration of 10 µM/mL for 1 h, and then infected EGFP-PEDV for 24 h. The fluorescence was analyzed by flow cytometry. *** p < 0.001.

Figure 4

No.7 and No.18 carbazole derivatives had low toxicity to vero-81 cells. (a–c) Cells were treated with increasing concentration (0, 10, 20, 40, 60, 80 µM) of No.5 (a), No.7 (b), or No.18 (c) carbazole derivatives for 24 h. The relative cell viability was evaluated by the CCK8 Kit. The dotted line indicates the 80% cytostatic concentration.

Figure 5

No.7, No.18 carbazole derivatives inhibited PEDV replication were in a dose-dependent manner. The vero-81 cells were incubated with increasing concentrates of carbazole derivatives (10, 20, 40 µM for No.7 (a,c,e); and 20, 40, 60 µM for No.18 (b,d,f)) for 1 h, and then infected with PEDV for 2 h. Then, the cells were treated with the same concentration of carbazole derivatives for 24 h. (a,b) Cells were collected for RNA extraction, and the relative vRNA level was determined by qRT-PCR. (c,d) Supernatants were harvested to determine viral titer by TCID₅₀. (e,f) Cells were lysed and subjected to western blot analysis. ** p < 0.01; *** p < 0.001.

Figure 6

Effects of No.7 and No.18 carbazole derivatives on PEDV replication were time-dependent. (a) Schematic representation of carbazole derivatives treatment time. (b,c) Vero-81 cells were incubated with PEDV (MOI = 0.1) for 1 h, followed by treatment with 40 µM No.7 (b) or No.18 (c) carbazole derivatives at the indicated time (hpi). The viral titer in cell supernatant was detected by TCID₅₀, and calculated by the Reed–Muench method. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

No.7 and No.18 carbazole derivatives inhibit PEDV attachment, but not entry. (a,b) Levels of vRNA in cells treated with 40 µM No.7 (a) or No.18 (b) carbazole derivatives during viral attachment and entry were determined by qRT-PCR.