Screening for anti-influenza virus compounds from traditional Mongolian medicine by GFP-based reporter virus

Abstract

Introduction: Screening for effective antiviral compounds from traditional Mongolian medicine not only aids in the research of antiviral mechanisms of traditional medicines, but is also of significant importance for the development of new antiviral drugs targeting influenza A virus. Our study aimed to establish high-throughput, rapid screening methods for antiviral compounds against influenza A virus from abundant resources of Mongolian medicine.

Methods: The use of GFP-based reporter viruses plays a pivotal role in antiviral drugs screening by enabling rapid and precise identification of compounds that inhibit viral replication. Herein, a GFP-based reporter influenza A virus was used to identify potent anti-influenza compounds within traditional Mongolian medicine.

Results: Our study led to the discovery of three active compounds: Cardamonin, Curcumin, and Kaempferide, all of which exhibited significant antiviral properties in vitro. Subsequent analysis confirmed that their effectiveness was largely due to the stimulation of the antiviral signaling pathways of host cells, rather than direct interference with the viral components, such as the viral polymerase.

Discussion: This study showcased the use of GFP-based reporter viruses in high-throughput screening to unearth antiviral agents from traditional Mongolian medicine, which contains rich antiviral compounds and deserves further exploration. Despite certain limitations, fluorescent reporter viruses present substantial potential for antiviral drug screening research due to their high throughput and efficiency.

Keywords: Cardamonin; Curcumin; GFP-based reporter virus; influenza A virus; kaempferide; traditional mongolian medicine.

Figure 1

Workflow for screening anti-IAV compounds using GFP-based reporter virus. The workflow was designed as followed. (A) Eight-Plasmid Schematic of GFP-IAV reverse genetics. (B) 293T cells were transfected with eight engineered plasmids, and the viruses harvested at 48 h post-transfection were subsequently inoculated into MDCK cells to be further amplified for a 72-hour duration. (C) The schematic diagram of GFP-IAV. GFP fluorescence could be detected when MDCK cells were infected with GFP-IAV. (D) Cells infected with GFP-IAV were treated with candidate anti-viral drugs. The expression of GFP would be reduced if the drug exhibited antiviral activity against IAV; conversely, higher expression GFP levels indicated a lack of antiviral efficacy. (E) Following the screening process, drugs with antiviral activity were subjected to further experimental validation. Statistical significance was calculated using two-way ANOVA (****p < 0.0001).

Figure 2

The construction and biological identification of the GFP-IAV. (A) The plasmid construction schematic of NA-2A-GFP. (B) MDCK cells were infected with the GFP-IAV and the expression of GFP was observed at 12, 18, and 24 h post-infection using a fluorescence microscope. (C, D) MDCK cells were infected with GFP-IAV (C) and wild-type IAV (D) (moi=0.001) and the supernatant was collected at 12, 18, and 24 h post-infection. The viral titers were then determined by plaque assay. (E–G) Correlation analysis of the titers (C), RNA copy numbers (D) and protein expression (E) between wild-type IAV and GFP-IAV at different time points during their proliferation process. (H) GFP expression of GFP-IAV after the first generation and six consecutive passages.

Figure 3

Three Mongolian medicine compounds with antiviral activity identified by GFP-IAV. (A) MDCK cells were infected with GFP-IAV and added natural compounds (Kaempferide, Curcumin and Cardamonin) to the supernatant at concentration of 50 μM and 25 μM, with Zanamivir as positive control and DMSO as negative control. GFP fluorescence was observed at 24 h post-infection. (B) MDCK cells were infected with GFP-IAV and added natural compounds (Kaempferide, Curcumin and Cardamonin) to the supernatant at different concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125μM and 1.5625 μM, with Zanamivir as positive control and DMSO as negative control. Virus titer was determined by qRT-PCR at different time points (12 h, 24 h, 48 h and 72 h post-infection) and converted to pfu numbers by standard curve. Virus growth curves were examined. (C) Chemical structures of Kaempferide, Curcumin, Cardamonin and Zanamivir. Statistical significance was calculated using two-way ANOVA (*p < 0.05; **p < 0.01; ****p < 0.0001).

Figure 4

Three compounds have significant inhibitory effects on the wild-type IAV. (A) MDCK cells were infected with the wild-type IAV (moi=0.001) and added natural compounds (Kaempferide, Curcumin and Cardamonin) to the supernatant at concentration of 50 μM, with Zanamivir as positive control and DMSO as negative control. The IAV titers were determined through plaque assays at 48 h post-infection. (B) MDCK cells were infected with wild-type IAV and added natural compounds (Kaempferide, Curcumin and Cardamonin) to the supernatant at different concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM and 3.125 μM, with Zanamivir as positive control and DMSO as negative control. Virus titer was determined by qRT-PCR at different time points (12 h, 24 h, 48 h and 72 h post-infection) and converted to pfu numbers by standard curve. Virus growth curves were examined. (C) 293T cells were co-transfected with pHW2000 plasmids encoding PB2, PB1, PA, and NP proteins, pHH21 plasmid expressing negative vRNA-like firefly luciferase (Fluc) RNA and plasmid encoding renilla luciferase (Rluc) as internal reference. The cells were then incubated with various concentrations (25 μM, 12.5 μM, 6.25 μM and 3.125 μM) of natural compounds (Kaempferide, Curcumin and Cardamonin), with Baloxavir marboxil as positive control and DMSO as negative control. Luciferase activity was measured at 24 h after transfection. Statistical significance was calculated using two-way ANOVA (**p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 5

Three compounds regulated the antiviral signaling pathways in host cells. (A, D, G) Scatter plots showing the genes that were differentially expressed within the A549 cells after Kaempferide (A), Curcumin (D) and Cardamonin (G) treatment, with DMSO as negative control. The red plots indicated the up-regulation genes, the green plots indicated the down-regulation genes and the grey plots indicated the un-changed genes. P value < 0.05 and |log2FoldChange| > 0. (B, E, H) KEGG pathways enrichment analysis of differential expressed genes associated with viruses after A549 cells treated with Kaempferide (B), Curcumin (C) and Cardamonin (D). (C, F, I) Heatmaps of genes enriched in the top ten up and down-regulated KEGG pathways enrichment analysis after A549 cells treated with Kaempferide (C), Curcumin (F) and Cardamonin (I). The colors depict the log2 fold change compared to the mock group. Each cell represents the average of the three samples.